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Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1331

Substance P EndopeptidasePurification and Characterizataion of Enzyme Activity andEvaluation of its Function during Stressful Condition

BY

KRISTER KARLSSON

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2004

Dissertation presented at Uppsala University to be publicly examined in B41, BMC, Uppsala, Thursday, April 29, 2004 at 09:15 for the degree of Doctor of Philosophy (Faculty of Medicine).

ABSTRACTKarlsson, K., 2004, Substance P Endopeptidase; Purifi cation and Characterization of Enzyme Activity and Evaluation of its Function during Stressful Condition, Acta Universitatis Upsaliensis, Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1331, 56 pp. Uppsala. ISBN 91-554-5908-0.

The purifi cation and biochemical characterization of the substance P (SP) hydro-lyzing enzyme, substance P endopeptidase (SPE), have been carried out; with sub-sequent orientation in neurobiological fundamental processes involved in opioid dependence, withdrawal, and heat-stress.SPE was purifi ed from rat spinal cord, human spinal cord and cerebrospinal fl uid (CSF), rat ventral tegemental area (VTA), and rat hippocampus. The enzyme activ-ity was found to release the biologically active fragments SP(1-7) and SP(1-8) as major products. The purifi ed enzymes were characterized with regard to their bio-chemical and kinetic properties. The typical SPE is neither inhibited by phosphor-amidon nor captopril nor phenylmethanesulfonylfl ourid (PMSF). In comparison to other known proteases SPE differed in characteristics regarding substrate specifi city, inhibition-profi le, cleavage pattern, and other kinetic parameters. The technically very delicate approach of micro purifi cation of SPE from the rat ventral tegemental area (VTA) (this is a very small tissue), turned out to be possible with the ÄKTA™-purifi er system. Studies revealed a crucial role of SPE in a series of clinically important neuropathological conditions, such as opioid tolerance, and withdrawal (SPE, increased); and heat-stress (SPE, increased). These fi ndings emerged from assessment of enzyme activity in hypothalamus, nucleus accumbens (NAc) periaq-ueductal gray (PAG), pituitary, striatum, substantia nigra (SN), VTA, spinal cord. Viewing the role of SPE in morphine tolerance, it was possible to note regional dif-ferences with a decrease in PAG, and striatum, whereas an increase was seen in SN, and VTA. After heat-stress treatment, SPE was raised in several regions (cerebral cortex, hippocampus, diencephalon, cerebellum, spinal cord), and the most precise observation of this was located to the hippocampus structure.

Krister Karlsson, Department of Neuroscience, Box 587, Uppsala University, SE-751 24, Uppsala, Sweden

© Krister Karlsson 2004ISSN 0282-7476ISBN 91-554-5908-0urn:nbn:se:uu:diva-4130 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-4130)

Printed in Sweden by University Printers, Uppsala 2004

This thesis is dedicated to:

Olle, Solveig, Katarina, Maria, Elsa†

andSeok-Jo, Kyung-Ka†, Nam-Sook, Seog-Hwon

†In memory of

PAPERS DISCOURSED

I. Karlsson, K., Eriksson, U., Andrén, P., and, Nyberg, F., (1997), Purifi cation and characterization of substance P endopeptidase activities in the rat spinal cord, Prep. Biochem. Biotechnol., 27(1), 59-78.

II. Karlsson, K., and, Nyberg, F., (1998), Purifi cation of substance P endopepti-dase (SPE) activity in human spinal cord and subsequent comparative studies with SPE in cerebrospinal fl uid and with chymotrypsin, J. Mol. Recogn., 11,266-269.

III. Karlsson, K., and, Nyberg, F., (2000), Purifi cation of substance P endopep-tidase activity in the rat ventral tegemental area with the Äkta-Purifi er chro-matographic system, J. Chromatogr. A, 893, 107-113.

IV. Zhou, Q., Karlsson, K., Liu, Z., Johansson, P., Le Grevés, M., Kiuru, A., and, Nyberg, F., (2001), Substance P endopeptidase-like activity is altered in vari-ous regions of the rat central nervous system during morphine tolerance and withdrawal, Neuropharmacol, 41, 246-253.

V. Karlsson, K., Sharma, H., and, Nyberg, F., (2004), Substance P endopeptidase in the rat brain - the activity of the enzyme is affected following heat stress, Submitted

ABBREVIATIONS

5-HT 5-hydoxytryptamine (=serotonin)ACE angiotensin converting enzymeACh acetylcholineACTH adrenocorticotropic hormoneAPN aminopeptidase N (/M)cAMP cyclic adenosine mono phosphateCCK cholecystokininCNS central nervous systemCSF cerebrospinal fl uidDA dopamineDP-IV dipeptidyl aminopeptidase IVEDTA ethylenediamine tetraacetic acidES electrosprayGABA gamma-aminobutyric acidGnRH gonadotropin-releasing hormoneHPLC high performance (/pressure) liquid chromatographyi.c.v. intra cerebroventriculari.p. intra peritonealMS mass spectrometryNAc nucleus accumbensNE norepinephrine (=noradrenaline)NEP neutral endopeptidase (/enkephalinase, /EC 3.4.24.1)NK-1 neurokinin-1 receptor (“substance P receptor”)NMDA N -methyl- D -aspartateNO nitric oxideNOS nitric oxide synthaseNPY neuropeptide YNT neurotrophinPAG periaqueductal grayPHMB p -hydroxymercuribenzoatePMSF phenylmethanesulfonyl fl uoridePPCE postproline cleaving enzymeRIA radioimmunoassays.c. subcutaneousSP substance PSP(1-7) substance P fragment (1-7)SPE substance P endopeptidaseTFA trifl uoroaceticacidTRH thyrotropin releasing hormoneVIP vasoactive intestinal polypeptideVTA ventral tegemental area

CONTENTS

1. INTRODUCTION................................................................................ 1

1.1. Neuropeptides as Transmitters ........................................................ 21.1.1. Background ..................................................................................................31.1.2. Modes of action ............................................................................................21.1.3. From biosynthesis to inactivation..................................................................31.1.4. Localization ..................................................................................................41.1.4.1. Distribution in the nervous system.............................................................41.1.4.2. Coexistence with classical transmitters .......................................................5

1.2. Drug Dependence, Reinforcement, and Withdrawal....................... 51.2.1. The anatomical basis of biological dependence .............................................51.2.2. Mechanism of reward and dependence .........................................................71.2.3. Withdrawal...................................................................................................81.2.4. Drug craving and relapse ..............................................................................8

1.3. Hyperthermia Induced Pathology.................................................... 91.3.1. Heat related illness........................................................................................91.3.2. Hyperthermia induced brain dysfunction.....................................................91.3.3. Stress component of heat pathology ............................................................10

1.4. Tachykinins, Substance P and Substance P Metabolites ................111.4.1. Background ................................................................................................111.4.2. Physiological role ........................................................................................111.4.3. Genes and peptide maturation ....................................................................121.4.4. Receptors ....................................................................................................121.4.5. Clinical settings ..........................................................................................131.4.6. Role in physical dependence and withdrawal ..............................................14

1.5. Inactivation and Conversion of Tachykinins by Peptidases .......... 151.5.1. Substance P degrading enzymes, in addition to SPE...................................151.5.1.1. Angiotensin Converting Enzyme ............................................................. 151.5.1.2. Endopeptidase 24.11 .............................................................................. 17

1.5.2. Substance P Endopeptidase.........................................................................171.5.3. Inhibition of Substance P Endopeptidase....................................................19

1.6. Strategies to Protein Purifi cation....................................................191.6.1. Basic approach and guidelines.....................................................................191.6.2. Three-phase strategy to purity................................................................... 20

2. AIMS OF THE THESIS ......................................................................21

3. EXPERIMENTAL PROCEDURES ................................................... 22

3.1. Purifi cation of Enzyme Activity..................................................... 223.1.1. Substance P endopeptidase (SPE) purifi cation from rat spinal cord........... 223.1.2. Substance P endopeptidase (SPE) purifi cation from human spinal cord.... 233.1.3. Substance P endopeptidase (SPE) purifi cation from rat ventral

tegemental area (VTA)............................................................................... 23

3.2. Assessment of Enzyme Activity ...................................................... 24

3.3. Sodium Dodecylsulphate-Polyacrylamide Gel Electrophoresis (SDS-page) With Silver Staining................................................... 24

3.4. Characterization of Enzyme Activity by High Pressure Liquid Chromatography (HPLC) .............................................................. 25

3.5. Characterization of Enzyme Activity by Mass Spectrometry(MS) 25

3.6. Animal Treatment and Preparation ............................................... 25

3.7. Heat-Stress Treatment ................................................................... 26

4. RESULTS AND DISCUSSION .......................................................... 27

4.1. Enzyme Purifi cation of Substance P Endopeptidase (SPE) ........... 274.1.1. Purifi cation from rat spinal cord .................................................................274.1.2. Purifi cation from human spinal cord ..........................................................314.1.3. Purifi cation from rat ventral tegemental area (VTA) ..................................33

4.2. Enzyme Chemical Characterization of Substance P Endopeptidase (Spe) ...................................................................... 36

4.2.1. Cleavage pattern ........................................................................................ 364.2.2. Substrate specifi city.....................................................................................374.2.3. Inhibition profi le.........................................................................................37

4.3. Functional Characteristics of Substance P Endopeptidase (SPE).. 394.3.1. Central nervous system distribution ...........................................................394.3.2. Pattern in morphine tolerance and withdrawal .......................................... 404.3.3. Behavior in heat-stress ................................................................................41

5. CONCLUSIONS ................................................................................. 43

6. ACKNOWLEDGEMENTS ................................................................ 44

7. REFERENCES.................................................................................... 45

1

Substance P Endopeptidase

1. INTRODUCTION

Substance P Endopeptidase (SPE) is an enzyme capable of degrading the neuro-active undeca-peptide Substance P (SP) (Fig. 1.1). SP is involved in a variety of biological functions such as gastric secretion, infl ammation, and pain transmission. Recently it has become evident that SP also plays a crucial role in neurodegenerative disease, memory, and affective disorders. SP has been shown to be involved in physi-cal dependence, tolerance, and withdrawal. The fact that the same neuropeptide performs such a vast variety of biological functions, depending on regional distribu-tion, is a common theme. Obviously the body uses these peptides to conduct differ-ent tasks in different parts of the body. It is therefore important to take the whole messaging scenario into account in order to understand the role of a given peptide in a certain area of the body. Peptides, may act as modulators of other transmitters’ function, or adapt a transmitter role, having intrinsic effects of their own, or act as hormones. In the nervous system, SP should be studied in perspective of neuronal structures, synthesis and release, co-transmission of classical transmitters, receptor proteins, and inactivating/converting enzymes. This thesis is developing the per-ception of SPE’s role in SP messaging functions in the CNS. Characterization is performed in regard to protein chemical properties, as well as integrative function in opioid physical dependence, and withdrawal, and heat-stress. Therefore, in the following the concept of opioid tolerance and dependence as well as stress in relation to heat will be discussed. However, this thesis summary will fi rst go through some general aspects with regard to biosynthesis of neuropeptides and their function as transmitters in peptidergic neurons.

Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2Fig. 1.1 Amino acid sequence of substance PThe eleven residue long peptide is amidated at the C -terminal. Belonging to the tachykinin peptide family. It contains the common C -terminal sequence Phe-X-Gly-Leu-Met-NH

2 where X repre-

sents Tyr or Phe.

1.1. Neuropeptides as Transmitters

1.1.1. Background

The concept of neuropeptides evolved during a long period of years. Some peptide-fractions had been discovered as early as in the beginning of the 20th century, such as the gut peptide secretin. Substance P was discovered in the 1930s. But it was not until the late 1960s and 1970s that the peptidergic nature of the hypothalamic releasing factors was discovered. In fact the peptide sequencing of substance P was not done until 1972 by Lehman et. al. In the same period it was observed that the common gut peptides (cholecystokinin (CCK), vasoactive intestinal polypeptide (VIP), and somatostatin) were also produced in the brain.

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Krister Karlsson

Neuropeptides exert their effects on neurons by acting as neurohormones, neuro-modulators, or neurotransmitters, via specifi c peptidergic receptors. This fact, that brain peptides have a direct effect on neurons, via specifi c peptidergic receptors, and that the peptides have highly specifi c physiological properties, were making the foundation to the neuropeptide concept[50; 125]. Neuropeptides can act on non-neu-ronal tissues, as well as on neurons, in so, integrating the functions of the brain and the rest of the body. Some of the widespread functions in which the neuropeptides are involved include: regulation of reproduction, growth, water and salt metabolism, temperature control, food and water intake, cardiovascular, gastrointestinal, and respiratory control, behavior, memory, and affective states. They also affect nerve development and regeneration, as well as apoptosis. Often they are co-localized with classical neurotransmitters such as acetylcholine (ACh) and the monoamines. The cloning of peptide receptors, and development of selective agonists/antagonists as well as the identifi cation of the hydrolyzing enzymes responsible for the regulation of peptide conversion/inactivation will probably contribute to a great signifi cance to the expansion of physiological knowledge leading to enhanced basic science, and clinical therapeutic applications for many pathological conditions.

1.1.2. Modes of action

Classical neurotransmitters (such as ACh, epinephrine, NE, DA, 5-HT, glutamic and aspartic acid, and GABA) exhibit a set of accepted characteristics, which are: specifi c affi nity binding to membrane receptors, Ca2+-dependent response, change in the membrane potential of the postsynaptic cell, release from synaptic vesicles in nerve terminals after nerve stimulation, and existence of an inactivating mechanism(s). The action by these transmitters is typically possible to counteract by application of an antagonist. Such a procedure clearly facilitate the ligand-recep-tor concept of transmitter action. Ligands acting through voltage-gated channels are called ionotropic neurotransmitters, and cause a rapid effect on the neuron. Metabotropic transmitters, on the other hand, act more slowly through second messengers. Synthesis of transmitters is rapid in the nerve terminals, so depletion of transmitter is unlikely. They are stored in small, clear vesicles in the nerve terminals. On the other hand neuromodulators do not evoke an effect on their own but alter the effects of a classical transmitter at the synapse. Their action is slow and long lasting as they act through second messengers. They are released in response to an action potential but their release requires a longer duration and higher frequency of stimulation. They are effective in very low concentrations but their biological activity may vary according to the length of the processed peptide. The inactiva-tion mechanism seems to be more generalized. Changes in second messengers affect membrane permeability to ions and thus can change the excitability of the postsynaptic membrane to transmitters. Neuromodulators may also affect receptor characteristics, whether the receptors are pre- or postsynaptic, or autoreceptors, and thus can inhibit or facilitate the release of other co-localized transmitter or neuro-peptides, or even their own release. Neuropeptides are stored in large, dense vesicles

3


which are not always limited to nerve terminals so that their action may be wide-spread. There is convincing evidence for SP[114] and GnRH (LHRH)[63; 110] as neu-rotransmitters. Vasopressin and oxytocin appear to act as excitatory transmitters in the CNS, but also as neuromodulators[136; 206; 280]. Somatostatin may act as either an excitatory or inhibitory transmitter depending on dosage, type of cell investigated, and the duration of application[62; 128; 153]. Thyrotropin releasing hormone (TRH) has both neurotransmitter and neuromodulator actions[77].

1.1.3. From biosynthesis to inactivation

Prior to release neuropeptides are synthesized fi rst as large preprohormones, i.e. poly-peptide chains including a signal sequence, which is removed by an endopeptidase action following translation (Fig. 1.2). The remaining peptide chain, the prohor-mone, is inactive and must be processed proteolytically into smaller fragments, both biologically active and inactive peptides. The prohormone is transported through the cis -face of the Golgi network to the trans -Golgi network where it is packaged into secretory granules, in which proteolytic processing by endoproteases (prohor-mone convertases), followed by exoproteases, continues, excising biologically active (and some inactive) sequences. Cleavage usually occurs at bibasic amino acid sites. Proteolysis begins at the trans -Golgi network and continues in the granules. During the transit through the Golgi network, the precursors may be subjected to enzymatic modifi cations such as glycosylation, phosphorylation, sulfatation, or hydroxylation. Regulated exocytosis of neuropeptides, which are contained in large dense core vesicles (LDCVs), is Ca2+-dependent. They may be released together with small, clear synaptic vesicles containing classical transmitters. Unlike the classical trans-mitters, neuropeptides are not recycled at the nerve terminals but have to be refi lled by newly synthesized neuropeptides delivered from the cell body. Again, unlike the relatively specifi c inactivation mechanisms for the classical neurotransmitters, there is little intracellular neuropeptide inactivation, apart from degradation to inactive metabolites in the RER or in the Golgi complex. Inactivation by peptidases occurs rapidly in plasma and in other cells.

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Krister Karlsson

Fig. 1.2 The peptidergic neuron, schematic representationThe neuropeptides differ from the classical transmitters in respect of their cellular location of syn-thesis. Neuropeptides begin their synthesis in the cell body (soma), and continue to mature while transported down the axon. Additionally, they are packed into “large dense core vesicles” and are inactivated or converted by peptidase action. Unlike the classical transmitters they are, as far as is known, not inactivated by any re-uptake mechanism.

1.1.4. Localization

1.1.4.1. Distribution in the nervous system

The richest sources of neuropeptides in the CNS are several hypothalamic nuclei, the preoptic area, and the pituitary gland[193]. However, many of the neurosecretory hypothalamic neurons have projection axons long enough to reach fairly distant areas of the CNS. Therefore, these neuropeptides can be found in high concentra-tion in nerve terminals in many regions of the brain and spinal cord. Peptidergic cell bodies are also found in many other brain areas. In addition to their concentration in hypothalamic nuclei and the pituitary gland, several neuropeptides such as soma-tostatin, TRH, neurotensin, substance P, insulin, CCK, VIP, and the enkephalins

5


are found in perikarya, generously distributed in certain areas of the CNS, areas that are rich in both peptide-immunoreactive cell bodies and terminals. These include the dorsal horn of the spinal cord, the dorsal vagal complex, the nucleus accumbens, the stria terminalis bed nuclei, the amygdala, and the periaqueductal central gray. Some neuropeptides appear to be restricted to the CNS, others are more widely distributed in various regions of the body, especially mucosal cells in the gastrointestinal tract, the pancreas, adrenal medulla, gonads, the placenta, and peripheral nerves, as well.

1.1.4.2. Coexistence with classical transmitters

Dale noted in 1935, that if the sensory peripheral terminals of a neuron secreted ACh, then it was likely that the central terminals of this neuron would secrete the same chemical. This concept was extended by Eccles [61] into what he termed “Dale’s principle”, stating that “any one class of nerve cells operates at all its synapses by the same chemical transmission mechanism”. In the beginning, it was blieved that only one transmitter could be synthesized by a neuron, but it is now know that several transmitters, including neuropeptides, can be produced and secreted by a neuron. It is still assumed that all branches of the neuron contain the same set of neurotrans-mitters, supporting Dale’s principle of the metabolic and functional unity of the neuron. It has been suggested that since the smaller neurotransmitter molecules are stored in small vesicles and the larger neuropeptide molecules are stored in dense-core vesicles, that there could be some selectivity of one axonal branch for the larger or smaller package[276]. Hökfelt et al., in a series of classic papers[94; 95; 96; 97],have clearly demonstrated the coexistence of neurotransmitters and neuropeptides within the same neuron, sometimes even within the same vesicle, in both central and peripheral neurons. Interestingly, there is little evidence for the coexistence of two or more classical neurotransmitters, although GABA has been shown to be co-localized with DA or 5-HT[12]. In some neurons, two or more neuropeptides may be present in the same or separate secretory granules depending on the processing of the prohormone.

1.2. Drug Dependence, Reinforcement, and Withdrawal

1.2.1. The anatomical basis of biological dependence

Addictive drugs are both rewarding and reinforcing[282], and the neuro anatomi-cal structures that convey this are referred as the brain reward pathways[13; 16; 21; 44;

67; 88; 134; 135; 210; 214; 215; 246; 247; 281; 283]. These include the nucleus accumbens (NAc; the major component of the ventral striatum), the basal forebrain (components of which have been termed the extended amygdala), and regions of the medial prefrontal cortex (Fig. 1.3). These structures receive rich dopaminergic innervation from the ventral tegemental area (VTA) of the midbrain. It appears that addictive drugs are rewarding and reinforcing because they act in brain reward pathways to enhance either dopamine release or the effects of dopamine in the NAc or related structures.

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Krister Karlsson

Drug-induced pleasurable states are important motivators of initial drug use. Drug actions that produce these states also produce associated, but ultimately undesir-able, changes in brain reward circuitry that promote future drug use. Another form of positive reinforcement involves the alleviation of unpleasant symptoms[37; 65; 106;

196; 213; 270], either from pre-existing states or caused by drug withdrawal, by means of drug use. Conditioned reinforcement, which occurs when previously neutral stimuli becomes associated with the pleasurable effects of drugs, is yet another type of positive reinforcement[107; 285]. All of these mechanisms contribute to repetitive drug taking that, in vulnerable individuals, may result in an addicted state. Labora-tory animals can learn behaviors necessary to self-administer such drug, and some of them will give up survival necessities, such as food and water, or work excessively, even to the point of death, in order to gain access to it. Another model used to inves-tigate drug reward in animal research is known as conditioned place preference. In this type of experiment, animals learn to associate a particular environment with passive drug exposure. With suffi cient training, the neutral cue becomes a condi-tioned reinforcer, or a desirable phenomenon that animals will exhibit appropriate behavior, such as pressing a lever, to obtain. Stimulation of the medial forebrain bundle and closely associated areas, results in the strongest reinforcement of paired behavior. The medial forebrain bundle consists of ascending and descending fi ber tracts that connect rostral basal forebrain and midbrain structures, and includes dopaminergic, noradrenergic, and serotonergic fi bers derived from monoamine nuclei of the brainstem[71; 76; 268]. Mesocorticolimbic dopaminergic projections (Fig. 1.3) originate in the VTA of the ventral midbrain and project through the medial forebrain bundle to limbic and forebrain structures[68]. In vivo micro dialysis stud-ies have indicated that addictive drugs cause selective elevation of extracellular DA levels in the medial subdivision of the NAc, known as the shell[89; 168].

7


Fig. 1.3 Neuroanatomical basis of drug reward illustrated by the rat brainAC, anterior commissure; AMG, amygdala; ARC, arcuate nucleus; Cer, cerebellum; C-P, caudate-putamen; DMT, dorsomedial thalamus; FC, frontal cortex; Hippo, hippocampus; IF, inferior col-liculus; LC, locus coeruleus; LH, lateral hypothalamus; NAc, nucleus accumbens; OT, olfactory tract; PAG, periaqueductal gray; RPn, raphe pontis nucleus; SC, superior colliculus; SNr, substantia nigra pars reticulata; VP, ventral pallidum; VTA, ventral tegemental area.

1.2.2. Mechanism of reward and dependence

There is a number of factors that motivate continuous opioid intake, such as their ability to relieve preexisting dysphoria, and abolish unpleasant symptoms of with-drawal, as well as, production of intense craving after long term use[17; 19; 38; 46; 109;

111; 118; 259; 293]. Opioids bind to the endogenous µ, δ, and κ-opioid receptors[57; 70].These receptors belong to the G-protein coupled receptor super family and exhibit signifi cant homology. Opioids are hypothesized to activate brain reward circuitry by means of: 1) disinhibition of the VTA, which results in dopamine release in the NAc, and 2) dopamine-independent activity in the NAc[57; 70]. Although the injec-tion of morphine, selective µ or δ opioid receptor agonists, or enkephalin analogs into several areas of the brain can be reinforcing, the VTA is particularly sensitive. Moreover, reinforcing effects of intravenous heroin can be partly attenuated by administration of an opioid receptor antagonist directly into the VTA or by lesions in dopaminergic neurons of the VTA[9]. Opiate activation of such neurons results from opiate inhibition of the GABAergic interneurons in the VTA that normally inhibit principal dopamine neurons[23]. Opiates also produce reinforcement through direct dopamine-independent actions on µ, and perhaps δ, receptors expressed in NAc neurons[7; 39; 49; 86; 255; 256]. These receptors are normally targets of the enkepha-linergic (and possibly endorphinergic) neurons that innervate this brain region. Within the NAc, opiates appear to directly inhibit the populations of medium spiny projection neurons that are inhibited by dopamine. Thus opioid and dopaminergic

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Krister Karlsson

systems appear to converge on a common efferent reward pathway in the NAc[266]. µand δ opioid receptor subtypes, both of which are present in the NAc and VTA, are important factors in opiate reinforcement. In contrast, κ opioid receptor activation is not reinforcing, rather aversive in both animals and humans.

1.2.3. Withdrawal

Withdrawal symptoms associated with physical dependence vary depending on the type of drug that has triggered dependence. Such symptoms are mediated by adapta-tions in neural substrates that are specifi cally mediated by a given drug[235]. It is more known about adaptations that produce tolerance and physical dependence, than about adaptations related to the emotional and motivational aspects of dependence. Tolerance and physical dependence are believed to result from homeostatic processes in the body, which are initiated to counteract the effects of continuous drug pres-ence, and to return the system to a normal. The emergence of withdrawal signs in response to cessation of drug use in a physically dependent individual, indicates that the body has adapted to the presence of the drug and, by virtue of the adaptation, requires it for normal function. During withdrawal, the overcompensated system is suddenly unopposed by the drug. Consequently, withdrawal symptoms appear that generally are opposite to the immediate effects produced by drug exposure[24;

78]. Opiate tolerance and dependence cannot be adequately explained by changes in endogenous opioid peptides or in opioid receptor affi nity or number[257; 292]. Rather, changes in intracellular signaling proteins have been implicated as important con-tributors to physical dependence, and possibly tolerance. The locus coeruleus (LC), located in the dorsal pons, is the major noradrenergic nucleus of the brain and is important for the regulation of attentional states and autonomic nervous system activity. The LC also has been implicated in the autonomic and stress-like effects of opiate withdrawal[104; 117; 179; 189; 197; 225; 229; 261; 267]. The activity and stress-like effects of opiate withdrawal. The activity of LC neurons is inhibited by opiate withdrawal. The excitation of LC neurons during opiate withdrawal is suffi cient to produce many of the signs and symptoms of physical withdrawal. These observations are coupled to the alterations in the cAMP-signaling pathway in LC neurons, which contribute to the changes in excitability that these neurons undergo.

1.2.4. Drug craving and relapse

Drug craving can be defi ned as the desire to reexperience the effects of a psychoac-tive substance. It may contribute to motivation of drug intake and dependence, and to trigger relaps of abuse in response to stress or conditioned stimuli. It is believed that relaps refl ects permanent synaptic remodeling in the brain, as opposed by the reversible adaptations that are seen during tolerance and withdrawal[58; 69; 72; 98; 131;

165; 180; 224; 258; 272]. It is proposed that sensitization of neural processes related to drug craving, or cues lead to the progressive increase in drug-seeking behavior. Cross-sensitization among reward stimulant drugs occurs, and is consistent with the involvement of common neurobiologic mechanisms[42; 74; 119; 138; 211]. Stimulation of drug-seeking behavior by drug-associated stimuli and stress is believed to be medi-

9


ated by dopamine-dependent and dopamine-independent mechanisms. Drug-asso-ciated stimuli are believed to activate circuits of the amygdala, which are implicated in memories of emotionally salient events. The amygdala, in turn, is believed to acti-vate dopaminergic neurons of the VTA directly, by means of glutamatergic or CRF inputs, and indirectly, through the prefrontal cortex. Stress is believed to activate the mesolimbic dopamine system through effects on the prefrontal cortex, amygdala, and hypothalamic-pituitary adrenal (HPA) axis. The effect of stress induced activa-tion of the HPA axis may be mediated by CRF or by increased levels of circulating glucocorticoids[130; 228; 239; 240; 274; 279]. Thus both sensitization and emotional cues seem to be involved in the craving mechanism.

1.3. Hyperthermia Induced Pathology

1.3.1. Heat related illness

Heat related illness can be divided into heat cramp, heat exhaustion and heat stroke[271]. Heat cramp is presented as painful cramping of skeletal muscles, and may appear after intensive workload. Heat exhaustion is associated with dehydration or salt depletion after prolonged work in high ambient temperature such as during heat waves. The heat exhausted exhibit intense thirst, fatigue, weakness, or anxiety and impaired judgment, which may lead to delirium and coma, hypotension, and tachy-cardia. Heat stroke is characterized by hyperpyrexia of more than 41 ºC associated with delirium, coma and anhidrosis. Heat stroke is the most severe of heat related illnesses, because it occurs in epidemic form and gives up to 60 to 80% mortality. The heat stroke survivors, often exhibit persistent mental disability.

There are a number of factors that increase the risk of heat related injury or illness. Since thermal balance is associated with circulation, the role of heart in the patho-genesis of heat stroke was recognized as early as in 1923 by Adolph who observed that patients with cardiovascular disease tolerate high environmental heat very poorly. Burch and his co-workers examined the effects of high temperature (32.2 ºC) and high humidity (75 %) on 23 patients in congestive heart failure [4; 5; 28; 29; 30;

55]. The outcome showed, that the cardiac output failed to rise normally and many subjects developed angina pectoris with electrocardiographic changes and some demonstrated a rise of venous pressure. Another risk factor in heat-stress, is chronic diseases, such as hypertension, diabetes mellitus, malnutrition and both acute and chronic alcoholism. These conditions increase the sensitivity to heat injury[132; 133].Impaired sweat production which occurs in advanced progressive systemic sclerosis (scleroderma), extensive dermal burns, cystic fi brosis, barbiturate intoxication, con-gestive heart failure, and pharmacotherapy with atropine, phenothiazines, antihis-tamines, anti-Parkinson’s disease drugs.

1.3.2. Hyperthermia induced brain dysfunction

Prolonged hyperthermia of any form is likely to cause irreversible tissue damage and cell death. However, the probable mechanisms of hyperthermia-induced brain

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Krister Karlsson

damage are not well known. Edema and micro hemorrhages in the brain damage is poorly studied. Nevertheless these are prominent fi ndings after hyperthermic insults to the brain in both clinical and in experimental observations[234].

Some animal models have been used to study focal brain heating. Those included the use of microwave heating, radio frequency, or water bath application[79; 80; 81; 245].The pathological fi ndings in these studies were hemorrhages, necrosis, infl ammation and gliosis. Lethality after hyperthermic insults to the brain was very common.

Pathological changes in the human brain have been documented from clinical cases[231]. Gross pathology reveals increase in the brain weight by several hundred grams. This indicates that edema is a common feature in heat-related illness. Edema and Microhaemorrhages are prominent in the leptomeninges and are confi ned to the perivascular space. Most pronounced hemorrhages can be seen in the para ventricular nucleus, the supra optic nucleus, medial parts of the ventromedial and dorsomedial hypothalamic nuclei. No hemorrhage is observed in the mammillary body. In pons and medulla oblongata the hemorrhage is mainly confi ned to the fl oor of the fourth ventricle and sometimes near the dorsal efferent nucleus of the vagus. The perifornical and septal regions and the medial portion of the thalamus and the caudal part of the hypothalamus are least affected. Light microscopy displays edema and congestion in the cerebral cortex with signs of degenerative changes in the neurons. The nerve cells often contain pyknotic nucleoli with chromatolysis of cytoplasm and vacuolation along with swollen dendrites. These changes are promi-nent in the frontal cortex 11 hours after heat injury. Eighteen hours after heat insult the neuronal cytoplasm and nuclei were hyperchromatic and the pericellular edema was apparent. In cases of 6 and 12 days of heat illnesses, the neuronal loss is most severe and the glial proliferation is quite distinct. Increase lipid content in nerve cells and in the perivascular space is common. These pathological changes are most pronounced in the upper layers of the cerebral cortex. Damage to the white matter, demyelination or vesiculation of myelin are absent. Pathological changes in the cer-ebellum are found in both the cerebellar hemispheres as well as in vermis. Edema of the Purkinje cell layer is most marked, however, the molecular and granular layers of the cerebellum are not affected. The dentate nucleus is the most sensitive structure of heat damage in the cerebellum and degeneration of nerve cells and hyperchro-matic reactions are present even 12 h after heat insult. The Purkinje cell layer is completely disappeared 24 h after heat injury and at this time, glial reactions are more evident in the Bergmann layer followed by the molecular layer. The Purkinje cell necrosis is common after 3 days of heat illness and hyperplastic changes are seen in the molecular and the Bergmann layers in cases of 12 days long heat injury.

1.3.3. Stress component of heat pathology

Acute heat exposure has the capacity to induce hyperthermia associated with the breakdown of the blood-brain-barrier (BBB) permeability, brain edema, and cell injury[232; 275]. The extent of the brain damage after heat exposure appears to be associated with the stress caused by heat and not only due to passive heating alone.

11


This is evident with the fact that the anaesthetized rats did not develop stress symp-toms. In these rats disturbances of the BBB permeability, brain edema, cerebral blood fl ow, or 5-HT levels were not observed[233]. There was no difference in the results obtained following heat-stress in rats anaesthetized either with urethane or Equithesin. Obviously, the feeling of discomfort and stress due to heat exposure is signifi cantly attenuated or prevented by anesthetics [230]. The effect of anesthetics are very similar to the anxiolytic or psychopharmacologic drugs in reducing perception of stress at the level of CNS[230]. Due to a reduction in the perception of stress, anes-thetic compounds apparently offer a certain degree of neuroprotection[223], a subject which encourage additional investigations.

1.4. Tachykinins, Substance P and Substance P Metabolites

1.4.1. Background

The tachykinin family members share the common C-terminal amino acid sequence Phe-X-Gly-Leu-Met-NH

2. Where X denotes Tyr, Phe, (aromatic) Val, or

Ile (hydrophobic). The tachykinins (tachys, swift) were early shown to evoke sharp contraction of the intestinal muscular layers. The three mammalian tachykinins (SP, NKA, NKB) are involved in different biological activities. SP was isolated 1931 by Euler and Gaddum, and because it was in powdered form, they named it substance P[198]. In view of SP pain transmitting qualities its name now also seems appropriate from a functional perspective.[192] Since the discovery of SP it took all until 1970s that SP was isolated and sequenced from bovine hypothalamus by Susan Leeman and her collaborators [34; 35; 147; 262], thereby defi nitely proving SP as a unique compound. Previous preparation most likely constituted a mixture of similar sub-stances. NKA and NKB were discovered in the 1980s[120; 159; 187].

1.4.2. Physiological role

Tachykinins are involved in many physiological processes, and widely distributed through out the body. Some of the functions include blood fl ow regulation and vascular permeability in infl ammation, salivary and intestinal excretion, and mic-turition, as well as smooth muscle contraction in the gastro-intestinal tract and respiratory system. In the neurous system they are released from pain transmitting pathways. Typically primary afferenting C-fi bers, synapsing in the dorsal horn of the spinal cord. In the brain, they transmit and modulate signals in sensory percep-tion (vision, olfaction, and audition) and pain, where they are broken down by vari-ous peptidases[155; 156; 157; 158].

SP can be found in the basal ganglia and nucleus accumbens, and to a somewhat lesser amounts in the cerebral cortex [41]. There is extensive evidence that SP interacts with nigrostriatal and various limbic nuclei DA neurons [90; 207; 208; 209] and forebrain nuclei [11]. Importantly SP seems to convey signals in the pain system such as peri-aqueductal gray, nucleus raphe magnus, and the nucleus reticularis gigantocellularis pars alpha[148]. SP is considerably present in the posterior hypothalamus, and basal

12

Krister Karlsson

forebrain [36; 151]. In relation to other tachykinins NKB seems to be more distributed to the anterior regions, however overlapping exists between with SP.[36; 152] In the spinal cord, SP and NKA are distributed in a similary pattern, whereas NKB is limited to lamina II (corresponding to interneurons of ascending pathways).[87]

1.4.3. Genes and peptide maturation

Tachykinins are expressed as large precursor proteins (preprotachykinins), and there are two genes encoding for such precursors, PPT-A and PPT-B. The preprotachy-kinin A (PPT-A) gene encodes for SP and NKA, and some extended variants of NKA (Neuropeptide K and neuropeptide-γ) (Fig. 1.4). After transcription this gene gives three different PPT-A mRNAs, (α, β, and γ) as a result of so called alternative splicing. α-PPT-A, β -PPT-A, and γ-PPT-A, can all give SP, however NKA is only achievable from the last two of these mRNAs[73]. The PPT-B gene encodes NKB, in two sequences, that can later be cut out during processing of the precursor[141].

Fig. 1.4 Preprohormones formed by alternative splicing of the mRNA from the PPT-A gene (a.k.a. “Substance P/NKA-gene”)Substance P is derived from all three transcripts, neurokinin A comes from β -PPT-1 mRNA, and γ-PPT-1 mRNA. Neurokinin B comes from the PPT-2 gene.

1.4.4. Receptors

There are three receptors to the tachykinins that have been cloned (NK-1, NK-2, and NK-3). A fourth (NK-4 or possibly a NK-3 subtype) seems to be discovered as well. They all belong to the heptahelical G-protein coupled receptors. Substance P is considered the natural ligand for the NK-1 receptor, and NKB the ligand for NK-3 receptor. However, NKA and NKB are fully effective agonists at the NK-1 receptors, SP and NKB are equally effective agonists at NK-2 receptors and, SP and

13


NKA are fully effective agonists at NK-3 receptors. The distribution and affi nities of these receptors are species- and tissue-specifi c, and there is a considerable overlap of function.

1.4.5. Clinical settings

The most obvious conditions in which the tachykinins play a role include pain, smooth muscle contraction, infl ammation and neurogenic activation of immune response. Chronic infl ammatory diseases such as asthma, rheumatoid arthritis, sys-temic lupus erythematosus, dermatomyositis, Wegener’s granulomatosis, Sjögren’s, progressiv systemic sclerosis, Crohn’s disease, ulcerative colitis, interstitial cystistis, etc., may be possible to affect by tachykinin receptor ligands. Neurogenic infl am-mation may also contribute to the emergence of such disease. Specifi c nonpeptide antagonists have been synthesized and have potential benefi ts in yet other con-dititions such as, chronic pain, Parkinson’s disease, depression, and migraine[129].Alzheimer’s disease exhibits altered SP signalling, and is also characterized by exten-sive neuronal degeneration, making it a a candidate for SP ligands. This should be of consensus regarding the evidence of SP’s role in neuroprotection/apoptosis[20; 56;

121; 254]. Recent evidence also point to the possible treatment of affective disorders by NK-1 antagonists[6; 8; 31; 137; 195; 216; 217; 222].

1.4.6. Role in physical dependence and withdrawal

Morphine and related opioid drugs are widely used as analgesics in the management of severe pain. However, repeated administration of these agent leads to the develop-ment of opioid tolerance and physical dependence, factors that limit their therapeutic usefulness. Opioid tolerance manifests as a loss in analgesic potency, whereas physi-cal dependence is indicated by the onset of a characteristic withdrawal syndrome precipitated by cessation of opioid drug treatment or a challenge with an opioid receptor antagonist such as naloxone. The mechanisms underlying the development and expression of opioid tolerance-dependence are not completely understood, how-ever recent evidence suggests that increased activity of spinal excitatory amino acid (L-glutamate/L-aspartate) and neuropeptide transmitters (calcitonin gene-related peptide (CGRP), substance P) may play an important role in these phenomena[113;

253]. During morphine treatment in rats, substance P and CGRP immunoreactiv-ity is considerably elevated in the spinal dorsal horn. Precipitation of withdrawal by naloxone decreases these levels. However, pretreatment with CGRP receptor antagonists, Substance P antagonists and cyclooxygenase inhibitors 30 min before naloxone challenge, is able to attenuate the withdrawal symptoms. Indeed, chronic co-administration of CGRP(8-37), SR14033, ketorolac, duP697, and nimesulide together with morphine makes the withdrawal symptoms less pronounced [260].Considerable attention has been focused on the role of excitatory amino acids and the activity of the NMDA receptor (an excitatory amino acid receptor sub-type) in the development of opioid tolerance and physical dependence. Several studies have shown that blockade of spinal NMDA receptors effectively inhibits development of tolerance to the antinociceptive actions of morphine [59; 60; 164; 236; 263; 264]. Addition-

14

Krister Karlsson

ally, blockade of this receptor also inhibits the expression of naloxone-precipitated morphine withdrawal [48; 59; 264]. These and other fi ndings have led to the proposal that chronic exposure to opioid drugs induces a latent increase in NMDA receptor activity that physiologically antagonizes the inhibitory effects of these agents and compromises the analgesic response [163; 164]. Cessation of drug treatment unmasks this increased NMDA receptor activity and gives rise to the autonomic and behav-ioral hyperactivity that constitutes the opioid withdrawal syndrome. In addition to the excitatory amino acid activity, the activity of sensory neuropeptide transmitters also contributes to the genesis of the opioid tolerant-dependent state [175; 176; 177; 202; 203;

204]. In nociceptive primary afferents that terminate in the superfi cial laminae of the spinal dorsal horn, L-glutamate is co-localized with CGRP and substance P [178]. It has been reported that repeated daily intra thecal administration of morphine sig-nifi cantly increased CGRP and substance P immunoreactivity in the rat spinal cord [177; 203] and in dorsal root ganglion neurons which gives rise to neuropeptide express-ing primary afferent fi bers [154]. Co-treatment with a CGRP receptor antagonist consistently blocked this effect and prevented the development of morphine toler-ance [203]. Some fi ndings indicated that treatment with an NK-1 receptor antagonist can also inhibit and reverse spinal morphine tolerance [203]. These fi ndings suggest that the activity of spinal CGRP and substance P contribute to the induction and expression of opioid analgesic tolerance, however their role in the development of opioid physical dependence is relatively unknown. A recent study demonstrating that morphine withdrawal is attenuated in CGRP defi cient transgenic mice supports the involvement of this neuropeptide in the genesis of opioid physical dependence [219;

220; 221]. Previous studies have also shown that blockade of the NK-1 receptor reduces the magnitude of opioid withdrawal associated contractions in isolated guinea-pig ileum [116] and inhibits some signs of morphine withdrawal in the rat [25; 160]. Interest-ingly, injection of SP(1-7) into the ventral tegemental area was found to modulate the levels of nucleus accumbens dopamine and dihydroxyphenylacetic acid in male rats during morphine withdrawal[291]. Thus, these studies suggest the involvement of CGRP and substance P in the development of opioid physical dependence. The mechanisms by which increased neuropeptide activity contributes to the develop-ment of opioid withdrawal are not known, but evidence form tolerance studies sug-gests an intermediary role of prostaglandins [202]. Activation of neuropeptide and amino acid receptor activity in the spinal cord results in prostaglandin release [105;

162; 166; 167]. Prostaglandins in turn act on terminals of primary afferents to further release CGRP, substance P, and glutamate, initiating a positive feedback loop [92; 162;

269]. This suggests that activation of CGRP and substance P receptors at the spinal level, contributes to the induction and expression of opioid physical dependence and that this activity may be partially expressed through the intermediary actions of prostaglandins [260]. The altered metabolism of substance P during morphine tolerance and withdrawal[289] is an additional evidence for the involvement of this peptide during opioid physical dependence, which should spur further biological research on drug dependence.

15


1.5. Inactivation and Conversion of Tachykinins by Peptidases

1.5.1. Substance P degrading enzymes, in addition to SPE

Following release and exertion of its effect, substance P is likely to be inactivated by various proteases (Table 1.1). Although no specifi c peptidase has yet been attributed to be the single one responsible for substance P inactivation, several enzymes capable of hydrolyzing the peptide have been described[199]. Some of these enzymes, e.g., dipeptidylaminopeptidase IV (DAP-IV) degrade the peptide sequentially form the N-terminal side[127], whereas others, e.g., angiotensin converting enzyme (ACE), hydrolyze substance P from its C-terminal part [66; 242; 243; 244]. Other proteases have been shown to hydrolyze substance P by an endopeptidase action [18; 126; 170; 171; 172; 185].Most of these enzymes , however, are known to potently act on several other pep-tides[184]. However, there is one enzyme originally isolated form human brain which seemed to be specifi c for substance P [146]. There has previously been reported a similar enzyme in human CSF [181; 183]. The CSF enzyme appeared to be very specifi c for the undeca-peptide, and was found to release products with retained biological activity, and this enzyme was named substance P endopeptidase (SPE).

Table 1.1 Opioid peptide and substance P converting and degrading enzymes

1.5.1.1. Angiotensin Converting Enzyme

Because ACE had long been considered a dipeptidyl carboxypeptidase or peptidyl dipeptidase requiring substrates with a free C-terminus, it was diffi cult to gain accep-tance for the reports that ACE inhibitors prevented the inactivation of substance P [32; 43; 145] and that substance P inhibited the hydrolysis of other substrates by ACE [174;

186; 237; 238; 286]. It was generally concluded that these effects were not due to hydrolysis

16

Krister Karlsson

of substance P by ACE, because this peptide has a blocked C-terminal amino acid that rendered it resistant to ACE. Nevertheless, the co-localization of ACE and substance P in the substantia nigra and globus pallidus was puzzling, although sub-stance P is immunohistochemically much more evident in the inner than the outer segment of the human globus pallidus [51; 52; 53]. The possibility that the specifi city of ACE was not restricted to its known action as a peptidyl dipeptidase was suggested by other experiments. For example, ACE cleaved a tripeptide from des-Arg9-brady-kinin[108] and it hydrolyzed the penultimate peptide bond of substrates in which the last amino acid was replaced by nitrobenzylamine[91]. Homogeneous human ACE cleaves substance P at two different sites, at Phe8-Gly9 and at Gly9-Leu10, to release either the C-terminal tri- or dipeptide [53; 194; 237; 286]. However, the release of the tri-peptide was clearly favored by a ratio of 4:1. After initial removal of the C-terminal di- or tri-peptide, ACE sequentially cleaved dipeptides form the remaining N-ter-minal fragment. Shorter C-terminal fragments of substance P were also cleaved by ACE. However, the tetrapeptide (substance P(8-11)) is not cleaved. When substance P and the free acid derivative was used as competitive inhibitors of furylacryloyl-Phe-Gly-Gly hydrolysis by ACE, a K

i value of 25 µM was found, for substance P

and 2 µM for the free acid. Thus, substance P fee acid interacts more favorably with the active site than does native substance P with a C-terminal amide group. Studies of the chemical modifi cation of ACE showed the active site to contain a positively charged arginine which presumably interacts with the negatively charged carboxyl group of most substrates [27; 99]. Because the free acid was a more favored substrate of ACE, we wondered whether the active site arginine is still required for binding and cleavage of substance P. After blocking the arginine residues of ACE with cyclo-hexanedion or butanedion, the hydrolysis of bradykinin, benzoyl-Gly-Phe-Arg and substance P was inhibited 80-93%. This indicates that the active site arginine is required for hydrolysis of substrates with both free and blocked C-terminal amino acids. It was hypothesize that the active site arginine of ACE interacts with the car-bonyl oxygen of Leu10 which, according to the proposed model of the active site [99],would allow the active site zinc to complex the carbonyl oxygen of Phe8. This results in the hydrolysis of the Phe8-Gly9 bond and releases the C-terminal tripeptide [115].The small amount of dipeptide released may come from a similar interaction of the active site arginine with the carbonyl oxygen of Met11-NH

2. Other studies has

also described the release of the C-terminal tripeptide by ACE [33; 286]. In addition, ACE inhibitors potentiated the sialogogue action of substance P in the rat [33] and inhibited the catabolism of substance P in the rat stomach wall [190]. One report [252],described two possible different variants of ACE, one preferably releasing the C-ter-minal dipeptide and the other preferably the tripeptide. These variants differed in Mr of about 10,000 (based on SDS-PAGE). Some contradictory studies have been reported indicating the possible existence of several ACE-like substance P hydrolyz-ing enzymes. Substrate specifi city of these ACE preparation has been diverging, and there is a clear lack of full characterization of the different ACE variants that seem to act on SP. [64; 101; 102; 103; 251; 252; 265; 287]

17


1.5.1.2. Endopeptidase 24.11

The specifi city of endopeptidase 24.11, hydrolyzing on the amino side of hydro-phobic residues, immediately suggests that substance P, containing two internal phenylalanin residues and a leucine, may be a favorable substrate for the enzyme. In 1983, it was shown that purifi ed preparations of kidney endopeptidase 24.11 were able to hydrolyze substance P at the predicted sites [169], and that synaptic membrane preparation hydrolyzed the peptide identically, both hydrolyses being inhibited by phosphoramidon [169]. Although other peptidases are able to hydrolyze substance P in vitro, for example dipeptidyl peptidase IV and ACE, data suggested that endopeptidase-24.11 is the one of the principal enzymes effecting this process in striatal synaptic membrane preparations [169]. In pig kidney microvillar membranes, an additional contribution due to dipeptidyl peptidase IV was detected, but ACE appeared to play no signifi cant role in this process [248; 249]. This is probably a refl ec-tion of the substantially higher K

cat/K

m value exhibited towards substance P by the

endopeptidase (Kcat

/Km=185 min-1 µM-1 for endopeptidase 24.11 and K

cat/K

m=9 for

ACE, (see, e.g. [248; 249]). Indeed, substance P and other tachykinins remain among the most favorable substrates identifi ed for the endopeptidase on a kinetic basis. In addition, endopeptidase-24.11 co localizes with substance P in a number of brain regions and endopeptidase inhibitors can protect from degradation substance P released form slices of rat substantia nigra [173]. It should be pointed out however, that the catabolism of substance P in the stomach wall of the rat is susceptible to inhibitors of ACE but not endopeptidase inhibitors [190]. Converting enzyme inhibi-tors can also potentiate certain of the peripheral actions of substance P, particularly salivation [33]. Thus, the relative contributions of different cell-surface peptidases to substance P degradation may differ at different tissue sites, and between spe-cies. The two other mammalian tachykinins, neurokinin A and neurokinin B, are also effi ciently hydrolyzed by endopeptidase-24.11 [100; 102; 103]. The amphibian tachykinin, physalaemin, is also hydrolyzed by the endopeptidase [171]. When the hydrolysis of the neurokinins by pig striatal synaptic membranes was examined, it was observed that a combination of phosphoramidon and bestatin was required to abolish hydrolysis. Captopril had no signifi cant effect. These data suggested that, like the enkephalins, the hydrolysis of the neurokinins could be attributed to both endopeptidase-24.11 and a bestatin-sensitive aminopeptidase [102], as well as other unknown enzymes.

1.5.2. Substance P Endopeptidase

An endoprotease capable of releasing both substance P(1-7) and SP(1-8) fragments from the parent peptide was identifi ed and purifi ed[122; 123; 124]. Interestingly, both these fragments have been found to possess biological activity [10; 26; 45; 75; 82; 83; 84; 90;

139; 149; 188; 218; 250; 273]. The enzyme named SPE thus hydrolyzes the undecapeptide within and at the carboxylic side of the double-Phe bond. It has a molecular weight of around 40 kDa and was characterized as a metal-dependent thiol-sensitive endoprotease [181]. It showed optimum activity at neutral pH and was inhibited by

18

Krister Karlsson

bacitracin. However, the most profound feature concerns its specifi city. SPE shows a surprisingly high preference for substance P. It also cleaves its C-terminal fragments SP(3-11) and SP(5-11) but at a much lower rate. Other tachykinins, lacking the double -Phe residues, such as neurokinin A and neurokinin B, were almost unaf-fected. Studies also revealed that SPE was strongly inhibited by calcitonin gene-related peptide (CGRP) [144; 182]. CGRP is co-localized with substance P in many primary sensory neurons. It has also been demonstrated that CGRP potentiates the action of substance P in several ways [47; 277; 278; 284]. It was observed that CGRP is also cleaved by SPE [143]. The enzyme was found to hydrolyze the Leu-Leu bond residing in CGRP, however, at a signifi cantly lower rate than that observed for substance P. With regard to substance P, it thus seems that SPE releases products with potent activity, i.e., it terminates the action of substance P by releasing a second signal from its structure. Indeed the levels of SP(1-7) in the brain was found to be affected during withdrawal of morphine tolerant rats[290]. By ion-exchange chromatography (DEAE-Sepharose CL-6B), at least three different SPE-like activities have been detected. One of these activities is inhibited by captopril (an inhibitor of angioten-sin converting enzyme, ACE) and one by phosphoramidon, (an inhibitor of neutral endopeptidase 24.11, NEP). The third SPE-like activity, representing a minor pool of the substance P(1-7)-generating activity in the rat CSF, was not affected by any of these inhibitors and could perhaps be identical to the human CSF variant of the enzyme. The presence of ACE in rat CSF has indeed been reported by others, and this enzyme may represent the major part of the SPE-like activity detected in CSF samples from this species [288]. However, in CSF collected from polyarthritic rats, where we see a change in SP(1-7)-generating activity, the level of ACE remained invariant and therefore it is possible that the observed alteration is due to SPE.

The observation that the substance P fragment (1-7) is present in the dorsal spinal cord [1] inspired the idea of an SPE-like activity in this area of the CNS. However, studies so far have only been covering whole cords. In the rat homogenates, where the major part of substance P(1-7)-generating activity appeared to be due to pro-teases other than SPE. Thus, in accordance with the fi nding in rat CSF, the major activity responsible for the release of the heptapeptide was sensitive towards the ACE inhibitor captopril and to some extent also, phosphoramidon, the above-mentioned inhibitor of neutral endopeptidase 24.11 (NEP, previously enkephalinase). Immu-nohistochemical and autoradiography studies have revealed that both ACE and NEP are also distributed throughout the CNS and PNS [54; 85; 201]. Both enzymes are known to act on a number of neuroactive peptides. For instance, NEP is considered to be one of the major peptidases involved in the physiological termination of the enkephalinergic signal, whereas ACE is known to hydrolyze both the enkephalins and substance P [66; 115; 241]. However, a recent study reported a membrane-bound substance P endopeptidase in the rat brain [15]. Except for a larger molecular weight this activity shows close similarity to the human SPE. SPE-like activity could be recovered both from the soluble and the membrane-bound fractions.

19


1.5.3. Inhibition of Substance P Endopeptidase

Substitution of the bis -phenyalanin moiety in substance P with a non-peptidergic fragment makes up a change in the structure of the undecapeptide, which yield a product which potently inhibit the endopeptidase. However, the modifi ed compound retained recognition of, and binding to, the substance P receptor (NK-1 receptor). Further development of these compounds i.e. three analogs of the bis -phenylalanin analogue JA48, showed inhibition of SPE but no affi nity for the NK-1 receptor [112].In additional studies a series of benzyl- or aryl-substituted 1,2,4-oxadiazole deriva-tives of phenylalanin was synthesized [112], and some of these compounds with an electron-defi cient aromatic ring, produced an inhibitory action on the enzyme. The development and use of selective SPE inhibitors may prospectively turn out to be very benefi cial in the treatment of various clinical conditions, as described above. At the moment though, questions regarding the pharmaceutical formulation and solu-bility, administration and compartment distribution in the body, as well as a more extensive biological action profi le examination, must be solved before the clinical use of such SPE inhibitors can be applied.

1.6. Strategies to Protein Purifi cation

1.6.1. Basic approach and guidelines

A systematic approach to protein purifi cation is essential and should include a number of considerations[2]. These should include objectives in regard to activity, quantity needed, and purity needed, properties of the target protein and critical impurities such as co-factors, isozymes etc. Furthermore, functional analytical assays, for fast and effi cient work, intermediate sample handling, time lengthy procedures, with activity recovery loss as a sequel, additives, at some point needed but best avoided if possible, protease elimination at early stage, and other critical damaging parameters should also be considered. Finally, integration of different techniques, advantage of combining characteristically differing techniques should be taken (size, charge, hydrophobicity, specifi city), use of only necessary steps, mini-mizing the loss of yield and time.

Of course this offers a complex decision process, and factors that may infl uence it include, economy, access to equipment, scaling up possibilities, activity, and time[22;

226; 227]. The usual limitations are obtaining suffi cient purity without losing specifi c activity. Purifi cation to “apparent homogeneity” in respect of commassie blue stain-ing may not be suffi cient in enzyme purifi cations because enzymes are such capable proteins that let us say a 5% contamination may not be acceptable on activity basis. On the other hand enzymes tend to drop their activity drastically when absolute purity is beginning to be reached. This is due to the loss of co-factors and stabiliz-ing chemical environment[2; 227]. Some properties of the target protein to take into account are:

20

Krister Karlsson

• Temperature stability• pH stability• Organic solvent stability• Detergent requirement• Ionic strength, and precipitation pattern• Co-factors for stability and activity• Protease sensitivity• Sensitivity to metal ions• Redox sensitivity• Molecular weight, charge properties, biospecifi c activity, posttranslational

modifi cations, and hydrophobicityA purifi cation schedule should include separation media, and analytical assays. An analytical assay such as an enzyme assay for enzymes should be rapid and reliable. Purity determination and protein measurement should be incorporated. Assays for impurities may also be necessary.

1.6.1. Three-phase strategy to purity

This very common setup features capture, intermediate purifi cation, and polishing, of an applied prepared sample[2; 3; 227]. The preparation often includes homogeni-zation, extraction, and clarifi cation by for instance centrifugation. The capturing phase may be optimized to isolate the target by concentration and stabilization. During the intermediate phase most of the bulk impurities such as other proteins, and nucleic acids are removed. In order to achieve fi nal purity the polishing phase is applied. Conforming to the predefi ned objectives the number of steps and choice of techniques may vary. For example each step can be optimized in regard to,

• resolution,• speed,• capacity,• recovery.

Every technique offers a balance between the above parameters and should be selected to meet the objectives for each purifi cation step. For example during the initial capturing phase high resolution is of high value allowing to appropriately separate the target. Depending of scaling trade off may be performed to recovery in order to get suffi cient amounts. At later steps capacity of the techniques tend to be weight against resolution. And at fi nal polishing the highest possible resolution should be applied without endangering the recovery. Speed is often of critical value for time/cost consuming reasons but also to avoid endangering the integrity of the sample[212].

21


2. AIMS OF THE THESIS

In order to gain further knowledge of the neurobiochemical picture of substance P messaging function in the CNS, we must integrate the peptidergic transmission regulation into the extensive role and distribution that this neuropeptide exhibit. Clearly the discovery of a substrate specifi c endopeptidase, acting on this peptide opened up for questions regarding what role this endopeptidase may play in regula-tion of substance P’s function. In order to answer these questions we formulated the following major aims:

• To purify and biochemically characterize SPE from rat spinal cord.• To purify and biochemically characterize SPE from rat VTA, allowing the

use of micro bio-analytical techniques to expand into the very intricate study of neurobiochemical processes.

• To purify and enzymatically compare SPE from human spinal cord and human cerebrospinal fl uid.

• To evaluate the SPE-activity in relation to spinal cord and cerebrospinal fl uid distribution as well as other enzymes like chymotrypsin.

• To investigate the distribution and role of SPE in CNS during morphine tolerance and withdrawal

• To elucidate the behavior of SPE in the brain after acute heat stress expo-sure, allowing an integrative view of the enzyme in such traumatic brain insult.

22

Krister Karlsson

3. EXPERIMENTAL PROCEDURES

3.1. Purifi cation of Enzyme Activity

3.1.1. Substance P endopeptidase (SPE) purifi cation from rat spinal cord

All procedures were carried out at 5 ºC. The tissue was thawed and homogenized in 20 mM Tris-HCl buffer, pH 7.8, (10 ml/g) for 1 min. the homogenate was extracted for 2 hours with continuous stirring. A subsequent centrifugation was performed in a Beckman J-21 centrifuge at 20,000 x g for 20 min. The supernatant was collected and the pellet resusspenden in the same Tris buffer. the suspension was stirred for 2 hours and then centrifuged at 20,000 x g for 20 min. The resulting pellet was again resuspended, this time with 1% Triton X-100 in 20 mM Tris-HCl buffer, pH 7.8. The suspension was stirred for 4 hours before additional centrifugation at 20,000 x g for 20 min. The supernatant was collected and the pellet was discarded. The Triton X-100 containing extract was applied directly on a DEAE-Sepharose CL-6B column (50 x 250 mm) and eluted with a linear gradient of NaCl (0-0.5 M) in 20 mM Tris-HCl buffer, pH 7.8, with 0.1% Triton X-100. Fractions of 20 ml were collected at a fl ow rate of 2.5 ml/min and assayed for protein content and enzyme activity. The active fractions were pooled and subjected to hydrophobic interaction chromatography on a Phenyl-Sepharose CL-4B column, equilibrated with 20 mM Tris-HCl buffer, pH 7.8, containing 0.1% Triton X-100 and 0.2 M NaCl. Elution was carried out with 20% acetonitrile in H

2O. The fl ow rate was 2.5 ml/min and

10 ml fractions were collected and assayed for enzyme activity. the active fractions were pooled and centrifuged at 20,000 x g for 10 min, which gave a small pellet. The supernatant was lyophilized and resuspended in 60 ml of 0.04 M NH

4HCO

3 before

application on a Sephadex G-50 column (5x90 cm) for molecular sieving. Elution was carried out with 0.04 M NH

4HCO

3 at a fl ow rate of 1 ml/min. Fractions of

10 ml were collected and those holding activity were pooled and concentrated by lyophilization. Two activities differing in size were obtained. The lyophilized fractions were each redissolved in 20 mM sodium phosphate buffer, pH 7.8, and subjected to a fi nal purifi cation by ion-exchange HPLC, using a Mono Q® column was equilibrated with 20 mM sodium phosphate buffer, pH 7.8. Following applica-tion of the sample, the column was washed with the same buffer and the adsorbed material was eluted with a gradient of 0-0.5 M NaCl in the same sodium phosphate buffer. The effl uent was collected in 100 µl fraction at a fl ow rate of 100 µl/min. The active material from this step was in each case collected for further analytical studies. For analytic purposes 50 µl of the purifi ed active fractions were analyzed on the SMART™-system using a Superdex® 75 column for molecular sieving. Elution was performed with 50 mM sodium phosphate buffer, pH 7.8, during collection of 100 µl fractions at a fl ow rate of 100 µl/min. The fraction containing the highest activity was analyzed by SDS-polyacrylamide gel electrophoresis with silver staining to check the purity and estimate the molecular weight.

23


3.1.2. Substance P endopeptidase (SPE) purifi cation from human spinal cord

All procedures were carried out at 5 °C. Frozen spinal cords (24 g) were thawed and homogenized in 20 mM Tris-HCl buffer, pH 7.8, (10 ml/g) for 1 min. The homogenate was extracted for 2 hours during continuous stirring. A subsequent cen-trifugation was performed in a Beckman J-21 centrifuge at 20,000 x g for 20 min. The supernatant was collected and the pellet resuspended in the same Tris buffer as before. The suspension was stirred for 2 hours and then centrifuged at 20,000 x g for 20 min. The resulting pellet was again resuspended, this time with 1% Triton X-100 in 20 mM Tris-HCl buffer, pH 7.8. The suspension was stirred for 4 hours before additional centrifugation at 20,000 x g for 20 min. The supernatant was collected and the pellet was discarded. The Triton X-100 containing extract was applied directly on a DEAE-Sepharose CL-6B column (50 × 250 mm) and eluted with a linear gradient of NaCl (0-0.5 M) in 20 mM Tris-HCl buffer, pH 7.8, with 0.1% Triton X-100. Fractions of 10 ml were collected at a fl ow rate of 2.5 ml/min and assayed for protein content and enzyme activity. The active fractions were pooled and subjected to molecular sieving on a Sephadex G-50 column (5 x 90 cm). Elution was carried out with 0.04 M NH

4HCO

3 at a fl ow rate of 1 ml/min. Frac-

tions of 10 ml were collected and those holding activity were pooled and concen-trated by lyophilization. The protein was resuspended in 50 mM Na-phosphate pH 7.8, and run on HPLC-molecular sieving using a Superdex_Peptide_HR_10/30 column (Pharmacia Biotech, Uppsala, Sweden). Elution was performed with 0.04 M NH

4HCO

3 at a fl ow rate of 0.70 ml/min collecting fractions of 1.40 ml. Those

holding activity were pooled and regarded as the spinal cord enzyme preparation. To manage the last purifi cation step a new HPLC-system was used, ÄKTA™-purifi er(Pharmacia Biotech, Uppsala, Sweden). ÄKTA™-purifi er is an automated HPLC-system scaled for preparative purifi cation and is equipped with a binary high perfor-mance gradient pump with two switch-valves. Flow rates can be up to 10 ml/min and pressures up to 25 MPa. A triple wavelength UV-Vis monitor is used online with a conductivity/pH monitor for detection.

3.1.3. Substance P endopeptidase (SPE) purifi cation from rat ventral tege-mental area (VTA)

All procedures were carried out at 5 °C. Frozen rat ventral tegemental areas from 40 animals (410 mg) were thawed and homogenized in 20 mM Na-phosphate, pH 7.8 (1 ml/g) with an ultrasonic probe equipment (Branson Sonifer cell disruptor B15) for 45 s. The homogenate was extracted in the same buffer for 5 min. before centrifugation at 18,000 x g for 10 min, (Eppendorf Centrifuge 5417, rotor F45-24-11, Beckman). The supernatant was collected and kept frozen (-70 °C) until use. At this stage the extract was aliquoted into three equal volumes. This means that we followed three identical preparation procedures from this point forward in the purifi cation scheme, which allowed us to retrieve statistical reliability. The extract

24

Krister Karlsson

was subsequently applied to size exclusion chromatography (SEC) through a 30 µl sample loop, using the column Superdex 75 HR 10/30 (Amersham-Pharmacia Biotech, Sweden) installed on the preparative HPLC-system ÄKTA™-purifi er 10 (Amersham-Pharmacia Biotech). Elution with 0.04 M NH

4HCO

3 at a fl ow rate

of 0.5 ml/min was carried out and 0.5 ml fractions were collected. The fractions ware scanned for enzyme activity, and those indicated to contain SPE-activity were pooled for further separation on anion-exchange chromatography. Again the ÄKTA™-purifi er 10 was used, now with a Resource Q, 1 ml column (Amersham-Pharmacia Biotech). In this chromatographic step the samples were eluted with 20 mM Na-phosphate, pH 7.4 using a linear gradient of 0-1 M KCl. The length of the gradient was set to 20 column volumes, and the fl ow was 4 ml/min. Fractions of 1.0 ml were collected and assayed for SPE-activity.

3.2. Assessment of Enzyme Activity

Enzyme activity was monitored by measuring the formation of SP(1-7), using sub-stance P as substrate. A radioimmunoassay (RIA) specifi c for SP(1-7) was utilized for this purpose. Substrate (0.05 µg) was incubated with 30 µl enzyme and a cock-tail of phosphoramidon/captopril (at a concentration of 15 µM) in a fi nal volume of 50 µl, at 37 ºC for 30 minutes. The reaction was terminated by adding 1.0 ml of ice-cold methanol/0.1 M HCl 1:1 followed by centrifugation (Beckman Microfuge B) at 1000 x g for 10 min and evaporation of the supernatant. The RIA was carried out in triplicates and was conducted as described in a preceding paper. Briefl y, the antibodies were raised in rabbits against the peptide-thyroglobulin conjugate and the iodinated peptide was used as tracer. The 125I-labeled Tyr0-SP(1-7) and the sample or unlabeled SP(1-7) (standard) were thus incubated with specifi c antibody against SP(1-7) in 1.5 ml Eppendorf tubes. The tubes were stored at 4 ºC until expected equilibrium had been established (>8 h). Then separation of bound pep-tide form unbound peptide was done based on the charcoal adsorption technique as described[142; 200]. The quantity of SP(1-7) was determined by measuring the radio-activity in a gamma-counter. The detection limit of the RIA and the cross reactivity of SP(1-7) related peptides in the RIA were as described[142], i.e. (<2%).

3.3. Sodium Dodecylsulphate-Polyacrylamide Gel Electrophore-sis (SDS-PAGE) with Silver staining

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was per-formed according to the procedure of Laemmli as described previously[40; 191]. The separated proteins were visualized by the silver staining method.

25


3.4. Characterization of Enzyme Activity by High Pressure Liquid Chromatography (HPLC)

RP-HPLC was used with the intention to identify the products that the peptidases were able to release from the substrate peptide. For this purpose the SMART™ system, equipped with a µRPC C2/C18, SC 2.1/10 column (Pharmacia Biotech), was used. The fragments were separated by a linear gradient of 0-60% acetonitrile in 0.14% trifl uoroacetic acid. The fl ow rate was 240 µl/min and 24 ml fractions were collected. The identity of the separated fragments was subsequently verifi ed by mass spectrometry.

3.5. Characterization of Enzyme Activity by Mass Spectrometry (MS)

Fraction from the SMART™ system were lyophilized and subsequently dissolved in 50 µl of 50% methanol/0.25% acetic acid. A Finnigan TSQ 7000 triple quadru-pole mass spectrometer equipped with a Finnigan electrospray (ES) source was used for the identifi cation of the peptides. the samples were continuously infused at a fl ow rate of 1 µl/min and the mass spectrometer was scanned from m/z 100-1000 in one sec. Standard ES conditions were as follows: needle voltage, 3.5 kV; heated capillary voltage, 20 V; heated capillary temperature, 175 ºC. In order to obtain structural and sequence information of the peptides contained in the collected SMART™ fractions tandem mass spectrometry (MS/MS) was performed. The fi rst quadrupole was set to transmit the precursor ion mass with an acceptance window of about ± 1.5 m/z units. The precursor ion was dissociated by Ar gas at 2.5 mT in the second quadrupole region. The resulting product ions were scanned in the third quadrupole and gave information about the primary structure of the peptide.

3.6. Animal Treatment and Preparation

Male Sprague-Dawley rats (200-220 g) were caged individually and allowed to habituate to the environment one week before the start of the experiment. The rats had free access to food and water and they were housed at ambient room tempera-ture with controlled humidity (60%) and 12 h light/12 h dark cycle. The animals were randomly divided into four groups, each consisting of 7 animals. two groups were treated with morphine (s.c., 10 mg/kg) twice daily, and the other two, used as control groups, were given injections (s.c.) of saline, twice daily. Using the Model 33 Tail Flick Analgesia Meter (iITC, Life Science, USA) tail-fl ick latencies were mea-sured every second day in order to assess morphine tolerance. Following eight days of morphine administration, when rats were completely tolerant to the opioid, one group of animals received morphine and one group of controls were given naloxone (s.c., 2 mg/kg). This dose was chosen as it is well known to be effi cient in eliciting withdrawal symptoms in this strain of rats (e.g.[93]). Immediately after challenge, the animals were placed individually into a perspex viewing box (820 cm x 20

26

Krister Karlsson

cm x 40 cm) and observed for a period of 1 h during which a variety of behaviors associated with opioid withdrawal (including “biting”, “wet dog shakes”, “digging”, “face washing” , “grooming”, “rearing”, “scratching”, “diarrhea”, and “ptosis”) were recorded. Subsequent to that, all animals were decapitated. The whole brains were removed and dissected on ice using a rat brain matrix (Activational Systems Inc., Forterra Drive, Warren, Michigan, USA). All tissues (including the spinal cord) were kept frozen at -70 ºC before further processing. The animal experiments were approved by the local ethical committee at Uppsala University, Uppsala, Sweden.

3.7. Heat-Stress Treatment

Experiments were carried out on male Sprague Dawley rats housed at controlled ambient temperature (21 ± 1 ºC) with 12 h light and 12 h dark schedule. The rat feed comprised of standard laboratory pellets and tap water were supplied ad libitumbefore the experiment.

All procedures were commenced between 8.00 and 9.00 A.M. Rats were exposed to 4 h heat-stress in a biological oxygen demand (BOD) incubator maintained at 38°C. The relative humidity 45-50% and the wind velocity 20-25 cm/sec were kept constant (Sharma 1982). Normal animals kept at room temperature served as controls. This experimental condition was approved by the ethical committee of Uppsala University, Uppsala, Sweden.

27


4. RESULTS AND DISCUSSION

4.1. Enzyme Purifi cation of Substance P Endopeptidase (SPE)

4.1.1. Purifi cation from rat spinal cord

Spinal cord extract containing the Triton X-100 soluble (“membrane bound”) frac-tion was subjected to ion-exchange chromatography, which resulted in the recovery of an endopeptidase activity capable of releasing the SP(1-7) fragment from SP (Fig. 4.1). The enzyme activity obtained from this step was highly purifi ed from contam-inants, since over 80% of protein was removed by this procedure (Table 4.1). The main protease activity was eluted in a relatively broad area including fractions 45-70 (Fig. 4.1). This activity could be further purifi ed by hydrophobic-interaction chro-matography (Table 4.1), where on protein basis the purifi cation was approximately 10-fold. The following molecular sieving on a Sephadex G-50 column (Fig. 4.2) yielded two activities, one which was eluted in the area of the void volume (fractions 49-54), and another later (fractions 62-74).

Table 4.1Purifi cation and Recovery of Substance P Endopeptidase from Rat Spinal Cord (58 g)

Step Total protein Total activity

Specifi c activ-ity

Purifi cation factor

mg pmol/min pmol/min/mgExtract 10400 8110 0.780 1DEAE-Sepharose 2050 19600 9.56 12Phenyl-Sepharose 107 1100 10.2 13Sephadex G-50 (A*) 7.11 233 32.8 42Sephadex G-50 (B**) 1.25 531 425 545

Mono Q® (A*) 0.21 46.6 1180 1516Mono Q® (B**) 0.0394 13.0 331 424* A refers to preparation A, and **B refers to preparation B derived from the fi rst and second activ-ity eluted from the Sephadex G-50 column, respectively. For further details, see text.

These two enzyme fractions were collected separately before application to the fi nal purifi cation step, HPLC ion-exchange chromatography. The result of that separa-tion is shown in Fig. 4.3 and the over all yield of the purifi cation procedure is shown in Table 4.1. The two preparations obtained, were used for subsequent character-ization, where A refers to the active material fi rst eluted on the Sephadex G-50 column, and B refers to the second active fraction. Upon analysis by molecular sieving (Superdex 75 column) preparation A was found to elute as a homogenous protein associated with enzyme activity (Fig. 4.4). However, with regard to prepa-ration B, some minor contaminants seem to be present although the main protein peak appeared homogenous and was found to account for the main enzyme activity

28

Krister Karlsson

(Fig. 4.4). The SDS-PAGE gave a molecular weight of 43 kDa for preparation B and 70 kDa for preparation A which is in accordance with data obtained from the calibrated Superdex® 75 column.Studies of the pH-optimum indicated a further difference between preparation A and B, where A showed a broad optimum ranging from pH 7 to 8.5 and B operated most effectively at pH 4.5.

Fig. 4.1 DEAE-Sepharose CL-6B chromatography of rat spinal cord extractThe detergent treated extract was applied on an ion exchanger (see text for details). The separation was performed in 20 mM Tris-HCl buffer at pH 7.8. A linear gradient 0-0.5 M NaCl in 20 mM Tris-HCl buffer pH 7.8, with 0.1% Triton X-100 beginning at fraction 36 was used to elute the protein. Enzymatic degradation of substance P to its SP (1-7) fragment was measured by radioim-munoassay in a 10-fold dilution. The UV-absorption at 280 nm was also measured on 10 fold diluted fraction-aliquots. Fractions 45-70 were pooled and subjected to hydrophobic-interaction chromatography.

Fig. 4.2 Molecular sieving on Sephadex G-50 columnThe pooled fractions from the Phenyl-Sepharose step was centrifuged at 20,000 x g for 10 min to remove a minor part of lipids and lyophilized before application on the column. Elution was done using 0.04 M NH

4HCO

3. Fractions 49-54 (A) and fractions 62-74 (B) were pooled and lyophilized

before the next purifi cation step (see Fig. 3).

29


Fig. 4.3 Ion-exchange HPLC on a Mono Q® PC 1.6/5 columnPanel a, From the molecular sieving step of the membrane bound activity (Fig. 4.2), the activity eluted fi rst (fraction 49-54) was applied on the Mono Q® column and eluted with a gradient 0-0.5 M NaCl in 20 mM phosphate-NaOH buffer pH 7.8. The eluted activity used in the subsequent procedures is marked as a fi lled segment under the curve. Panel b, The second activity (fraction 62-74) from the same step as in panel a (Fig. 4.2), was applied on the Mono Q® column and eluted with a gradient 0-0.5 M NaCl in 20 mM sodium phosphate, pH 7.8. The fi lled segment under the curve corresponds to the enzyme activity used in the further treatment.

30

Krister Karlsson

Fig. 4.4 HPLC molecular sieving on a Superdex® 75 PC 3.2/30 columnPanel a, The separated material marked in Fig 4.3a was applied on a Superdex® column to get an even more purifi ed preparation (here denoted A). 50 µl was applied at a fl ow 100 µl/min and 100 µl fractions were collected and tested for substance P degrading activity. Panel b, The peptidase recovered from the Mono Q® chromatography indicated in Fig 4.3b was purifi ed on the Superdex®

column. Conditions were as described for panel a.

Two enzymes acting on substance P were, thus, purifi ed to apparent homogeneity from the membrane-bound fraction of the rat spinal cord. Both enzymes were capa-ble of hydrolyzing the undecapeptide at the Phe7-Phe8 and the Phe8-Gly9 bonds. However, a minor cleavage also seemed to occur at the Gln6-Phe7 bond. This latter cleavage was most pronounced for the preparation A. With regard to their protein chemical characteristics, the two purifi ed enzymes differed from each other. The most obvious divergence between the two proteases is refl ected by the difference in pH for their activity optimum and their inhibitory profi les. A pH optimum at around 4.5 as indicated for preparation B suggests an intracellular or vesicular origin of this protease. The pH optimum recorded for preparation A (in the range of 6-8) is compatible with a protease located in the cell membrane exposed to the extracellu-lar compartment. With respect to their sensitivity towards group-specifi c inhibitors PHMB, PMSF and EDTA but also to captopril and phosphoramidon it was con-cluded that preparation A is closely related to neutral endopeptidase (NEP), whereas preparation B resembles the substance P endopeptidase (SPE), previously identifi ed in human CSF. A possible identity between preparation B and SPE was further sup-ported by the observed cleavage pattern and the estimated molecular weight.

Enzymes which would act on substance P in a similar fashion to preparation B are to be found among the cathepsins, e.g. cathepsin D. Both cathepsin D and prepara-tion B have their pH-optimum at acidic pH and both enzymes are capable of hydro-lyzing substance P in the vicinity of its aromatic amino acid residues. However,

31


there are several properties, such as molecular weight, that differ the present enzyme from cathepsins. Also the preparation B activity differs with regard to several other kinetic and biochemical properties as compared to the cathepsins. Cathepsin D has been reported to degrade SP, that appeared to be restricted to the Phe7-Phe8 bond, while our preparation B also shows marked ability to cleave the Phe8-Gly9 bond. Moreover, in experiments performed at neutral pH we noted that at prolonged incubation, preparation B was fully capable of hydrolyzing substance P in a similar fashion as at pH 4.5. This observation suggests that if the enzyme could be released into the extracellular fl uid it may still effectively convert its substrate.

Another substance P degrading enzyme, which resembles the present preparation B is the above mentioned human brain endopeptidase. This enzyme (designated SPDE) was also identifi ed in the rat spinal cord using an immunocytochemical technique. The rat enzyme, however, appeared to be associated with a molecular weight of around 70 kDa, a value which differs from that determined for prepara-tion B. The kinetic properties (including cleavage specifi city, pH-optimum, K

m,

etc.) for the rat SPDE were not described.The high specifi city of preparation B for substance P (K

m=5 µM) is of interest.

It suggests that it may have a regulatory function of the substance P level in the spinal cord area, and thereby control the action of the peptide in, for instance, pain-processing pathways. Previous studies have shown that the activity of a SPE-like enzyme in the rat CSF is affected during chronic pain condition. In human CSF the activity of SPE was seen to be affected in certain states of chronic pain. Also during conditions of opioid tolerance the activity of SPE in the rat CSF seemed to be affected.

It is clear that both enzymes purifi ed in this study could be important for the con-trol of the substance P level in the spinal cord and thus affect the pain signal in this area. So far we have only focused on whole spinal cord and further characterization of the indicated SPE-activity would be essential for the elucidation of SPE’s role in pathophysiological conditions. The development of selective inhibitors will facilitate a wider research view of the spinal SPE activity.

4.1.2. Purifi cation from human spinal cord

The Triton X-100 soluble fraction (“membrane bound”) of the spinal cord extract was subjected to an ion-exchange chromatography, which resulted in the recovery of an endopeptidase activity capable of releasing the SP(1-7) fragment from SP. The enzyme activity obtained from this step was purifi ed from contaminants, since over 90% of the protein was removed by this procedure (Table 4.2). The protease activity was eluted in an area corresponding to 170 ml in the NaCl range 0.33 M to 0.46 M of the gradient. This activity was further purifi ed by molecular sieving on a Sephadex G-50 column, which yielded an activity over 15 times purifi ed from the previous step on protein basis (Table 4.2). The pooled active fraction from the Sephadex G-50 column was concentrated and applied to the new HPLC-system, ÄKTATM-purifi er (Pharmacia Biotech) for HPLC-molecular sieving. The peak associated with the enzyme activity was considered as the spinal cord preparation.

32

Krister Karlsson

The result from this step and the over all purifi cation is shown in Table 4.2. The specifi c activity in the crude extract was diffi cult to estimate due to the many con-taminating protease activities present, however, the ion-exchange step performed on the extract did probably at least purify the specifi c activity of the extract to a factor of 10 in magnitude, giving the over all purifi cation-factor to be above 500.

To verify the cleavage of SP by the three enzymes RP-HPLC separation was con-ducted as described [122]. The major cleavage sites as determined, were at the Phe7-Phe8 bond and at the Phe8-Gly9 bond. This observation distinguishes SPE from the cathepsins, which cleave SP restrictively at the Phe7-Phe8 bond [14].

An enzyme acting on substance P was partially purifi ed from the membrane-bound fraction of the human spinal cord. A substance P endopeptidase activity was also recovered from human cerebrospinal fl uid by the method described[181].Both enzymes were capable of hydrolyzing the undecapeptide at the Phe7-Phe8

bond. With regard to their molecular sizes, and inhibition profi le, the two purifi ed enzymes differed from each other. The most obvious divergence between the two SPE activities is refl ected by the difference in sensitivity to thiol-blocking agents and pepstatin A (Table 4.5). The spinal cord SPE seemed much more sensitive than the CSF enzyme toward PHMB. Regarding the NK-1 receptor agonist/antagonists GR-82334, it had a less potent inhibitory action on the CSF SPE, while the opposite was seen for L-703,606 which is a non peptide compound. The non-peptidergic NK-1 receptor compound WIN 51,708 potently inhibits all three enzymes, chy-motrypsin included. The tachykinin C-terminal mimicking compound GR-73632 gives rise to a clear inhibition of the enzyme activity for both the SPE preparations and chymotrypsin, suggesting that the C-terminal sequence which is common for all tachykinins (Phe-X-Gly-Leu-Met-NH

2) is important for recognition (Table

4.5). It is true that SPE shares some features with for instance cathepsin D, but as previously shown [122] several properties, such as molecular weight, cleavage specifi c-ity, kinetics, and stability at neutral pH, distinguish from those of the cathepsins. At neutral pH in this study, Km was determined by means of SP turnover to be 2 µM, 5 µM and 12 µM for the spinal cord peptidase, the CSF enzyme, and chy-motrypsin, respectively. This high affi nity for the substrate is in the same order of magnitude as previously reported for a SPE-like activity [122; 181]. Another substance P degrading enzyme (designated SPDE), which resembles the present preparations, is a human brain endopeptidase [146], which also has been reported to be present in the rat spinal cord [205]. The present SPE may have a regulatory function on the sub-stance P level in the spinal cord area, and thereby control the action of the peptide in for instance pain processing pathways. Previous studies have shown that the activity of a SPE-like enzyme in the rat CSF is affected during chronic pain condition [199].In human CSF the activity of SPE was seen to be affected in certain states of chronic pain [150]. Also during conditions of opioid tolerance the activity of SPE in the rat CSF seemed to be affected [199]. It is clear that both enzymes purifi ed in this study could be of importance for the control of the substance P level in the spinal cord and thus affect the pain signal in this area.

33


Table 4.2Purifi cation and recovery of substance P endopeptidase activity from human spinal cord (24 g)Step Total protein Total activity Specifi c activity Factor

mg pmol/min pmol/min/mgExtract 852 - - -DEAE-Sepharose 80 227 2.83 1Sephadex G-50 5.2 73.9 14.2 5Superdex 75 1.05 151 143 51

4.1.3. Purifi cation from rat ventral tegemental area (VTA)

Fig. 4.5. shows the distribution pattern of protein content and enzyme activity obtained when the soluble fraction from the extracted rat VTA was subjected to size exclusion chromatography. As shown, the SPE activity was resolved in three major activity peaks one appeared at fractions 16-17, another at fraction 21, and a third at fractions 25-27. All these three peaks were pooled into three fractions which were separately applied to HPLC-anion-exchange chromatography. The result from this separation indicated that the major activity seen after ion-exchange chromatography originated from the early eluting material (fraction 16-17) in the gel fi ltration step. Only very little activity was recovered from ion-exchange chromatography of frac-tions 21 and 25-27. In the case of the activity in fractions 25-27 it is likely that this is due to interference of low molecular compounds (e.g. salts, or even endogenous SP(1-7)) in the RIA procedure. The enzyme activity and protein profi le recorded in this step are shown in Fig. 4.6. As can be seen in the fi gure, the major SPE activ-ity was found to elute ahead of gradient. At least two separate SPE-like fractions were observed. Additional studies based on the inhibitor testing of activity found in the size-exclusion step suggested that a more phosphoramidon sensitive activity was present in fraction 16-17, whereas a captopril sensitive activity was recovered in fraction 21. Regarding the two activity peaks found in fraction 3 and 6 in the ion-exchange step (referred to preparation A and preparation B, respectively) the sen-sitivity towards phosphoramidon remain only in preparation A. Preparation B was neither sensitive toward phosphoramidon nor captopril. By phosphoramidon (15 µM) prep. A was inhibited 30% compared to control but was not affected by capto-pril. These fi ndings indicate that a NEP-like phosphoramidon activity (prep. A) was possible to discern from an SPE-like activity (prep. B). By SDS-PAGE a major band was stainable for the SPE-like preparation B (Fig. 4.7). Molecular weights (Mr) values of 90 kDa and 76.3 kDa were estimated for the enzyme preparations A and B, respectively. Mr for preparation A was determined from a commassie stained gel of poor quality, not shown. Based on protein assay, the amount of protein in the crude extract originating from 410 mg tissue was estimated to 1 804 µg. In the purifi ed preparation A and B the protein content was assessed to 30 and 250 ng, respectively. The purifi cation factor based on specifi c activity were almost 7,500 for the SPE-like activity (preparation B), see Table 4.2. The corresponding fi gure for preparation A was calculated to 45,000.

34

Krister Karlsson

Fig. 4.5 Size exclusion chromatography of the soluble fraction of rat ventral tegemental homog-enate.The extract was applied to a Superdex 75 HR 30/10 column and eluted with 0.04 M NH

4HCO

3 at

a fl ow rate of 0.5 ml/min. Fractions of 0.5 ml were collected.*Peak I fractions (16-17), and **peak II fractions (21) were pooled separately, and each pool was further processed by anion-exchange chromatography as described in the text.

Fig. 4.6 HPLC-anion exchange chromatography on the material present in peak I from the size exclusion step (Fig. 4.5).The pooled material was applied on a Resource Q (1 ml column) through a 1000 µl sample loop. The fl ow rate was 4 ml/min. and fractions of 1.0 ml were collected during the linear elution gradi-ent of 0-1.0 M KCl, 20 mM Na-phosphate, pH 7.4. The enzyme activity was assayed as described in the text.*Preparation A (corresponding to fraction No. 3), and **preparation B (corresponding to fraction No. 6) were analyzed on SDS-PAGE (Fig. 4.7.).

35


Fig. 4.7 Sodium Dodecyl Sulfate Polyacrylamide gel electrophoresis (SDS-PAGE).Analysis was performed on the preparation A and preparation B from the anion-exchange step (Fig. 4.6.). The preparations were lyophilized and resolved in 10 µl of sample buffer (62.5 mM Tris-HCl, pH 6.8; 10% (v/v) Glycerol; 2% (w/v) SDS; 0.05% Bromphenol blue; H

2O ad. 8.0 ml)

before run.Lane I Bio-Rad Kaleidoscope, Cat. No. 1610324, Lane II 0.5 µg Bovine Serum Albumin (BSA), Lane III 0.1 µg BSA, Lane IV 0.25 µg BSA, Lane V Prep B.

Two enzyme activities capable of degrading the undecapeptide substance P were recovered. One of these activities was to a part (30%) sensitive toward the NEP inhibitor phosphoramidon, while the other activity was not affected neither by phosphoramidon nor captopril. An ACE-like activity was detected after the gel per-meation step but was only detected in negligible amounts after ion-exchange chro-matography. These enzymes seem to resemble the substance P degrading activity previously found in the rat spinal cord, where two different SPE-like activities were found. One of which was inhibited by phosphoramidon and captopril and another that was not affected by either phosphoramidon or captopril. As shown for the enzymes in the spinal cord, SPE in VTA is also present as a soluble protein. Thus, it is likely from this study that SP is to a large extent inactivated in the rat VTA by a NEP-like enzyme, but also that an SPE-like activity signifi cantly contribute to the hydrolysis of SP. Previous studies have shown SPE to be more specifi c for SP and it could well be at least as signifi cant for the SP regulation in that area, as suggested for the spinal cord.

It is also possible to conclude that the ÄKTA™-purifi er is a powerful system for small amounts of enzyme activity. An about 7,000-fold (preparation A) or 40,000-fold (preparation B) purifi cation factor in a 2-step procedure demonstrates an impressing capacity of this system. Dealing with minute amounts of protein may complicate the handling of enzymes. Attachment of active material to the wall of the incubation vials and loss of enzyme material during the process may contribute to loss of activity and enzyme stability. Also during purifi cation, the optimal condi-tions for enzyme activity may change.

36

Krister Karlsson

The functional relevance of the purifi ed enzymes preparations still remains to be clarifi ed. However, as both of them have the ability to release SP(1-7) from SP it is obvious that both can modulate the signal of SP. In the case of morphine withdrawal, where the content of the fragment seems to increase it appears that the enzyme activity releases a compound counteracting the effect of the parent peptide. Thus, SP potentiates the intensity of the withdrawal reaction, while the fragment SP(1-7) has the opposite effect.

As a conclusion, the ÄKTA™-purifi er was used to purify two enzyme activities from rat VTA. Both enzymes hydrolyze SP to yield its bioactive SP(1-7) fragment. One of these peptidases resembles NEP whereas the other exhibits characteristics similar but non-identical to SPE. The chromatographic system appears useful for this purpose but to fully elucidate the structure of the studied activities more tissue needs to be processed.

4.2. Enzyme Chemical Characterization of Substance P Endo-peptidase (SPE)

4.2.1. Cleavage pattern

Conversion studies with larger amount of substrate were guided by reversed-phase HPLC (Fig. 4.8) and it was observed that the SPE preparation A from the rat spinal cord was capable of releasing the fragments SP(1-6), SP(1-7), SP(8-11), SP(1-8) and SP(9-11) as major products from substance P. Furthermore, a similar pattern was seen for preparation B, except that a more specifi c cleavage between the Phe7-Phe8 and Phe8-Gly9 bonds seemed to occur. The products generated were all con-fi rmed by mass-spectrometry.

Fig. 4.8 Reversed phase High Pressure Liquid Chromatography (RP-HPLC).Panel a, RP-HPLC of the converted fragments after 20 h of substance P (2.83 µg) incubation with the enzyme (preparation A). The reaction was carried out with 6.3 µg of protein in a total volume of 30 µl. The marked fragments were verifi ed by mass spectrometry (ES-MS/MS, see text for details). Panel b, RP-HPLC of the converted fragments after 5 h of substance P incubation with the enzyme (preparation B). Incubation was performed as described for preparation A with 5.9 µg of protein, with subsequent MS analysis, in a line with the above mentioned.

37


4.2.2. Substrate specifi city

In conversion studies (Table 4.3) using various tachykinins and substance P deri-vates as substrates, it was noticed that both preparations A and B showed a high activity for substance P conversion, but a much lower action on neurokinin B and (MePhe8,Sar9)-SP. Preparation B was able to convert eledoisin in comparable amounts as with substance P. These observations indicate that the substrate specifi c-ity for our preparations is closely related to the substance P structure.

Table 4.3HPLC Recorded Conversion of Substance P and Other SubstratesPeptides (1,4 nmol) were incubated with the enzyme (4 µg) in a total volume of 30 µl for 5 h at 37 °C. Reaction mixtures were analyzed by reversed-phase HPLC. See text for a more detailed description.Substrate preparation A*

% conversionpreparation B**

% conversionSubstance P 62 57Neurokinin B 18 0Eledoisin 27 60(MePhe8,Sar9)-SP 12 0*A refers to preparation A and **B to preparation B. For a more detailed description see text.

4.2.3. Inhibition profi le

The Michaelis-Menten constant, Km, as estimated for the release of SP(1-7) from SP

was 15 µM for preparation A and 5 µM for preparation B. The inhibition profi le for the rat spinal cord derived SPE differed between the two preparations with regard to sensitivity to group-specifi c inhibitors and activators (Table 4.4). Preparation A was inhibited by the thiol-blocking agent p -hydroxymercuribenzoate (PHMB) and the serine protease inhibitor phenylmethanesulfonyl fl uoride (PMSF), while prepara-tion B remained unaffected by PMSF but was rather activated by PHMB. A striking feature of preparation A is its inhibition by high concentrations of phosphoramidon, as well as captopril. This may indicate an activity similar to neutral endopeptidase (EC 3.4.24.11) or angiotensin converting enzyme (ACE). Both preparations were strongly inhibited by the chelating agent EDTA.

38

Krister Karlsson

Table 4.4Effects of Various Agents on Enzyme ActivityAbbreviations: EDTA, ethylenediamine tetraacetic acid; PHMB, p -hydroxymercuribenzoate; PMSF, phenylmethanesulfonyl fl uoride.Agent Conc. preparation A*

% Controlpreparation B**% Control

mMPhosphoramidon 0.1 61 98Captopril 0.1 69 107EDTA 5 17 10PHMB 1 54 142PMSF 1 55 103Ca2+ 1 251Co2+ 1 110 53Cu2+ 1 25 8Fe2+ 1 34 30K+ 1 60 102Mg2+ 1 68 121Na+ 1 71 93Zn2+ 1 49 80*A refers to preparation A and **B to preparation B. For closer descrip-tion see the text.

Using SPE from human spinal cord, studies were performed in order to characterize the enzymes’ substrate-recognition sites. Four NK-1 receptor agonists/antagonists were used: GR-82334; L-703,606; WIN 51,708; GR-73632 (RBI Research Bio-chemicals International, Natick, MA, USA). The compounds were incubated together with substrate to test the sensitivity of the enzyme activity. Interestingly the two SPE preparations differed in their inhibition profi le to these agents (Table 4.5). The peptidergic NK-1 receptor agonist GR-73632 potently decreased the SP turnover rate for all three enzymes, while the cerebrospinal fl uid SPE activity appeared more insensitive than both the spinal cord SPE and chymotrypsin for the peptide NK-1 receptor antagonist GR-82334 (Table 4.5). The non-peptide (andro-stanol-derivative) NK-1 receptor antagonist WIN 51,708 seemed to be robust at inhibiting the peptidase activity of all three enzymes with a striking similarity between the spinal cord SPE and chymotrypsin. The non-peptidergic NK-1 recep-tor antagonist L-703,606 appeared to be the least potent of the four NK-1 receptor substances to inhibit the SPE activity. However, since chymotrypsin was inhibited, this compound distinguishes chymotrypsin from the SPE preparations. The three enzymes were also tested for other agents (Table 4.5). The inhibition profi les of the peptidases for these agents were similar as far as the metal dependency is concerned showing moderately but clear inhibition by the chelating agent EDTA. The sensitiv-ity towards thiol-specifi c inhibitors/activators seemed to be slightly less pronounced for the cerebrospinal fl uid SPE preparation. To verify the cleavage of SP by the three enzymes RP-HPLC separation was conducted as described [122].

39


Table 4.5Effects of various agents on enzyme activity.The abbreviations used are: EDTA, ethylenediamine tetraacetic acid; PHMB, p -hydroxymercuribenzoate;

Agent Conc. SPE human spinal cord

SPE human cere-brospinal fl uid

Chymotrypsin bovine pancreas

mM %Control %Control %ControlGR-82334 0.2 42 74 41L-703,606 0.2 81 53 38WIN 51,708 0.2 19 37 19GR-73632 0.2 44 44 28Aprotinin 1 51 51 25PHMB 1 16 54 30Pepstatin A 1 18 86 33EDTA 5 52 59 40

4.3. Functional Characteristics of Substance P Endopeptidase (SPE)

4.3.1. Central nervous system distribution

SPE-like activity was assessed and detectable in many areas of the rat CNS (Table 4.6). In controls, saline treated rats, high enzyme activity was found in the spinal cord, hypothalamus, PAG, and striatum, whereas moderate activity was measured in e.g. VTA and nucleus accumbens. Following eight days of daily morphine injec-tions (s.c.) a slight increase in SPE-like activity was observed in substantia nigra and VTA, whereas in the striatum the opioid induced a signifi cant decrease in the enzyme activity (Table 4.6). A tendency to a decrease in the enzyme activity was also found in the PAG of morphine treated animals. During naloxone precipitated withdrawal, the SPE-like activity was signifi cantly enhanced in PAG, the pituitary, substantia nigra, VTA and spinal cord. In VTA an approximately 4-fold increase in enzyme activity was recorded. As mentioned in Material and methods (statistical analysis) it is not possible to compare data shown in the upper part of the table with those presented in the lower part (from naloxone treated groups), as animal groups were treated differently.

40

Krister Karlsson

Table 4.6Substance P endopeptidase-like activity in male rat central nervous system during mor-phine tolerance and withdrawalSPE-like activity (pmol/min/mg)Tissue control morphineHypothalamus 29.1±4.7 33.9±33.9Nucleus accumbens 18.1±3.0 18.2±3.9Periaqueductal gray 35.2±12.6 19.9±1.6Pituitary 2.8±0.7 3.3±0.9Striatum 39.6±5.7 20.3±1.7Substantia nigra 9.1±1.1 13.3±1.6VTA 18.6±5.1 25.1±4.4Spinal cord 83.9±35.2 76.5±15.0

control + naloxone morphine + naloxoneHypothalamus 30.3±7.1 36.4±8.0Nucleus accumbens 12.4±3.7 20.5±6.6Periaqueductal gray 12.5±2.1 36.3±6.9**Pituitary 1.9±0.4 6.0±1.5*Striatum 39.6±5.5 47.0±4.2Substantia nigra 9.8±1.3 22.5±2.2***VTA 26.4±5.9 93.0±29.0*Spinal cord 96.5±19.4 323.0±75.1***P<0.05, **P<0.01, ***P<0.001, morphine+naloxone treated animals versus the control+naloxone group; ††P<0.01, morphine treated animals versus controls.

4.3.2. Pattern in morphine tolerance and withdrawal

By comparing the levels of SPE-like activity with scores recorded in the behavioral assay it is evident that the enzyme activity correlated with some of the withdrawal signs. A signifi cant negative correlation was observed between the enzyme level in VTA and signs of “wet dog shake” (r2=0.568, p<0.05). In contrast, a signifi cant positive correlation was observed between SPE activity in VTA and “chewing” (r2=0.656, p<0.05). A positive correlation between grooming behavior and SPE-like activity was observed in substantia nigra (r2=846, p<0.01). Despite a low frequency of “scratching” behavior observed during withdrawal (Table 4.7), signifi cant corre-lation between the signs of this behavior and the SPE-like activity in the spinal cord was calculated (r2=0.597, p<0.05). No signifi cant correlation between withdrawal behavior and enzyme activity in any other tissue examined was observed.

Table 4.7Correlation coeffi cients between SPE-like activity and morphine withdrawal signsWithdrawal signs Tissue P R2 Type of correlationWet dog shake VTA 0.05 0.568* NegativeChewing VTA 0.03 0.656* PositiveGrooming Substantia nigra 0.003 0.864** PositiveR=correlation coeffi cient; VTA=ventral tegemental area; *P<0.05, **P<0.01

41


4.3.3. Behavior in heat-stress

The assessment of SPE activity in crude extracts from the various brain extracts indicated that enzyme activity was present in all tissues examined. In untreated con-trol rats high levels of SPE activity were recorded in the brain stem, cerebral cortex, hippocampus and spinal cord. Following 4 h heat stress a signifi cant increase in SPE activity was observed in all tissues except for the brain stem (Table 4.8).Extracts originating from the rat hippocampus were pooled and the SPE activity was purifi ed by gel permeation chromatography. In accordance with the purifi cation scheme previously outlined for the recovery of SPE-like activity from the rat VTA [124], the fi rst peak was further processed using anion-exchange chromatography. Also in this chromatographic step the SPE activity was separated in two distinct fractions. According to the previously published scheme the fi rst peak in this fi gure is to be regarded as a phosphoramidon-sensitive substance P endopeptidase-like activity. We denoted this as “preparation A”. The second peak, “preparation B”, would correspond to the non-phosphoramidon sensitive substance P endopepti-dase-like activity. As we continued to investigate how the heat-shock treatment infl uenced the SPE-like activity in the hippocampus area of the rat, we used the combined fraction of preparation A and preparation B in order to have suffi cient amounts of enzyme. We found out that the heat-shock treated group of animals exhibited a striking 27% increase in SPE-like activity (Table 4.8). This result was also in a line with the testing of the soluble fraction of the crude extract, where an even higher increase (95%) in SPE activity was found (Table 4.8). While the testing of the purifi ed SPE-activity was done without inhibitor, the SPE-activity in the crude extract was measured with the inhibitor-mixture has described in the methods section. This difference may explain the discrepancy in activity observed between the two SPE-containing fractions.

Table 4.8Changes in SPE-activity in various central nervous system regions of heat-stressed rats

Tissue *SPE-like activity control(pmol·mg-1·min-1)

*SPE-like activityheat-stress(pmol·mg-1·min-1)

Change in SPE-like activity compared to control (%)

Spinal Cord 1.97 4.28 +117Brain Stem 2.29 1.81 -21Cerebellum 0.53 2.48 +368Diencephalon(Thalamus + Hypo-thalamus)

1.55 3.85 +148

Hippocampus 2.65 5.18 +95Cerebral Cortex 3.19 4.44 +39Hippocampus(highly purifi ed SPE)

46,800 63,700 +27

*Data from the pooled tissues of six animals is shown. This was done for preparative reasons, and results were derived from triplicates of RIA measurements.

42

Krister Karlsson

The present study provides evidence that heat stress affects the activity of SPE in several brain regions. The high levels of SPE-like activity in extracts from the hip-pocampus prompted us to purify this enzyme activity for further characterization. The SPE activity was purifi ed according to the schemes generally laid out in preced-ing papers [122; 123; 124]. It is now evident that that scheme is successfully applied for the recovery of SPE or SPE-like activities from a number of different CNS regions. As our recent study concluded, it is possible to obtain SPE from very small regions of the brain such as the ventral tegemental area (VTA) of the rat. For this purpose the Amersham Bioscience’ preparative chromatographic system ÄKTA™-purifi er is used. Indeed, the methodology of micro purifi cation seems possible to apply when studying neurobiological events, which must be considered a highly sophisticated technical state, and very useful in the gain of knowledge in the fi elds of brain and central nervous system biology. Up to the present we have performed the purifi ca-tion of SPE in various CNS areas such as spinal cord, VTA, and hippocampus using this technique [124]. In this work we also noted increase of SPE-like activity in hip-pocampal fractions purifi ed from heat stressed animals. The increase of this enzyme activity during heat stress may have several implications. Its substrate peptide, SP, has previously been shown to be involved in brain processes important in opioid withdrawal in rats [161]. Studies have also demonstrated that SP may enhance the intensity of opioid withdrawal, an effect which is counteracted by its heptapeptide fragment SP(1-7) [140]. Both the level of SP(1-7) and the SPE activity in VTA are shown to be elevated during morphine withdrawal. SP and its heptapeptide, as well, are shown to affect memory processes and the functional anatomy of memory and cognitive function are suggested to be associated with hippocampus. Therefore, it is inviting to speculate upon the possibility that the increased activity of SPE may produce an imbalance in the level of SP and its fragment, which may be involved in cognitive disabilities seen in heat stressed individuals. The consequences of altera-tions in SPE activity seen in other brain structures (Table 4.8) is not known but clearly it may be of importance for several behaviors associated with SP in the actual brain areas.

43


5. CONCLUSIONS

This thesis presents evidence for the existence of the substrate specifi c neuropeptide endopeptidase “substance P endopeptidase” (SPE), acting on substance P (SP). SPE was purifi ed and biochemically characterized from a variety of central nervous system (CNS) regions, including rat spinal cord, rat ventral tegemental area (VTA), human spinal cord, human cerebrospinal fl uid (CSF), rat hippocampus, and other brain regions such as brain stem, cerebellum, diencephalon (hypothalamus and thalamus), and cerebral cortex. Preliminary purifi cation schemes were also done for nucleus accumbens, striatum, periaqueductal gray, and the pituitary.

By enzymatic comparison SPE was possible to distinguish from other known peptidases such as angiotensin converting enzyme (ACE), neutral endopeptidase (NEP), cathepsin D, and chymotrypsin.

SPE was biochemically mapped in CNS, and distribution was assessed in spinal cord, CSF, brain stem, VTA, periaqueductal gray, hypothalamus, thalamus, hip-pocampus, striatum, nucleus accumbens, cerebellum, cerebral cortex, and pituitary, using direct enzyme activity measurements.

SPE activity turned out to be affected during morphine tolerance and withdrawal, and is probably an important parameter in these states. SPE activity was substan-tially increased after heat stress treatment in rat brain hippocampus, suggesting a regulatory role in this area under heat traumatized conditions.Hence, it is possible to state that:

• SPE was successfully isolated, purifi ed and extensively identifi ed as a sig-nifi cant SP neurotransmission/neuromodulation regulatory enzyme in the CNS.

• SPE was recovered using delicate high technology micro-preparation methodology, proving that this new technique provides an excellent tool in studying enzymatic processes.

• SPE plays an important role during morphine physical dependence, toler-ance, and withdrawal.

• SPE is likely to contribute to the SP involvement after traumatic brain injury, such as acute heat stress.

• The physiological function of SPE is now possible to evaluate with direct biochemical assessment of its activity.

• The increased activity seen in SPE during opioid withdrawal and heat stress may enhance the release of a peptide fragment from SP, counteracting the expression of the actual stress symptoms. Thus, SPE is a possible target for future pharmacological therapy.

44

Krister Karlsson

6. ACKNOWLEDGEMENTS

The presentation of this thesis is possible through the contribution by many people. Grati-tude to all teachers and tutors, who have shared their skills and knowledge through out the years. In addition, the following are specifi cally recognized for their efforts:Prof. Fred Nyberg, for smooth tutoring and sharing the vast scientifi c skillsProf. Lars Oreland, for medical tutoring and understandingly overseeingDr. Pierre LeGrevés, for co-tutoring and collaborating, especially molecular biologyProf. Per Andrén, for co-authoring and sharing the science of mass-spectrometryProf. Hari Shanker Sharma, for co-authoring and introduction to the fi eld of hyperther-miaDr. Qin Zhou, for co-authoring and great collegial chatsDr. Liu Zhurong, for co-authoring and co-laboration and a lot of creativityUlrica Eriksson, for co-authoring and co-laboration in the early graduate phaseDr. Dan Henrohn, for inspiration to pursue the education as physician and good friend-shipDr. Michael Fountoulakis, and Dr. Christer Norstedt, for scientifi c tutoring at F. Hoffman La Roche AG, Basel, SwitzerlandDr. Adlan Elhassan, for co-working and medical chatsAll other current and previous colleagues @Biol. Res. on Drug Dependence: Dr. Anna Kindlundh, Dr. Katarina Sanderson-Nydahl, Dr. Pia Johansson-Steensland, Agneta Berg-ström, Alexandra Nystrand, Barbro Synnergren, Britt-Marie Johansson, Francesca Bianchi, Hugo Nilsson, Madeleine LeGrevés, Mattias Hallberg, Milad Botros, Mina Pourmousa, Per-Anders Frändberg, Petra Villani, Rebecka Klintenberg, Tobias Johnsson; @Biochemi-cal Pharmacology: Prof. Ernst Oliw, Dr. Chao Su, Dr. Tina Kunz, Erica Johansson, Kata-rina Stark, Lydia Schauer, Mirela Cristea; @Pharmaceutical Pharmacology: Prof. Ingrid Nylander, Prof. Jarl Wikberg, Prof. Lena Bergström, Dr. Anne-Lie Svensson, Dr. Jonas Lindblom, Dr. Karolina Ploj, Agneta Hortlund, Aleh Yahorau, Amanda Raine, Ann-Marie Hedlund, Annika Häger, Erika Roman, Felikss Mutulis, Ilona Mandrika, Ilze Mutule, Hans Darberg, Kjell Åkerlund, Lisa Gustafsson, Magnus Jansson, Maris Lapinsh, Marita Berg (also for M.D. student affairs), Petris Prusis, Ramona Petrovska, Santa Veiksina, Sergei Kopanchuk, Svetlana YahoravaAmersham Bioscience (former Amersham-Pharmacia Biotech), for impressive range of products and numerous service encounters as well as workshops, and tipsBioRad Sweden for top of the line proteomics equipmentSwedish medical research council, for grants: 9459, and 11565All the fellow medical students, for inspirations and big ambitionsAll fantastic friends, for being nice, especially: Mikael Agge, Gustaf Götmar, Lisa Mietinen, Jörgen Möller, Isa Tacacho, Annelie Hultberg, Maria Eriksson, Susanne Lindberg (also for proof-reading), Lina Karlsson, Borislava Brkovic’, Sara Rostedt, Emma Elmgren, Daniel Johansson, Daniel Hultander, Mattias Larsson, Daniel Hagström, Johan Lans, Lenakim Arctaedius-Svenungsson, Tobias Hübinette (and all of AKF), Johan Högberg (and all of AR), Åsa Johansson, Christian Lindberg, Sandra Sporrenstrand, Lina & Jonas Nimmersjö, Samuel Hector, etc. ...please forgive me for memory blackout.Above all I thank GOD, in the name of the LORD Jesus Christ. Thanks for medical science.

45


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Acta Universitatis UpsaliensisComprehensive Summaries of Uppsala Dissertations

from the Faculty of MedicineEditor: The Dean of the Faculty of Medicine

Distribution:Uppsala University Library

Box 510, SE-751 20 Uppsala, Swedenwww.uu.se, [email protected]

ISSN 0282-7476ISBN 91-554-5908-0

A doctoral dissertation from the Faculty of Medicine, Uppsala University,is usually a summary of a number of papers. A few copies of the completedissertation are kept at major Swedish research libraries, while the sum-mary alone is distributed internationally through the series Comprehen-sive Summaries of Uppsala Dissertations from the Faculty of Medicine.(Prior to October, 1985, the series was published under the title “Abstracts ofUppsala Dissertations from the Faculty of Medicine”.)

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