regional distribution of pyroglutamyl peptidase ii in rabbit brain, spinal cord, and organs

Peptides, Vol. 13, pp. 255-260, 1992 0196-9781/92 $5.00 + .00 Printed in the USA. Copyright © 1992 Pergamon Press Ltd.

Regional Distribution of Pyroglutamyl Peptidase II in Rabbit Brain, Spinal Cord, and Organs

M I G U E L A N G E L VARGAS, M I G U E L CISNEROS, J O R G E H E R R E R A , PATRICIA J O S E P H - B R A V O A N D JEAN-LOUIS C H A R L I l

Departamento de Bioqulmica, Instituto de Biotecnolog{a, Universidad Nacional Autrnoma de M~xico, A.P. 510-3, Cuernavaca, Mor., 62271, Mexico

Received 28 May 1991

VARGAS, M. A., M. CISNEROS, J. HERRERA, P. JOSEPH-BRAVO AND J.-L.CHARLI. Regional distribution ofpyroglutamTl peptidase 11 in rabbit brain, spinal cord, and organs. PEPT1DES 13(2) 255-260, 1992.--Pyroglutamyl peptidase II (PPII) is a narrow specificity ectoenzyme that degrades thyrotropin-releasing hormone (TRH). We detected the enzyme in the brain of various mammals, with highest specific activity in rabbit brain. In this species, activity was heterogeneously distributed in the central nervous system. There was a 28-fold difference between regions of highest and lowest PPII activity. Enzyme activity was highest in the olfactory bulb and posterior cortex. In the spinal cord, activity was low but unevenly distributed, with highest values detected in the thoracic (T) region. Segments T I and T2 activities were particularly high. Other organs contained low or undetectable levels of activity. The levels of TRH-like immunoreactivity (TRH-LI) in spinal cord segments were greatest in T3-T4 and lumbar L2-L6. Low concentrations were found in TI and T9-TI2. There was a partial correlation between the distribution of PPII activity and TRH receptors but not with TRH-LI levels. These results demonstrate that PPII is predominantly a central nervous system enzyme, and they support the hypothesis that PPII is responsible for degrading TRH released into the synaptic cleft.

Thyrotropin-releasing hormone Autonomic nervous system Thoracic co rd Sacral cord

Pyroglutamyl peptidase II Spinal co rd Rabbit Thyroliberinase Synapse Motor neurons Cord segment Cervical cord Lumbar cord

Testis

SEVERAL peptidases degrade thyrotropin-releasing hormone (pGlu-His-Pro-NH2). From these, pyroglutamyl peptidase I (PPI, E.C. 3.4.19.3) and prolylendopeptidase (PE, E.C. 3.4.21.26) are cytosolic enzymes present in neurons and glia [for review see (20)]. They have a broad substrate specificity (3), and their involvement in TRH metabolism has been questioned (4). Another pyroglutamyi peptidase, present in serum (thyroliberinase), dif- fers substantially from PPI in several biochemical characteristics, including its narrow substrate specificity (24).

In the brain, a pyroglutamyl peptidase (PPII, E.C. 3.4.19.-), similar to thyroliberinase, is found in the plasma membrane of nerve endings (19,25). It is an ectoenzyme (5), localized in neuronal cells (9), and its specific inhibition allows increased recovery of TRH released from brain slices (6). These evidences, together with its narrow specificity characteristics (27), make PPII the likely candidate for the inactivator of TRH at the synaptic cleft.

PPII has been detected in guinea pig, rat, and rabbit brains (5,19,27). In the rat, the brain has the highest specific activity compared to other organs ( l 1), although it varies within different regions (26). However, the distribution in the spinal cord is not yet known. TRH is present in the gray matter of the spinal cord (10). It appears to be localized in at least 3 different pathways: one originating in the medullary raphe nuclei projecting to dif-

ferent segments of the spinal cord. These neurons densely in- nervate motoneurons of the ventral horn. Other TRH neurons originating in the nuclei interfascicularis hypoglossi and para- gigantocellularis project to the intermedio-lateral column. Fi- nally, intrinsic neurons are present in the dorsal horn (18). Dorsal gray matter (Rexed laminae I and II) and ventral gray matter (laminae IX and X) are the regions most enriched in TRH receptors of the guinea pig, rat, and rabbit cord (18). The thoracic region contains the greatest density of receptors (23). TRH fa- cilitates the excitation of motoneurones, may act as atrophic agent on lower motor neurons, and modulates the spinal reflexes (18). Determination of TRH-degrading activity in the spinal cord is important due to the known beneficial effects of TRH on the outcome of experimental spinal cord injury and amyotrophic lateral sclerosis (ALS) (18).

In the present work we have measured PPII activity and TRH levels in isolated segments of rabbit spinal cord. Since previous distribution studies were made in the rat, we also performed measurements of PPII activity in different organs and brain regions of the rabbit and compared them to the rat. The whole brain specific activity of rabbit, hamster, guinea pig, pig, rat, and mouse PPII was also quantified. Our data demonstrate that PPII is heterogeneously distributed in the

Requests for reprints should be addressed to Jean-Louis Charli.

255

256 VARGAS ET AL.

spinal cord and that it is mainly a central nervous system enzyme.

METHOD

Materials

[L-proline-2,3,4,5,-3H]TRH ([3H-Pro]TRH) (100 Ci/mmol) was purchased from New England Nuclear Co., Boston, MA. Peptides were from Peninsula Laboratories, Belmont, CA. All other chemicals were from J. T. Baker or Sigma. Ion exchange paper chromatography was performed on Whatman cellulose phosphate P81 sheets. N-l-carboxy-2-phenylethyl(N im benzyl)- histidyl-fl-naphthylamide (CPHNA), a specific inhibitor of PPII (6), was a gift of Dr. S. Wilk.

Animals

Adult male New Zealand white rabbits, hamsters, Wistar rats, CDI mice and guinea pigs, kept under controlled lighting conditions (light on: 0700-1900) and fed ad lib, were used in all experiments. Fresh heads of pigs were obtained from a local slaughterhouse.

Dissections

Regions from brain, cervical, thoracic, lumbar and sacral spinal cord, as well as other organs, were rapidly dissected, frozen on dry ice, and kept at -70°C until assayed for PPII activity or TRH-L|. When segmental PPI| activity or TRH-LI was measured, spinal cord regions were thawed out, placed on ice, and dissected in segments.

Preparation o[ Enzyme Source

Spinal cord segments and brain tissues were homogenized with a Potter Elvehjem homogenizer and other organs with a polytron homogenizer (model PT 10-35, Kinematica), in 10% (w/v) 0.05 M sodium phosphate buffer, pH 7.5. All steps were performed at 4°C. The homogenate was centrifuged at 1000 X g for 15 rain. The pellet was homogenized with half the amount of buffer and centrifuged again. The two supernatants were pooled and centrifuged at 12,000 × g for 15 rain. The final supernatant was discarded, while the pellet was resuspended in sodium phosphate buffer and used for PPII quantification.

PPII Assay

Assay was performed essentially as described (26) but at 10 -6

M substrate concentration. Briefly, membranes were preincubated for 5 min at 37°C in 25 #1 0.05 M sodium phosphate buffer, pH 7.5, containing 2.5 mM bacitracin and 2.5 mM N- ethylmaleimide. At time 0, 100,000 cpm [3H-Pro]TRH (10 6 M final concentration) (6 #1) were added and further incubated. Three different enzyme dilutions were used to ensure linearity and the best approximation to initial velocity. At 0 and subse- quent times, 5 ~1 aliquots were immediately spotted on cellulose phosphate paper. The ascending chromatography was developed in I M acetic acid. After drying the paper, His-[3H]-Pro-NH~ was eluted from the first centimeter and counted by liquid scintillation. Zero time values were substracted from the other time values and each determination was performed in duplicate. To test the effect of CPHNA, the assay buffer contained CPHNA 3 X 10 4 M plus bacitracin and N-ethylmaleimide.

TRH Radioimmunoassay and High Pressure Liquid Chromatography (HPLC)

Spinal cord segments and the entire lumbar region were homogenized in 20% acetic acid, centrifuged at 12,000 X g for 10 min at 4°C, and the supernatant evaporated. The residue obtained after dryness was extracted with 1 ml 90% methanol, vortexed, the supernatant dried, and the residue subjected to radioimmunoassay (RIA). Serial dilutions of each segment ex- tract presented an immunoreactivity parallel to the standard curve. For TRH-LI characterization, the lumbar regions were reconstituted in 0.01% acetic acid and subjected to HPLC as described (17). Fractions of I ml were collected during 20 rain, evaporated, and reconstituted in RIA buffer. A main peak of TRH-LI corresponding to synthetic TRH was detected in fractions 8-1 l and amounted to 82 + 7% of total immunoreactivity (n - 3). The remaining immunoreactivity was spread over the other fractions.

Proteins

Proteins were determined according to Lowry et al. (15) after membrane digestion with l N NaOH at room temperature for 24 h.

Statistical Analyses

Statistical analyses were performed using Student's t-test. For multiple comparisons, analysis of variance (Fisher) was followed by the multiple comparisons Duncan's test when a significant F ratio was present.

RESULTS

Pyroglutamyl peptidase II activity was stable at least for several months when intact mouse brain or rabbit membrane fractions (kept in sodium phosphate buffer) were stored at -70°C (Table 1), but not if membranes were suspended in buffer containing bacitracin and N-ethylmaleimide (not shown). All reported determinations were performed on tissue kept for less than 4 months at - 70 °C.

Brain PPII specific activity was measured in six species (Table 2). The highest activity was found in rabbit brain compared to the other species that presented similar values. Incubation of brain membranes with a specific inhibitor of PPII, CPHNA at 3 × 10-4 M, reduced enzyme activity to 43-68% of control values (Table 3). A lower concentration of inhibitor (10 4 M) caused a smaller inhibition (15-43%). These data suggest that in all species a similar enzymatic activity is present.

Distribution of PPII in rabbit brain was heterogenous: olfactory bulb and posterior cerebral cortex had the highest activity, the lowest being in medulla oblongata and hypophysis. Specific activity measurements (in identical conditions) (Table 4) gave similar values between rabbit and rat brain regions except for thyroliberinase activity, which was higher in the rat, as previously reported (2).

The main interest of this work was to define the segmental distribution of PPII and TRH in the spinal cord. PPII levels in the spinal cord were lower than in the brain; there was a 28-fold difference between olfactory bulb activity and the spinal cord region with lowest activity (S 1 ). Figure 1 shows that highly significant differences in PPII distribution exist throughout this region, F(27,70) = 2.089, p < 0.025. The thoracic segments, in particular T1 and T2, were the most active, while PPII was not detected in sacral segments $3 and $4. PPII specific activity for each entire region was higher in the thoracic region (Table 5).

D I S T R I B U T I O N OF P Y R O G L U T A M Y L PEPTIDASE II 257

TABLE 1

PPII STABILITY UPON STORAGE AT -70 ° OF WHOLE BRAIN (MOUSE) OR BRAIN MEMBRANES (RABBIT)

Time Frozen (month) Mouse Brain

PPII Specific Activity

Rabbit Brain Membranes

0 100 _+ 6 (3) 100 (2) 0.25 107 + 8 (3) 90 (2) 0.75 109 _+ 4 (3) 98 (2) 1.00 101 (2) 3.50 110 _+ 14 (3) 107 (2) 4.50 93 (2) 6.00 108 (2) 9.00 139 _+ 12 (3)

Four groups of intact mouse brains or rabbit brain membranes, resuspended in 0.05 M sodium phosphate, pH 7.5, as described in the Method section, were kept frozen at -70°C until assayed for PPII activity. Membranes from mouse or rabbit tissues were preincubated at 3 different dilutions in 0.05 M sodium phosphate, pH 7.5, containing 2.5 mM bacitracin and 2.5 mM N-ethylmaleimide for 5 min at 37°C. 100,000 cpm [3H-Pro]TRH (10 -6 M) were then added and incubation continued for up to 15 min. The His-[3H]-Pro-NHz produced was separated by ion exchange paper chromatography and quantified by liquid scintillation. We usually detected 160 cpm His-[3H]-Pro-NH2 per mg rabbit brain membrane protein in 10 min. Amount of protein in the assay tube for rabbit brain membrane was 60 ug. Specific activity was derived from the number of cpm His-[3H]-Pro-NH#min/mg protein, as well as the known amounts ofcpm [3H-Pro]TRH (100,000 cpm) and pmol TRH present in the assay tube (31 pmol). Results indicate the mean enzyme activities normalized to control (fresh mouse brain tissue or rabbit membranes). The number of independent determinations is indicated in parentheses. Mouse brain values expressed as mean +_ SEM.

The dis t r ibut ion of TRH-LI in the rabbi t spinal cord was heterogenous, F(25,45) = 15.33, p < 0.005 (Fig. 1). The highest levels of TRH-LI were associated with segments of thoracic and lumbar cord, T 2 - T 4 and L2-L6, respectively. The lowest con- centra t ions were found in T 1 and T 8 - T 12. TRH-LI con ten t per region was higher in the l u m b a r region (Table 5).

Brain PPII activity was very high compared to other organs (Table 6). In decreasing order, PPII was detected in the testis, spleen, prostate, adrenal, heart, skeletal muscle, lung, and kidney; the liver, thymus, and pancreas were totally devoid of activity.

TABLE 2

PPII SPECIFIC ACTIVITY IN THE BRAIN OF SIX MAMMALIAN SPECIES

Species PPII Specific Activity

Rabbit 5.20 _+ 0.88 (4) Hamster 3.50 +_ 0.62 (3) Guinea pig 3.33 _+ 0.86 (3)* Pig 3.23 + 0.07 (3)* Rat 3.23 _+ 0.66 (3)* Mouse 2.97 _+ 0.39 (3)t

PPII activity was determinated as described in the legend of Table 1. Specific activity is expressed as pmol His-Pro-NH2/ min/mg protein _+ SEM. Number of independent determinations is in parentheses. Statistical significance: *p < 0.05 and tP < 0.025 with respect to rabbit value.

TABLE 3

EFFECT OF CPHNA ON BRAIN PPII SPECIFIC ACTIVITY OF SIX SPECIES


Species Protein* Control t CPHNAt % Inhibition

Rabbit 0.19 7.51 4.26 43 Guinea pig 0.19 4.58 2.36 48 Hamster 0.15 4.41 2.07 53 Pig 0.17 4.62 1.74 62 Rat 0.14 3.35 1.22 63 Mouse 0.10 3.90 1.25 68

Enzyme activity was measured as described in Table 1. Membranes were preincubated for 5 min with CPHNA 3 × 10 -4 M (plus bacitracin and N-ethylmaleimide) before addition of substrate. Data are the mean of two determinations. *mg protein in assay tube. tpmol His-Pro-NH2/ min/mg protein.

The lack of activity in rabbit liver, as opposed to what was described for the rat (11), led us to compare frozen and fresh tissues, since a difference of storage stability could occur compared to brain tissue. PPII activity was undetectable in m e m b r a n e s pre- pared from freshly dissected rabbit liver or pancreas. In the rat, activity was detectable in fresh or frozen liver m e m b r a n e s but not in fresh or frozen pancreat ic m e m b r a n e s (data not shown).

DISCUSSION

On the basis of a l imited dis t r ibut ion study in the rat, Fried- man and Wilk (11) suggested that PPII is a brain-specific enzyme, and our results in the rabbit substant iate this. Several groups

TABLE 4

DISTRIBUTION OF PPII IN RABBIT AND RAT BRAIN REGIONS AND HYPOPHYSIS


Region Rabbit Rat

Olfactory bulb Posterior part of cerebral

cortex Hippocampus Cerebellar vermis Cerebellar hemisphere Nucleus accumbens-lateral

septum Mesencephalon Median eminence Hypothalamus minus median

eminence Pons Medulla oblongata Adenohypophysis Hypophysis Serum

6.58 +_ 1.02 (4) 6.33 _+ 0.29 (4)

6.50_+ 0.37 (4) 4.98 _+ 0.77 (6) 5.05 + 0.58 (4) 4.64_+ 0.73 (5) 3.78 _+ 0.21 (5)

2.20 _+ 0.25 1.90_+ 0.31 (3) 1.53 _+ 0.28 (3)

1.25 _+ 0.25 (4) 1.13 _+ 0.05 (4) 0.90_+ 0.07 (3)

(3) 2.80 _+ 0.04 (4)

1.12_+0.11 (3) 0.50 _+ 0.05 (5)

0.23 _+ 0.06 (4) 0.02 +_ 0.002 (3) 0.11 _+ 0.04 (3)

PPII was assayed as described in the legend for Table 1. Data are mean specific activity (pmol His-Pro-NH2/min/mg protein) _+ SEM. Number of independent determinations is in parentheses.

258 V A R G A S ET AL.

1100-

9 0 0 -

7 0 0 -

.c_ m 0 &

z s o o - p-

E c~

CERVICAL THORACIC LUMBAR

0

._= oJ o

0 4 -

._c

{ 0 3 -

i

Z

O 2 - E a.

0,1-

tt

C[RVICAL = JI TPIORACIC • ~ LUMBAR = ~SACRA~

FIG. I. Segmental distribution of TRH and PPII specific activity in rabbit spinal cord. Upper panel: TRH-LI was extracted by acid followed by methanolic extractions and quantified through the use of a specific RIA as described in the Method section. Data are mean pg TRH/mg protein _+ SEM. Number of determinations are 3, except for C7, T7, T9 and L5 with n = 2. Statistical significance: p < 0.05 for C2-T3, T4-TI0, and L4-T12 comparisons. Lower panel: PPII activity was measured as described in legend for Table 1, except that incubations were carded out for up to 1 h. Data are mean pmol His-Pro-NHdmin/mg protein _+ SEM. Number of independent determinations are: n - 3 for C3, C5, C6, T2, T3, T9, TI0, T11, LI, L6, L7, SI, $2, $3, $4; n = 4 for C1, C2, C4, C7, T 1, T6, T7, T8, T 12, L4; n = 5 for T4, T5, L2, L3, L5. Statistical significance: p < 0.05 for C1-C2 and C7-T1 comparisons; p < 0.01 for L7-S1 comparison.

TABLE 5

REGIONAL DISTRIBUTION OF PPII ACTIVITY AND TRH-LI IN RABBIT SPINAL CORD

Region PPll * TRH-L1 t

Cervical 0.332 _+ 0.010 (3)$ 319 _+ 66 (3)§ Thoracic 0.423 + 0.018 (3) 268 _+ 10 (3)¶ Lumbar 0.322 _+ 0.022 (4)$ 582 _+ 85 (3) Sacral 0.193 +_ 0.032 (3)$

PPII activity and TRH-LI were assayed as described in the legend for Fig. 1.

*$ Data are mean pmol His-Pro-NH2/min/mg protein _+ SEM. Statistical significance: Sp < 0.005 with respect to thoracic region.

t§¶ tPg TRH-LI/mg protein _+ SEM. Statistical significance: §p < 0.05; ¶p < 0.005 with respect to lumbar cord.

have reported that PPII activity is present in brain tissue of some m a m m a l i a n species (5,19,27). PPII in rabbit, guinea pig, and rat brains was conf i rmed and was shown to be also present in mouse, hamste r and pig brains. Its characteristics in the central nervous system might be similar between species, at least in mammals , since activities and susceptibility to C P H N A inhi- bi t ion were very similar in all species tested; fur thermore, the regional dis t r ibut ion of PPII in the rabbi t resembled that of the rat brain. As ment ioned , a l though there is coexistence of TRH, T R H receptors, and PPII [(26) and this study] in regions such as olfactory bulb, nucleus accumbens-lateral septum, and hypothalamus, no strict correlation exists between these parameters.

The spinal cord presented two peaks of TRH-LI activity, T 3 - T 4 and L2-L6, with the highest T R H levels in the lumbar segment. Fone et al. ( I0) have reported that the level of TRH- LI in the rabbit thoracic segment was greater than in the lumbar segment, but they only compared T 4 - T 5 vs. L1-L2. Our results coincide with those previously reported in the rat (12), monkey (14), and h u m a n (13). PPII activity was highest in the thoracic

TABLE 6

DISTRIBUTION OF PPII IN RABBIT ORGANS

Organ PPII Specific Activity

Brain 5.2 _+0.88 (4) Testis 0.34 _+ 0.013 (6) Spleen 0.15 (2) Prostate 0.14 (2) Adrenal 0.10 (2) Heart 0.05 (2) Skeletal muscle 0.04 (2) Lung 0.03 (2) Serum 0.02 _+ 0.002 (3) Kidney 0.02 (2) Liver <0.001 (2) Thymus <0.001 (2) Pancreas <0.001 (2)

Membranes were obtained as described in the Method section. Enzyme activity was determined as indicated in the legend for Table 1, except that incubations were carded out for up to 2 h. Values are mean pmol His-Pro-NH2/min/mg protein _+ SEM of the number of independent determinations shown in parentheses.

DISTRIBUTION OF P Y R O G L U T A M Y L PEPTIDASE II 259

region, as is the case for TRH receptors (23), suggesting that they could act in concert on TRH physiology. Although it is difficult to speculate on a possible function for PPII, particularly high enzyme values in the upper thoracic region are consistent with the postulated involvement of TRH in autonomic regulation of some functions of the cardiovascular system (18). This could lead to the design of analogues resistant to PPII action or specific inhibitors which could be effective in the treatment of diseases, including ALS. It has been shown that in ALS, TR H receptors in lamina IX decrease (18), TRH levels do not change, while histidyl proline diketopiperazine (His-Pro-DKP), a product of pyroglutamyl aminopeptidase activity on TRH, increases (13). In the brain, PPII degrades TRH released in vitro [(6), Charli et al., unpublished). In consequence, at least part of His-Pro-DKP could come from PPII action on TRH. The high levels of His- Pro-DKP in ALS may be due to increased PPII activity and/or increased TRH release. Previous studies had demonstrated that PPI and PE are present in the spinal cord, and that their levels are raised in Wobber mice but not in human motor neuron disease (8). However, TRH is probably not a substrate of these enzymes in vivo (4). PPII properties (in particular, whether it is a pre- or postsynaptic enzyme) must be defined in order to obtain a better understanding of TRH pathophysiology in the spinal cord. Except for the central nervous system, the highest activity was found in the spleen, testis, and prostate; these last two organs are rich in TRH-LI (21), although not all the immunoreactivi ty may correspond to authentic TRH (7).

Pancreatic membranes did not degrade TRH. High levels of TRH are present transiently early during postnatal development (16). Failure to detect PPII in adult rabbit pancreas is probably not due to inhibition of its expression in adulthood since, in the

rat, it is similarly absent in the first days after birth (Vargas et al., submitted). Thus, in some instances, TRH degradation by PPII may not be the TR H inactivation mechanism, or else TRH target cells are not in the pancreas. His-Pro-DKP, however, is present in cells which do not contain TRH (18). Since TRH degradation by PPII (this study) or by PPI (4) cannot generate His-Pro-DKP in this tissue, its presence is either due to uptake of circulatory His-Pro-DKP generated by thyroliberinase action or to production by a mechanism differing from TRH degradation (18).

PPII activity was not detected in rabbit liver, results that dis- agree with previous reports in the rat [(11,22), and this study]. This discrepancy is not due to loss of liver PPII activity when membranes are kept frozen, because the same results were obtained with fresh liver membranes. However, this species difference could explain the low thyroliberinase activity found in rabbit serum compared to rat serum, if thyroliberinase is generated in the liver through differential expression of PPII gene.

The limited tissue distribution of PPII contrasts with the broad distribution of peptidases postulated to be involved in the degradation of numerous peptides. In conclusion, this work gives further support for a PPII role in some of the neurotransmitter functions of TRH, as has been suggested for its neuroendocrine role (1). Its presence in the spinal cord makes it a potential ther- apeutic target in some motor neuron diseases.

ACKNOWLEDGEMENTS

We are grateful for the technical assistance ofE. Mata and S. Gonzalez, and for typing of manuscript by M. Sifuentes. We also acknowledge the gift of CPHNA from Dr. S. Wilk. Work supported in part by grants from Fundacirn Miguel Aleman A.C. and Third World Academy of Sciences.

REFERENCES

1. Bauer, K. Degradation and biological inactivation of thyrotropin releasing hormone (TRH): Regulation of the membrane bound TRH degrading enzyme from rat anterior pituitary by estrogens and thyroid hormones. Biochimie 70:69-74; 1988.

2. Brewster, D. Species variation in TRH inactivation: Advantages of stable analogues. In: Griffiths, E. C.; Bennet, G. W., eds. Thyrotropin releasing hormone. New York: Raven Press; 1983:109-118.

3. Browne, P.; O'Cuinn, G. An evaluation of the role ofa pyroglutamyl peptidase, a postproline cleaving enzyme and a postproline dipeptidyl aminopeptidase, each purified from the soluble fraction of guinea- pig brain, in the degradation of thyroliberin in vitro. Eur. J. Biochem. 137:75-87; 1983.

4. Charli, J. L.; Mrndez, M.; Joseph-Bravo, P.; Wilk, S. Specific inhibitors of pyroglutamyl peptidase I and of prolyl endopeptidase do not change the in vitro release of TRH or its content in rodent brain. Neuropeptides 9:373-378; 1987.

5. Charli, J. L.; Cruz, C.; Vargas, M. A.; Joseph-Bravo, P. The narrow specificity pyroglutamate aminopeptidase degrading TRH in rat brain is an ectoenzyme. Neurochem. Int. 13:237-242; 1988.

6. Charli, J. L.; Mrndez, M.; Vargas, M. A.; Cisneros, M.; Assai, M.; Joseph-Bravo, P.; Wilk, S. Pyroglutamyl peptidase II inhibition spe- cifically increases recovery of TRH released from rat brain slices. Neuropeptides 14:191-196; 1989.

7. Cockle, S. M.; Aitken, A.; Beg, F.; Smyth, D. G. A novel peptide, pyroglutamylglutamylproline amide, in the rabbit prostate complex, structurally related to thyrotropin releasing hormone. J. Biol. Chem. 264:7788-7791; 1989.

8. Court, J. A.; McDermott, J. R.; Gibson, A. M.; Marshall, E.; Blox- ham, C. A.; Perry R. H.; Edwardson, J. A. Raised thyrotropin releasing hormone pyroglutamylpeptidase and proline endopeptidase are present in the spinal cord of Wobber mice but not in human motor neurons disease. J. Neurochem. 49:1084-1090; 1987.

9. Cruz, C.; Charli, J. L.; Vargas, M. A.; Joseph-Bravo, P. Neuronal localization of pyroglutamate aminopeptidase II in primary cul- tures of fetal mouse brain. J. Neurochem. 56:1594-1601; 1991.

10. Fone, K. C. F.; Bennet, G. W.; Marsden, C. A. Regional distribution of substance P and thyrotrophin releasing hormone like immunoreactivity and indoleamines in the rabbit spinal cord. J. Neurochem. 48:1027-1032; 1987.

I 1. Friedman, T. C.; Wilk, S. Delineation of a particulate thyrotropin releasing hormone degrading enzyme in rat brain by the use of a specific inhibitor of prolylendopeptidase and pyroglutamyl peptide hydrolase. J. Neurochem. 46:1231-1239; 1986.

12. Harkness, D. H.; Brownfield, M. S. Segmental distribution of thyrotropin releasing hormone in rat spinal cord. Brain Res. Bull. 17: 11-19; 1986.

13. Jackson, I. M. D.; Adelman, L. S.; Munsat, T. L.; Forte, S.; Lechan, R. M. Thyrotropin releasing hormone and histidyl proline diketopiperazine in the spinal cord and cerebrospinal fluid. Neurology 36: 1218-1223; 1986.

14. Lechan, R. M.; Snapper, S. B.; Jacobson, S.; Jackson, I. M. D. The distribution of thyrotropin releasing hormone (TRH) in the rhesus monkey spinal cord. Peptides 5:185-194; 1984.

15. Lowry, O. H.; Rosenbrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265-275; 1951.

16. Martino, E.; Seo, H.; Refetoff, S. Ontogenetic patterns ofthyrotropin releasing hormone-like material in rat hypothalamus, pancreas, and retina: Selective effect of light deprivation. Proc. Natl. Acad. Sci. USA 77:4345-4348; 1980.

17. Mgndez, M.; Joseph-Bravo, P.; Cisneros, M.; Vargas, M. A.; Charli, J. L. Regional distribution of in vitro release ofthyrotropin releasing hormone release in rat brain. Peptides 8:291-298; 1987.

260 VARGAS ET AL.

18. Metcalf, G.; Jackson, I. M. D. Thyrotropin releasing hormone: Biomedical significance, vol. 553. New York: Annals of the New York Academy of Sciences; 1989.

19. O'Connor, B.; O'Cuinn, G. Localization of a narrow specificity thyroliberin hydrolysing pyroglutamate aminopeptidase in synaptosomal membranes of guinea-pig brain. Eur. J. Biochem. 144:271-278; 1984.

20. O'Cuinn, G.; O'Connor, B.; Elmore, M. Degradation ofthyrotropin releasing hormone and luteinising hormone releasing hormone by enzymes of brain tissue. J. Neurochem. 54:1-13; 1990.

21. Pekary, A. E.; Meyer, N. V.; Vaillant, C.; Hershman, J. M. Thy- rotropin releasing hormone and a homologous peptide in the male rat reproductive system. Biochem. Biophys. Res. Commun. 95:993- 1000; 1980.

22. Scharfmann, R.; Morgat, J. L.; Aratan-Spire, S. Presence of a particulate thyrotropin releasing hormone degrading pyroglutamate aminopeptidase activity in rat liver. Neuroendocrinology 49:442-448; 1989.

23. Sharif, N. A.; Burt, D. R. Receptors for thyrotropin releasing hormone (TRH) in rabbit spinal cord. Brain Res. 270:259-263; 1983.

24. Taylor, W. L.; Dixon, J. E. Characterization ofa pyroglutamyl aminopeptidase from rat serum that degrades thyrotropin releasing hormone. J. Biol. Chem. 253:6934-6940; 1978.

25. Tortes, H.; Charli, J. L.; Gonz~,lez-Noriega, A.; Vargas, M. A.; Joseph- Bravo, P. Subcellular distribution of the enzymes degrading thyrotropin releasing hormone and metabolites in rat brain. Neurochem. Int. 9:103-110; 1986.

26. Vargas, M. A.; Mrndez, M.; Cisneros, M.; Joseph-Bravo, P.; Charli, J. L. Regional distribution of the membrane bound pyroglutamate aminopeptidase degrading thyrotropin releasing hormone in rat brain. Neurosci. Lett. 79:311-314; 1987.

27. Wilk, S.; Wilk, E. Pyroglutamyl peptidase II, a thyrotropin releasing hormone degrading enzyme: Purification and specificity studies ot the rabbit brain enzyme. Neurochem. Int. 15:81-90; 1989.

regional distribution of pyroglutamyl peptidase ii in rabbit brain, spinal cord, and organs

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