Naanyn-Schmiedeberg's Arch Pharmacol (~ 985) 330:130-135 Na unyn~Schmiedeberg's

Archives of Pharmacology �9 Springer-Verlag 1985

In vivo and in vitro actions of mammalian tachykinins* U. Holzer-Petsche, E. Schimek, R. Amann, and F. Lembeck Department of Experimental and Clinical PharmacoIogy, University of Graz, Universit~itsplatz 4, A-8010 Graz, Austria

Summary. Recently, two kassinin-like tachykinins have been isolated from mammalian nervous tissue. The potencies of these peptides, substance K (neurokinin e) and neuro- medin K (neurokinin fl) were compared with those of sub- stance P, eledoisin, and kassinin in various pharmacological systems in vivo and in vitro.

In contracting the isolated guinea-pig ileum and rabbit jejunum the potencies of eledoisin, kassinin, substance K, and neuromedin K were 1 3 - 8 0 % that of substance P. In the rat vas deferens substance K and neuromedin K potentiated the electrically induced contractions with potencies similar to those of eledoisin and kassinin; they were 4 6 - 2 3 6 times as potent as substance P.

In stimulating salivation in the rat after intravenous in- jection, eledoisin, kassinin, and substance K were ,re- spectively 2.3, 1.3 and 0.33 times as potent as substance P. In contrast, neuromedin K exhibited negligible activity. Each peptide tested led to a short fall in blood pressure after intravenous injection in the rabbit, substance P being 1 2 - 250 times as potent as the other peptides. Substance P was 20 times as potent as substance K or neuromedin K in inducing vasodilatation in the rat hind paw in vivo.

Of the peptides tested, only substance P (10 nmol/min) significantly increased the release of histamine from the rat isolated hindquarter preparation.

The results are discussed with respect to several theories of tachykinin receptor heterogeneity.

Key words: Substance K - Neuromedin K - Neuro- kinin - Substance P - Tachykinin receptors

Introduction

The tachykinins are a family of small peptides which have a similar C-terminus: Phe-X-Gly-Leu-Met.NH2 (Fig. 1). Until recently, substance P was thought to be the only tachykinin present in the mammalian nervous system. Other tachykinins, among them eledoisin and kassinin, were isolated from octopod salivary gland or from amphibian skin (Erspamer 1981). Although they have not been shown to occur in mammals, they exhibit higher potencies than substance P on several biological test systems, e.g. the rat

* The work was supported by the Austrian Scientific Research Funds, grant No. P5616, by the Austrian National Bank, grant No. 2216, and the Pain Research Commission of the Austrian Academy of Sciences

Send offprint requests to U. Holzer-Petsche at the above address

vas deferens, whereas in other tissues they are equiactive with substance P (Lee et al. 1982). These observations and the multiple rates of recovery seen following desensitization in the guinea-pig ileum led Lee et al. (1982) to postulate the existence of two different types of substance P receptors: a "SP-P" receptor where all tachykinins are approximately equiactive, and a "SP-E" receptor where eledoisin and kassinin are considerably more potent than substance P.

Maggio et al. (1983) reported on the presence of a kassininqike peptide in bovine spinal cord. They named it substance K. Independently, in two other laboratories kassinin-like peptides were isolated from mammalian ner- vous tissue, which were named neurokinin e and ]~ (Kimura et al. 1983), neuromedin K (Kangawa et al. 1983) and neuromedin L (Minamino et al. 1984). Neurokinin e and neuromedin L were subsequently shown to be identical to substance K, neurokinin/~ to neuromedin K (Fig. 1). Nawa et al. (1983) identified two DNAs for substance P precursors fi'om bovine brain, one of which also contains the nucleotide sequence corresponding to substance K.

The present study was undertaken in order to compare the potencies of substance K and neuromedin K with those of substance P, eledoisin and kassinin in various organ systems. The results are discussed in the light of the existing theories on different tachykinin receptors.

Methods

Isolated guinea-pig ileum and rabbit jejunum. Guinea-pigs of either sex (300-400 g) were fasted overnight. They were killed by a blow on the neck. Rabbits of either sex (approximately 2.5 kg) were killed by an overdose of pentobarbitone. The ilemn (guinea-pig) or jejunum (rabbit) was rapidly excised and kept in aerated Tyrode solution (Holzer and Petsche, 1983) at room temperature. For the guinea-pig ileum the Tyrode solution also contained 0.6 gM atropine. Segments of tissue, 1 - 2 cm long, were suspended in a silanized glass organ bath containing aerated Tyrode solution at 37 ~ C. Contractions were measured isotonically. Guinea-pig ilea were standardized with a maximally effective dose of histamine (9 gM), and the rabbit jejunum, which does not react to histamine (von Euler and Gaddum 1931), by 1.8 mM acetylcholine. Cumulative dose-response curves were constructed with all tachykinins, increasing doses of peptide being added to the bath every 30 s. With this schedule no tachyphylaxis to substance P is observed (Holzer and Petsche 1983). In order to compensate for any changes in sensitivity to substance P with time, dose-re- sponse curves to the different tachykinins were alternated

Substance P

Eledoisin

Kassinin

Substance K (Neurokinin r Neuromedin L)

Neuromedin K (Neurokinin /3 )

A r g - P r o - L y s - P r o - G i n - G i n - P h e - P h e - G l y - L e u - M e t . N H 2

p G l u - P r o - S e r - L y s - A s p - A l a - P h e - l i e - G l y - L e u - M e t . N H 2

A s p - V a l - P r o - L y s - S e r - A s p - G i n - P h e - V a l - G l y - L e u - M e t . N H 2

His - L y s - T h r - A s p - S e r - P h e - V a l - G l y - L e u - M e t . N H 2

A s p - M e t - His - Asp - Phe - P h e - V a l - G l y - L e u - M e t . N H 2

Fig, 1 Amino acid sequences of the five tachykinins used

131

with dose-response curves to substance P. The dose-re- sponse relationship was expressed as probit contraction against log dose by linear regression and the EDs0 was calculated for each curve. The relative potency of each peptide was calculated as the average EDs0 for substance P divided by the EDso of the peptide tested.

Isolated rat vas deferens. Male Sprague-Dawley rats (300- 350 g) were killed by a blow on the neck. The vasa deferentia were excised and 2 cm of the middle portion were suspended in a silanized glass organ bath containing oxygenated Krebs- bicarbonate solution (Lee et al. 1982) at 37~ The vas deferens was stimulated at 0.1 Hz by double pulses of 1 ms width, 75 ms delay, at maximal voltage. Contractions were measured isotonically. The relative potencies of the taehykinins in potentiating the electrically induced contractions were evaluated by a 2 x 2 assay against substance P (Schild 1942). Each assay was repeated 3 times in different preparations and the results expressed as 2 + SEM.

Rat salivation in vivo. Male Sprague-Dawley rats (300- 350 g) were anaesthetized with urethane (1.2 g/kg, i.p.). The trachea was cannulated. Further cranially the trachea and the oesophagus were ligated. One jugular vein was cannulated for peptide injections. Rectal temperature was kept near 37~ with a table lamp. The saliva secreted was absorbed by ophthalmic swabs placed in the rat's mouth for consecutive 2 rain periods. After two control periods peptides were injected i.v. and the secretion of saliva monitored for three further 2 min periods. Only one dose of peptide was tested in each animal. The testing of the peptides was designed as a 2 x 2 assay (Schild 1942), each dose being repeated in five animals. The results were calculated according to Saunders and Fleming (1971).

Rabbit blood pressure. Rabbits of either sex (2 .5-3.0 kg) were anaesthetized with urethane (1.5 g/kg i.p.). The trachea was cannulated as were the right carotid artery for blood pressure monitoring and the left jugular vein for peptide injections. Rectal temperature was kept near 39~ by an electric pad. Evaluation of peptide potency was performed by a 2 x2 assay against substance P (Schild 1942), which was repeated in three different animals for each peptide.

Vasodilatation in the rat hind paw. Hindpaw vasodilatation was measured in male Sprague-Dawley rats (300-400 g) as described by Lembeck and Holzer (1979). Briefly, the rats were anaesthetized with pentobarbitone (50 mg/kg i.p.) and one superficial epigastric artery was cannulated for continu- ous retrograde infusion of Tyrode solution into the femoral artery (0.03 ml/min). For administration of peptides the in-

fusion was switched to peptide solutions during 3 rain. The various doses of peptides were infused in random order. An interval of at least 30 rain was allowed between two consecutive doses of peptides in order to avoid tachyphylaxis. The outflow from the saphenous vein was measured by a drop recorder. Throughout the experiment systemic blood pressure was kept constant by infusion of heparinized rat blood into one jugular vein.

Histamine release from the isolated hindquarter of the rat. Male Sprague-Dawley rats (300- 350 g) were anaesthetized with urethane (1.2 g/kg i.p.). Perfusion of the isolated hindquarter of the rat was performed as described by Erjavec et al. (1981) except for the Krebs-Ringer solution which contained 2.5 mM CaC12 (Skofitsch et al. 1983). After two control periods of 5 rain each the peptides were perfused i.a. during the 3rd 5min period. After derivation with o-phthaldialdehyde the histamine content of the perfusates was measured by high performance liquid chromatography with fluorescence detection (Skofitsch et al. 1981). Substance P caused a prolonged histamine release, which continued for 5 min after the end of the perfusion period. Thus, the average release of histamine during the 3rd and 4th period was calculated.

Substances. All peptides were purchased from Peninsula. They were dissolved in 0.01 N acetic acid at a concentration of 1 mg/ml except for neuromedin K which, due to its other- wise low solubility, had to be dissolved in 70% (12 N) acetic acid. Further dilutions were made with 0.9% NaC1 containing 1% gelatine or, for the vasodilatation experi- ments with Tyrode, and for the rat hindquarter with Krebs- Ringer solution. Atropine sulphate was obtained from Merck, histamine dihydrochloride from Serva. All o the r chemicals were of analytical grade and purchased from commercial sources.

Results

Guinea-pig ileum and rabbit jejunum

In the guinea-pig ileum and rabbit jejunum the ECso of substance P was 2.7 and 9 nM, respectively. Eledoisin, kassinin and neuromedin K were slightly less active than substance P (Fig. 2, Table 1). In these preparations sub- stance K was the least active of the tachykinins tested, its potency being respectively only 13% or 27% that of sub- stance P. In some preparations of guinea-pig ileum, sub- stance K did not elicit the same maximal contraction as substance P, whereas in the rabbit jejunum all peptides ex- hibited the same efficacy as substance P.

It has already been reported that the contractions of some smooth muscles to kassinin are less rapid than those

132

to substance P (Falconieri Erspamer et al. 1980). Here also, neuromedin K elicited a slightly slower contract ion than did substance P, especially at doses below the EDso.

slight increase in saliva secretion, a l though at such doses the rats showed pronounced hyperaemia o f their paws and ears, tachycardia , and seemed to breathe heavily.

Rat vas deferens

Substance P potent iates the electrically induced contract ions of the isolated ra t vas deferens at micromolar concentra- tions. In this system, eledoisin and kassinin have al ready been described to be far more potent than substance P (Table 1) (Lee et al. 1982; Growcot t et al. 1983; Watson et al. 1983; Nawa et al. 1984). In the present study, substance K and neuromedin K were also more active than substance P, substance K being the more potent of the two.

Rat salivation

At doses of 1 and 3 nmol /kg i.v. substance P, eledoisin and kassinin induced marked salivation in anaesthetized rats in vivo. Substance K was considerably less active (Table 1). Neuromedin K at doses of up to 30 nmol /kg caused only a

Rabbit blood pressure

The rabbi t was extremely sensitive to i.v. injections of substance P. Doses as low as 0.2 pmol /kg caused a marked fall in b lood pressure (see Bernatzky 1981). Maximal ly effective doses of 2 0 - 3 0 pmol /kg led to a fall in b lood pressure of 4 0 - 6 0 mm Hg depending on the initial value. All other peptides tested were far less active than substance P (Table 1), a l though each had the same quali tat ive effect and was able to elicit the same maximal response as substance P. Even after twice the maximal ly effective doses no tachyphylaxis could be observed to any of the peptides.

Vasodilatation in the rat hind paw

Int raar ter ia l ly infused substance P dose-dependent ly in- creased the b lood flow in the ra t hind paw with an EDs0 of 1.7 pmol /min (Fig. 3, Table 1). Both substance K and

75

o

5O o o ~e

28

G U I N E A - P I G I L E U M

1 0 0

0 I I I I

10 9 8 7

p e p t i d e c o n c e n t r a t i o n ( - l o g M)

'~176 I 75

50

o o

R A B B I T J E J U N U M

I I I I tO 9 8 7

p e p t i d e c o n c e n t r a t i o n ( - l o g M)

Fig. 2. Dose-response curves for substance P (�9 eledoisin (O), kassinin ([3), substance K ( � 9 and neuromedin K (A) in the guinea-pig ileum and the rabbit jejunum. The symbols are g _ SEM forn = 6

!,0

I I I I I 0 . 5 1 .5 5 15 5 0 1 5 0

p m o l / r n i n

Fig. 3. Dose-response curves for substance P (O), substance K ( � 9 and neuromedin K (A) with respect to vasodilatation in the hind paw of the rat. The symbols are 2 + SEM for n = 6-- 8

TabLe 1. Relative potencies of eledoisin (EL), kassinin (KASS), substance K (SK), and neuromedin K (NMK) with respect to substance P (SP)

SP (EDso)

Relative potencies (SP = 1)

EL KASS SK NMK

Guinea-pig ileum 2.7 nM 0.80 Rabbit jejunum 9.0 nM 0.42 Rat vas deferens 3.5 IxM a 58 __. 7

Rat salivation 1.5 nmol/kg b 2.3 (1.6-3.3)*

Rabbit blood pressure 0.75 pmol/kg c 0.08 • 0.01

Rat hindpaw vasodilatation 1.7 pmol/min -

0.61 0.13 0.55 0.38 0.27 0.63 230 _+ 27 236 _ 37 46 +_ 8

1.3 0.33 < 0.03 (1.0-1.7)* (0.16-0.57)* 0.08 • 0.009 0.04 • 0.006 0.004 • 0.001

-- 0.05 0.05

+ SEM (n = 3)

2 • SEM (n = 3)

" Lee et al. (1982) b Hanley et al. (1980) ~ Bernatzky (1981) * Limits of error at P = 0.05

133

1000 F

,o o l-

"i 500

'E-- 250 0") t-

O K-R SP KASS SK (6) (4) (4) (4)

Fig. 4. Histamine release from the rat isolated hindquarter by perfusion with Krebs-Ringer (K-R) or 10 nmol/min substance P (SP), kassinin (KASS), or substance K (SK). ff +_ SEM, number of determinations in parentheses. * P < 0.01 compared with K-R by Student's t-test

neuromedin K elicited the same effect, although at much higher doses: the EDso for substance K was 31, for neuromedin K 32 pmol/min. At the doses tested, no effects on systemic blood pressure were observed.

Histamine release

During the two control periods the basal histamine release from the rat isolated hindquarter amounted to 263 _+ 32 ng/ 5 min (n = 18). All peptides were perfused at a dose of 10 nmol/min. Only substance P was able to increase the histamine release significantly above the value obtained in control preparations with Krebs-Ringer perfusion (Fig. 3). Eledoisin has already been reported not to release significant amounts of histamine from the isolated rat hindquarter preparation (Erjavec et al. 1981). Neuromedin K was not tested, because the solution to be perfused would have contained approximately 7% acetic acid; 0.4% acetic acid has already been noted to cause marked vasoconstriction in the rat hindquarter (Holzer-Petsche, unpublished observa- tion).

Discussion

The potency ratios of the tachykinins in the guinea-pig ileum and rabbit jejunum, and the rat vas deferens correspond well with the concept of "SP-P" and "SP-E" receptor subtypes respectively proposed by Lee et al. (1982). In contracting the guinea-pig ileum there are no dramatic differences in potency between the various tachykinins tested (Lee et al. 1982; Growcott et al. 1983; Hunter and Maggio 1984a; Minamino et al. 1984; Nawa et al. 1984), which corresponds to an action via "SP-P" receptors. The same holds for the rabbit jejunum. The rat vas deferens, on the other hand, is a typical example of a "SP-E"-type tissue, where eledoisin and kassinin are far more potent than substance P (Lee et al. 1982; Growcott et al. 1983; Hunter and Maggio 1984a; Nawa et al. 1984). Here, substance K and neuromedin K have similar potencies to kassinin and eledoisin (see also Hunter and Maggio 1984a; Nawa et al. 1984). It is unclear why substance K elicited the same maximal effect as substance P only in two of the six guinea-pig ileum prep- arations. As the same schedule for constructing the dose-response curve was followed in all instances, it can only be speculated that some pieces of guinea-pig ileum are more

prone to tachyphylaxis than others. Recently, Hunter and Maggio (1984b) observed a similar flattening of the dose- response curve to substance P in half of their preparations of guinea-pig urinary bladder.

The action of the tachykinins on the rat salivation has also been attributed to a stimulation of "SP-P" receptors, although the lack of effect of neuromedin K shown here is difficult to reconcile with its activity in other "SP-P" systems.

The hypotensive effects of the tachykinins in the rabbit also do not fit into either of the proposed systems. Here, substance P is active at doses ofpmol/kg, whereas each of the other tachykinins tested exhibits at most 8% of the potency of substance P. Similarly, in inducing vasodilatation in the rat hindpaw, substance K and neuromedin K were only 5% as potent as substance P.

It is the in vivo models where the potency ratios of the tachykinins do not fit neatly into either the "SP-P" or "SP-E" system. In the circulation substance P is rapidly inactivated by enzymes (Lembeck et al. 1978). It cannot be ruled out that such peptidases degrade other tachykinins faster than substance P and that this could contribute to their apparently low potencies. It must also be noted that in both in vivo systems neuromedin K is the least active of the peptides tested. At physiological pH neuromedin K is the most hydrophobic of the peptides tested. Therefore it may well be less soluble in plasma than the other peptides and might be bound to plasma proteins immediately after i.v. injection. This would lead to a decrease in the free, active concentration of neuromedin K and thus decrease its appar- ent potency. This explanation seres to be contradicted by neuromedin K being equally potent as substance K in in- ducing vasodilatation. However, these two peptides seem also to be equally potent (and about 10 times less potent than substance P) in inducing plasma extravasation in the rat hind paw after injection into the femoral artery (Holzer- Petsche, unpublished), whereas neuromedin K is about 10 times more potent than substance P or substance K in in- ducing plasma extravasation after intracutaneous injection in the rat (Gamse in press). Thus, it appears as if the potency of neuromedin K to induce plasma extravasation is reduced by intravascular administration. In view of these arguments the finding that neuromedin K seems to be less potent than substance P in inducing vasodilatation has to be regarded with caution.

Of all the tachykinins tested, only substance P (10 nmol/ min) was able to cause a significant release of histamine from the isolated hindquarter of the rat. Eledoisin (Erjavec et al. 1981 ; Fewtrell et al. 1982) and kassinin (Piotrowski et al. 1984) have also been shown not to release histamine from peritoneal mast cells. The histamine releasing property of substance P has been suggested to be based on a co-opera- tion between the basic N-terminal tetrapeptide and the C-terminal heptapeptide (Mazurek et al. 1981). The two Phe-residues in positions 7 and 8 were said to be the most important part of the latter fragment (Fewtrell et al. 1982). Thus, it is not unexpected that eledoisin (Erjavec et al. 1981), kassinin and substance K (which are neither basic nor have a Phe-residue in position 8) were inactive in releasing hista- mine from the isolated hindquarter of the rat. In the skin, substance P releases histamine from mast cells (H/igermark et al. 1978), although the mechanism of action of substance P on skin mast cells has not been (and may not easily be) studied as thoroughly as on peritoneal mast cells. In any case, the binding site mediating substance P-induced hista-

134

mine release seems to differ f rom either the "SP-P" or "SP- E" receptor subtype.

Besides the efforts o f Lee et al. (1982) various attempts have been made to classify tachykinin receptors. Different receptor types have also been postulated using fragments o f substance P. On such a basis Piercey et al. (1982) proposed a SP1 receptor insensitive to N-terminal fragments of substance P (e.g. in the guinea-pig ileum) and a SP2 receptor mediating rat salivation or mouse hindlimb scratching, where N-terminal fragments retained some activity. F rom the experiments o f Teichberg et al. (1981) a third receptor type might be postulated, occurring in the cow pupillary sphincter and guinea-pig bladder, where the C-terminal octapeptide o f substance P is more potent than the complete molecule.

A different pattern again emerges when looking at the specificity of substance P.antagonists. Recently, several substance P analogues with substitutions of D-amino acids have been described which do not have any partial agonist activity (Mizrahi et al. 1982; Bj6rkroth et al. 1982; Rosell et al. 1983a). Testing o f such antagonists against various tachykinins in different tissues yielded results which point to different types of tachykinin receptors, al though the classification according to tissue and agonist selectivity o f antagonists did not always coincide with the classification according to studies with agonists only (Featherstone et al. 1985; Rosell et al. 1983 a, b). It should be mentioned also that different patterns again are revealed by tachykinin binding assays (see Watson, 1984).

Several explanations have been proposed to reconcile the divergent receptor classifications: (i) Apparent differences in potency might be caused by different kinetic properties of the various agonists. (ii) Differences in lipophilicity between agonists might influence diffusion into a particular tissue and access to the receptor sites. (iii) Tissue-inherent proper- ties may be important in modulating the apparent potency of an agonist: diffusion of an agonist may vary in different tissues. (iv) Watson (1983) showed that tachykinins are in- activated at widely differing rates in various tissues. Tachykinin fragments or antagonists might also influence the enzymatic inactivation of the agonists. The proposit ion has also been raised that there exists only one tachykinin receptor comprising different binding sites for different agonists (Growcott et al. 1983), al though the reason for one or the other agonist exhibiting higher potency remains to be established.

The present investigation shows that a classification of receptor subtypes by binding studies or in vitro experiments cannot be extrapolated easily to in vivo conditions. In order to reach a conclusion about the physiological relevance of a particular tachykinin in a particular tissue it will be necessary to combine the various methods.

Acknowledgements. The authors thank Dr. S. Bailey for critically reading the manuscript, Mr. W. Schluet for preparing the drawings and Mrs. I. H6rzer for typing the manuscript.

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Received January 11, 1985/Accepted May 6, 1985

Note added in proof

Recently, Quirion proposed the existence of three classes of tachykinin receptors, namely a substance P-, substance K-, and neuromedin K-receptor subtype. [Quirion R (1985) Multiple tachykinin receptors. TINS 8 : 183-185]