Classification of tachykinin receptors in muscularis mucosae of opossum oesophagus

Transcription

1 Br. J. Pharmacol. (1989), 97, Classification of tachykinin receptors in muscularis mucosae of opossum oesophagus 'E.E. Daniel, S. Cipris, Y. Manaka, P. Bowker & *D. Regoli Smooth Muscle Program, Department of Neurosciences, Health Sciences Centre, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada, L8N 3Z5 and *Department of Pharmacology, Universite Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N 1 Muscularis mucosae of the distal oesophagus of the opossum contracts in response to substance P and to a variety of tachykinins. To delineate the nature of the receptors present in this tissue, we evaluated contractile responses to substance P, neurokinin A, neurokinin B, eledoisin and analogues believed to be highly selective for NK-1, NK-2 and NK-3 receptors. In addition, the effects of prolonged exposure to each of these agents (10-M or 10-5M) on contractile responses to substance P and to itself were evaluated. Similarly effects of prolonged exposure to the various tachykinins and their analogues on the field-stimulated responses of this muscle were studied. 2 All naturally occurring tachykinins were full agonists and differed in potency (comparing ED50 values) by less than ten fold. In nearly all cases there was cross tachyphylaxis between substance P and the other tachykinins and each reduced tonic responses to field stimulation, a response previously shown to be mediated by a substance P like agent. Eledoisin failed to cause tachyphylaxis under the conditions of these experiments. 3 When highly selective tachykinin analogues were used, only that believed to activate NK-1 receptors was a full agonist. [Ii-Ala,Sar9,MetO2I 1](- 1) was also only slightly less potent than substance P. In contrast, an agonist selective for NK-2 (NK-A) receptors, [Nlel'](-10), and [f-asp,mephe7](l10) were unable to produce a one selective for NK-3 (NK-B) receptors, response equal to 50% of the maximum even at 10- M. However, all three selective tachykinin analogues reduced responses to substance P but not to carbachol. They usually reduced both phasic and tonic responses to field stimulation. We conclude, based on this and earlier study, that the tachykinin receptors of opossum oesophagus muscularis mucosae recognize all naturally occurring tachykinins but may represent only NK-1 receptors. The ability of analogues selective for other types of tachykinin receptors to reduce responses to substance P raises the possibility that their selectivity depends in part on diminished efficacy rather than totally on diminished affinity at some classes of receptor. Introduction The muscularis mucosae of opossum oesophagus has numerous nerves containing substance P- immunoreactive material (Domoto et al., 1983; Daniel et al., 1987) and responds to field stimulation of its nerves by a tonic (following phasic) contraction which appears to be mediated by substance P () or a related tachykinin (Domoto et al., 1983; Robotham et al., 1985). The tachykinin or tachykinins present in this tissue or released on nerve stimulation have not been identified but resemble substance P (Daniel et al., 1987). An initial attempt to identify the tachykinin receptor present gave results interpreted as showing a non-selective receptor (Daniel et al., ' Author for correspondence. 198). This conclusion was reached because eledoisin, physalaemin and kassinin, all full agonists, were similar in potency to. Eledoisin and physalaemin were originally used to differentiate -E and -P receptors (Lee et al., 1982). This classification of receptors is no longer accepted (Henry, 1987). Recently, three classes of tachykinin receptors have been recognized, corresponding to three naturally occurring members of the tachykinin family: a receptor for substance P (), for neurokinin A (), and for neurokinin B () (Henry, 1987). It is recognized that these naturally occurring tachykinins are not highly selective for their own receptors (Iversen et al., 1987). Furthermore eledoisin, physalaemin and kassinin all The Macmillan Press Ltd 1989

2 101 E.E. DANIEL et al. interact with all three receptors with varying but disputed potencies (for example, see Iversen et al., 1987). Thus identification of receptor type with the natural agonists may not be straight-forward. However, recently synthetic tachykinins have been synthesized which are more selective for each of these receptor classes (Drapeau et al., 1987a,b) based upon studies in tissues thought to contain only or predominantly one class of receptor. The objective of this study was to utilize the naturally occurring tachykinins (,, and eledoisin) as well as the synthetic, more selective tachykinins [#-Ala, Sar9, MetO1](_11) for, [Nle'0](10) for, and [f-asp,mephe7]- (jo) for receptors, to characterize tachykinin receptor types present in opossum oesophagus muscularis mucosae. Methods Opossums were killed by an overdose (5mgkg-') of pentobarbitone, the oesophagus was rapidly removed and the mucosa including the muscularis mucosae, separated. The mucosal folds were trimmed off and the thickened distal region of the mucosa used to prepare eight 2-3-mm wide longitudinal strips. These consisted mostly of muscularis (see Domoto et al., 1983). These strips were mounted under 1 g tension in 3 ml muscle baths containing Krebs Ringer solution (composition in mm: NaCl 115.5, KCI.1, CaC12 2.5, MgSO 1.1, NaHCO3 21.9, NaH2PO3 1.1, glucose 11.1). The bath solution was bubbled with 95% 02: 5% Co2 and maintained at a temperature of 37 C. The tissues were mounted on glass rods to which concentric Pt electrodes were attached. Tissues were tied by a silk thread to a hook at one end of the glass rod and pulled through the concentric electrodes and attached at the other end by a silk thread to a Grass FT.03 strain gauge transducer. Tension records were made on an eight channel Beckman R 11 oscillograph. After 1 h for recovery from dissection, the viability of tissues was assessed by field stimulation at 5 and at 0Hz (0.5ms square pulses at OVcm-'). Only tissues which showed typical phasic and tonic contractions were used (Domoto et al., 1983). Then 10-M carbachol was administered to determine the maximum response. This was washed out and cumulative tachykinin concentration-response curves obtained (concentration range 10-0 to 10- or 10-5M). Usually 2 or strips were exposed to each peptide. The protocol for obtaining concentration-response curves usually involved 10 fold increases in concentration with each step, adding the next highest concentration as soon as the response to the preceding one had reached a plateau. This protocol avoided tachyphylaxis (demonstrated by repeating the exposure to concentrations of peptide producing about half maximal and maximal responses 0min after washout of peptide), reduced the time for completion of experiments (since both contraction and relaxation were slow and since the range of effective concentrations is wide) and provided reproducible results. In some experiments, attempts were made to produce selective tachyphylaxis to various peptides. In one protocol using naturally occurring tachykinins to generate the concentration-response curves, the maximum concentration of each peptide was left in the bath until the response had faded. Then the bath was rapidly washed, responses to field stimulation were tested and another concentrationresponse curve was generated. The same peptide or another one was used in some of the muscle strips in each experiment. This procedure did not affect responses to the subsequent addition of carbachol. Another protocol involved desensitization to 10 -M by the selective agonists or itself. Tissues were first exposed to the selective agonist (10-M), (10-M) or the equivalent solution without tachykinin. After the maximum response was achieved, the bath was rinsed rapidly with Ringer solution and added. After the maximum response was achieved the bath was again rapidly rinsed and carbachol 10-M added. Contractile responses were expressed as % of the maximum response to carbachol. Concentrationresponse curves were plotted as logarithm of concentration versus response. The ED5o values for each strip were determined for each peptide. Means of ED50 values and their standard errors were calculated after transformation of the value to a logarithmic scale. In addition the maximum response (usually occurring at 10-M) was determined for each peptide as well. Mean values for various tachykinins were compared using unpaired comparisons and two tailed t tests of their significance; P values less than 0.05 were considered significant. Drugs used were: substance P, neurokinin A, neurokinin B, eledoisin and carbachol from Sigma and the synthetic analogues from Dr D. Regoli. Results In the first series of experiments, potencies and maximum responses were compared when different peptides were used. Table 1 summarizes the findings. Substance P was slightly but consistently more potent than and, and roughly equipotent with eledoisin. All of these peptides were full agon-

3 TACHYKININ RECEPTORS IN OPOSSUM MUSCULARIS MUCOSAE 1015 Tabe 1 ED5o values and % maximum responses for tachykinins in opossum muscularis mucosae vs Exp. 1 Exp. 2 Exp. 3 Exp. Exp. 5 Exp. Overall mean vs Eledoisin Exp. 1 El Exp. 2 Overall mean El vs Exp. 1 Exp. 2 Overall mean n ED5o (M) x x 10-8* x x 10-8* x x 10-8* x x 10-8* x x x x x x x x x x x x x x 10-7* 3.5 ± 1. x x 10-8* 1.0 x lo-.0 x 10-8 Max ± ± ± 2.5t ± 2.t ± ± ± ± ± * Significantly different (P < 0.05) from corresponding value for. t Significantly different from 100%., substance P;, neurokinin A;, neurokinin B. ists. There was thus no clear basis for distinguishing the class or classes of receptors present from use of the natural agonists. From these data one might conclude that there was a mixture of receptors selective for and substance P or a non-selective receptor. Therefore, more selective agonists were tested. Table 2 compares these results. The results of this study were clear cut. Only the agonist selective for NK-1 receptors (ASM-_10) was a full agonist. Its a 12 g 1 min CCh 10- M M b 9 12 g 1 mi 10-9M CCh 10*M * -8* NP 10-9 M g M CCh 10- M ASM or M NL M AMP M Figre 1 Typical responses to several agonists used in this study. In each case, an initial response to 10-M carbachol (CCh) is shown to define the maximum contraction of the strip and later a cumulative concentration-effect curve is shown. Tension is in g and time in minutes. (a) Compares cumulative responses to substance P () and neurokinin A (), 10' to 10-M. (b) Compares cumulative responses to and, 10-9 to 10-5M. (c) Compares cumulative responses to [fl-ala', Sai9, Met(02)1 1]-(-- 1) (ASM 10), (NL) and (AMP) - [l-as", MePhe7]-(10) 10-9 to 10-5 M. potency was slightly less than that of substance P. However, agonists selective for NK-2 or NK-3 receptors seemed to be partial agonists; i.e., at 10-5M they produced less than 50% of the maximum response. Figure 1 illustrates these findings. In several experiments, after completion of the concentration-response curve the maximum concentration (10- M) of each peptide was left in the bath for 2 to 2.5 h while the contractions gradually faded.

4 101 E.E. DANIEL et al. Table 2 A Actions of selective tachykinin peptides on opossum muscularis mucosae Peptide [Pi-Alat,-Sar',MetOVl]_j [Nle10]_10 [P-Asp',MePhe7]lo B n ED50 (M) 8.3±3.3x x 10-8 > 10- > 1-0 Effect of exposure to 10- M tachykinin on response to 10- M substance P Agent n % CCh maximum None Ffl-Ala,-Sar',MetO"l] 11 [Nlel ]1o [f_-asp,mephe7]0o Values are mean ± s.e.mean. t Significantly different (P < 0.05) from 100%. tt Significantly different (P < 0.05) from no treatment. CCh, carbachol ±Ott ± 0.tt ± 11.tt ± 13.5tt Max response 92.5 ± ±.t 35.5 ± 5.0t Then responses to field stimulation at 5 and 0Hz were tested before and just after washing of the bath. Afterwards, the concentration-effect curves for various peptides were repeated. In all cases, some strips were exposed to the same peptide as was used for producing tachyphylaxis while other strips were exposed to other peptides to determine if cross-tachyphylaxis had occurred. Table 3 summarizes these results. It shows that there was cross tachyphylaxis between and. produced tachyphylaxis to both and eledoisin, but the latter was relatively ineffective against both and itself. produced tachyphylaxis against both itself and and also caused tachyphylaxis to. Thus there was no evidence that the naturally occurring tachykinins caused selective desensitization to NK-2 or NK-3 receptors compared to NK-1 receptors. Desensitization by more selective peptides was tested and compared to that by. Exposure to 10-M of each caused reduction of the responses to 10-m substance P. The data are summarized in Table 2B. Addition of itself abolished the responses to a subsequent exposure. None of these agonists affected the response to 10-m carbachol added immediately afterwards. When effects of prolonged exposure to each peptide on tonic responses to 0 Hz field stimulation were compared (Table ), it was evident that all of Table 3 Effects of prolonged exposure to tachykinins (10 M) on responses Test peptide Exposed to n ED50 (M) Eledoisin Eledoisin Eledoisin.5 ± 2.2 x10-t 1.2 ± 2.5 x10-'t 1.3 ± 2.3 x10-t 1.0±9.2x10-t 2 8. x x 10-7tt x x ± 1.2 x10-7t 1. ± 0.17 x 10-7t 9.±2.0x10-t.7 ± 1.5 x10-7t Max(%) 3.8 ± 10.9t t t 50.8 ± 8.5t 5tt 1tt ± ± ± 10.5t 7.8 ± 2.t t Significantly different from control values in Table 1. tt Outside the range of values observed in experiments summarized in Table 1.

5 TACHYKININ RECEPTORS IN OPOSSUM MUSCULARIS MUCOSAE 1017 Table Effect of prolonged exposure to tachykinins on responses to field stimulation (0 Hz) Experiment 2 3 Exposed to 10-M 10-M 10-5M 10-5M 10IM Eledoisin 10- M Decrease in response (%) ± them were capable of reducing the response. In the case of the highly selective peptides, none was capable of selectively inhibiting the tonic responses (compared to the phasic response) to field stimulation. In most cases, non-selective peptides inhibited both phasic and tonic contractions even though they did not reduce the response to 10-M carbachol. The mechanism of this effect has not been investigated further but may reflect a presynaptic action of these substances. Discussion n These results confirm and extend our previous finding (Daniel et al., 198) that tachykinin receptors in muscularis mucosae of opossum oesophagus appear to be non-selective in the sense that they do not discriminate between the putative naturally occurring agonists for three postulated classes of receptors; NK-1, NK-2 and NK-3. Thus in this tissue, substance P,, eledoisin and physalaemin are all full agonists and differ in potency by less than 1 log unit. However, when agonists designed to be more selective than the natural agonists were used (Drapeau et al., 1987a,b), it was found that only the one highly selective for NK-1 receptors was a full agonist. Furthermore, the naturally occurring agonists produced tachyphylaxis of responses to substance P and to tonic responses to high frequency (0 Hz) field stimulation, but the agonists highly selective for various NK receptors reduced both phasic and tonic responses to field stimulation. There are two possible explanations for these findings: (1) these receptors are one class of receptors which recognize several naturally occurring tachykinins with roughly similar potencies; or (2) these receptors are a mixture of all three classes of receptor, present in sufficient densities to permit maximal contractions when each class was occupied by naturally occurring tachykinins. If the second explanation was correct and some of these agonists were truly selective, then selective desensitization to receptors for each class should be possible; i.e., there should be no or minimal cross desensitization between agonists acting on different classes of receptors. In fact there was cross desensitization between most natural tachykinin peptides (Table 3) and even selective peptides for NK-2 and NK-3 receptors caused some desensitization to substance P, but did not depress responses to carbachol. Therefore it is possible that these substances achieve selectivity by low efficacy at some receptors but still recognize all receptors. Thus the present findings support that neurokinin receptors in this tissue may represent a predominant NK-1 population which retains the ability to recognize several synthetic and naturally occurring tachykinins. However, these findings do not rule out the existence of some NK-2 and NK-3 receptors in this tissue which are insufficient in number or efficacy to cause a maximum response when selectively activated but which do contribute to the maximum response. Ligand binding studies and ultimately receptor isolation and biochemical characterization are required to confirm whether only one receptor is present and if this receptor is identical to that found in other tissues. However, comparison of these results to those reported by Regoli et al. (1987) suggest that in fact this tissue contains a single type of receptor which resembles other NK-1 receptors. Regoli et al. reported (1987) that this receptor type in canine carotid artery was characterized by potency ratios such that substance P was more potent than and even more potent compared to (i.e., > > ). The ED50 values were four times higher and twelve times higher than that for substance P; very similar to those found in this study (four and eight times higher). However, in carotid artery, eledoisin was about 1/3 as potent as substance P and physalaemin equipotent while kassinin was 1/ as potent as substance P. Our previous data (Daniel et al., 198) suggested that physalaemin was equipotent to substance P in muscularis mucosae but that eledoisin and kassinin were slightly less potent than substance P. In the present study which followed a different protocol, eledoisin, was equipotent with substance P. Previous studies of the C terminal fragments of substance P on putative NK-1 receptors in the carotid artery (Regoli et al., 1987) and in our previous study of muscularis mucosae (Daniel et al., 198) also revealed some quantitative differences, but all were full agonists in both studies. These discrepancies may reflect minor differences in the receptor or in the experimental conditions, including susceptibility to degradation. The responses of the tissue to all agonists studied were slow in onset and in offset compared to those to muscarinic agonists (Daniel et al., 198). There appeared to be no basis for distinguishing their

6 1018 E.E. DANIEL et al. effects based on duration of action in contrast to observations in the rabbit iris sphincter (Too et al., 1988). Similarities in the onset and offset of actions could depend on the kinetics of interaction of each agonist with the receptor. If this is the case and there is only one receptor subtype with which most agonists interact with similar affinity then all might be expected to have similar durations of action. However, potencies of the selective agonists varied considerably. If their potencies were determined by their affinities for the receptor, then their common time course of action is surprising. The duration of agonist actions can, of course, be determined by variation in rates of agonist degradation, receptor down-regulation or the time course of the system coupling receptor occupation to response. We propose that a common coupling mechanism determines the time course of these agonist since degradation rates of the agonists are unlikely to be similar and the extent of desensitization also varied. Overall, the findings of this and the earlier study (Daniel et al., 198) are consistent with the conclusion that opossum muscularis mucosae contains only one major class of tachykinin receptor, NK-1. Its interaction with tachykinin antagonists has not been studied quantitatively. The basis for persistence in evolution of an NK-1 receptor which still recognizes all naturally occurring tachykinins is unclear. This preparation may be useful for binding studies to NK-1 receptors and such studies will help clarify the small differences between this preparation and dog carotid artery. Supported by the Medical Research Council of Canada. References DANIEL, E.E., JURY, J. & ROBOTHAM, K.H. (198). Receptors for neurotransmitters in opossum muscularis mucosae. Br. J. Pharmacol., 88, DANIEL, E.E., NAGAHAMA, M., SATO, H., JURY, J. & BOWKER, P. (1987). Immunochemical studies on Substance P release from muscularis mucosae (MM) of opossum oesophagus. In Substance P and Neurokinins, Proceedings of Symposium, Montreal, Quebec, ed. Henry, J.L., Couture, R., Cuello, A.C., Pelletire, G., Quirion, R. & Regoli, D. pp New York: Springer Verlag. DOMOTO, T., JURY, J., BEREZIN, I., FOX, J.E.T. & DANIEL, E.E. (1983). Does substance P co-mediate with acetylcholine in nerves of opossum esophageal muscularis mucosae? Am. J. Physiol., 8, G19-G28. DRAPEAU, G., D'ORLEANS-JUSTE, P., DION, S., RHOLEB, N.E., ROUISSI, N.E. & REGOLI, D. (1987a). Selective agonists for substance P and neurokinins. Neuropeptides, 10, 3-5. DRAPEAU, G., D'ORLEANS-JUSTE, P., ROHLEB, N.E., DION, S. & REGOLI, D. (1987b). Specific agonists for neurokinin B receptors. Eur. J. Pharmacol., 13, HENRY, J.L. (1987). Discussion of nomenclature for tachykinins and tachykinin receptors. In Substance P and Neurokinins. Proceedings of 198 Symposium, Montreal. ed. Henry, J.L., Couture, R., Cuello, A.C., Pelletier, G., Quirion, R. & Regoli, D. pp New York: Springer Verlag. IVERSEN, L.L., FOSTER, A.C., WATLING, K.J., McKNIGHT, A.J., WILLIAMS, B.J. & LEE, C.M. (1987). Multiple receptors and binding sites for tachykinins. In Substance P and Neurokinins. Proceedings of 198 Symposium, Montreal. ed. Henry, J.L., Couture, R., Cuello, A.C., Pelletier, G., Quirion, R. & Regoli, D. pp. 0. New York: Springer Verlag. LEE, C.M., IVERSEN, L.L., HANLEY, M.R. & SANDBERG, B.E.B. (1982). The possible existence of multiple receptors for substance P. Naunyn-Schmiedebergs Arch. Pharmacol., 318, REGOLI, D., DRAPEAU, G., DION, S. & D'ORLEANS-JUSTE, P. (1987). Receptors for neurokinins in peripheral organs. In Substance P and Neurokinins, Proceedings of 198 Symposium, ed. Henry, J.L., Couture, R., Cuello, A.C., Pelletier, G., Quirion, R. & Regoli, D. pp New York: Springer Verlag. ROBOTHAM, H., DANIEL, E.E. & JURY, J. (1985). Capsaicin effects muscularis mucosae of opossum oesophagus; substance P release from afferent nerves? Am. J. Physiol., 28, G55-G2. TOO, H.P., UNGER, W.G. & HANLEY, M.R. (1988). Evidence for multiple tachykinin receptor subtypes in the rabbit iris sphincter muscle. Mol. Pharmacol., 33, -71. (Received August 1, 1988 Revised March 1,1989 Accepted April 7,1989)