Neurokinm receptors in the rabbit iris sphincter characterised by novel agonist ligands

European Journal of Pharmacology, 199 (1991) 9-14 © 1991 Elsevier Science Publishers B.V. 0014-2999/91/$03.50

ADONIS 0014299991003983 EJP 51897

Neurokinin receptors in the rabbit iris sphincter characterised by novel agonist ligands Judith M. Hall, D e b r a Mitchell and Ian K. M. M o r t o n Pharmacology Group, Dit,ision of Biomedical Sciences, King's College London, Manresa Road, London SW3 6LX, U.K Received 10 September 1990, revised MS received 12 March 1991, accepted 19 March 1991

We have used novel selective agonist ligands to examine neurokinin receptors mediating the contractile response to tachykinins in the rabbit iris sphincter preparation in vitro. The selective NK-1 receptor agonist 6-amino valeryl-[L-Pro9,N-Me Leu 10]SP-(7-11) (GR73632) and the NK-3 receptor-selective agonist succ-[Asp 6,N-Me-Phe 8]SP-(6-11) (senktide) were bo~ very active (concentration range 0.032 pM-10 nM and 0.1 pM-32 nM respectively), and were 933 and 16.6 times more potejtt than substance P, respectively, in contracting the iris. In contrast, the NK-2 selective agonist [Lys3,GiyS-R-y-lactam,Leug]NKA-(3-10) (GR64349) was active only at the highest concentrations tested (3.2 nM-32 /.tM), and had 0.054 the activity of substance P. The presence of several peptidase inhibitors was without effect on the concentration-response relationship to substance P, GR73632, GR64349 or senktide. Tachykinins differed in their offset kinetics. Responses to GR73632, GF64349 and senktide were rapid in offset (times to reach half maximal responses were 1.5, 1.1 and 5.1 min, respectively), whereas responses to substance P were very much more prolonged in duration (time to reach half maximal response was 35.3 min). These results suggest the presence of both NK-1 and NK-3 receptors mediating contraction of the rabbit iris sphincter preparation. In addition, differences in response offset kinetics seem not to be due to differences in peptide metabolism, and suggest a property of substance P not shared by the other tachykinins used in this study. Neurokinin receptors; Iris (rabbit); Tachykinins; NK-1 receptors; NK-3 receptors; Peptidase inhibitors; g-Amino valeryl-[L-Prog,N-Me-Leul°]SP-(7-11); [Lys3,Giy8-R-y-lactam,Leug]NKA-(3-10); Senktide

1. Introduction

Ocular injury in the rabbit eye results in symptoms of neurogenic inflammation including vasodilation, plasma extravasation, breakdown of the blood-aqueous barrier and miosis. These effects are thought to be a consequence of antidromic reflexes in sensory fibres originating in the trigeminal ganglion (Bruce, 1910; Perkins, 1957; Stjernschantz et al., 1979; Butler and Hammond, 1980), and considerable evidence favours the involvement of neuropeptides including neurokinins in mediating these responses. For example, in vivo electrical or mechanical stimulation of the trigeminal nerve releases neurokinin-like immunoreactivity into the aqueous humour with concomitant signs of inflammation (Bill et al., 1979). Further, in vivo exogenous tachykinins mimic (Bill et al., 1979) and neurokinin receptor antagonists block (Wahlestedt et al.,

Correspondence to: J.M. Hall, Pharmacology Group, Division of Biomedical Sciences, King's College London, Manresa Road, LOndoll SW3 6LX, U.K.

1985) contractile responses to trigeminal nerve stimulation. Extensive work has been carried out using the isolated rabbit iris sphincter pupillae preparation in vitro in order to characterise the post-junctional receptors mediating the neurokinin-mediated contractile response to trigeminal nerve stimulation (Ueda et al., 1986; Too et al., 1988; Hosoki e t a . , 1985; 1987; Magbagbeola et al., 1988b,c). Such studies have, however, produced apparently anomalous results regarding the subtype of neurokinin receptor involved. In particular, naturally occuring tachykinin agonist relative potencies and rank orders of potency are not consistent with any established single neurokinin receptor subtype (Too et al., 1988; Hosoki et al., 1987; Magbagbeola et al., 1988b, and neurokinin receptor antagonists such as [D-Argl,D-Pro2,D-Trp7,9,Leu II ]SP-(1-11) show agonistdependent antagonist potency estimates (Muramatsua et al., 1987; Hosoki et al., 1987; Too et al., 1988; Magbagbeola et al., 1988c) with non-unity Schild plots (Hosoki et al., 1987). These observations have led authors to propose the presence of mixed, or novel, neurokinin receptors in the rabbit iris sphincter (Hosoki

et al,, 1987: Too et al., 1988; Magbagbeola et al., |988b,c), Such conclusions are supported by studies involving determination of agonist onset and offset kinetics, receptor cross-desensi+isation and irreversible receptor alkylation techniques (Hosoki et al., 1987; Too et al., 1988). The recent availability of several novel neurokinin receptor agonists having proposed subtype selectivity and metabolic stability prompted this present study concerned with the further characterisation of neurokinin receptors in the rabbit iris sphincter preparation in vitro. Thus, activities of the NK-l-selective agonist ~-amir~o valeryl-[L-Pro~,N-Me-LeuHqSP-(6-11) (GR73632), the NK-2-selective agonist [Lys3,GlyS-R-), lactam,Leu°]NKA-(3-10) (GR64349) (Hagan et al., 1989) and the NK-3-selective agonist succ-[Asp6,N-Me Phe~]SP-(6-11) (senktide, Wormser et al., 1986) were determined along with substance P. Experiments were also carried out to investigate the effect of peptidase inhibitors on tachykinin actions, as peptide metabolism has previously been reported to distort tachykinin potency estimates and response kinetics in some peripheral preparations (Sekisawa et al., 1987; Stephens-Smith et ai., 1988; Hall et ai., 1990) including the rabbit iris sphincter (Magbagbeola et al., 1988a). Differences in peptide metabolism may, in part, account for the apparently anomalous results regarding neurokinin receptor subtypes obtained by other workers.

hexamethonium (10 p.M), atropine, mepyramine, cimetidine, guanethidine and ibuprofen (all 1 p.M). Z2. Experimental protocols

2. Materials and methods

Cumulative concentration-response curves to substance P, GR73632, GR64349 and senktide were obtained in each individual preparation. Three of these agonists were applied in a random order within preparations, with 30 min intervals between each agonist, but with substance P concentration-response curves determined always at the end of experiments in view of the prolonged washout time of responses to this agonist (see Results). Experiments were carried out in parallel in the absence, or combined presence, of the peptidase inhibitors phosphoramidon, enalaprilat and mergetpa (all 1/.t M) to inhibit neutral endopeptidase (E.C.3.4.24. 11), angiotensin-converting enzyme (E.C.3.4.15.11 ) and certain carboxypeptidases, respectively. In a separate series of experiments, the presence of the aminopeptidase inhibitor bestatin (100 /~M) was investigated on cumulative concentration-response curves to neurokinin A, an agonist known to be degraded by bestatin-sensitive aminopeptidases (Nau et al., 1986). One iris acted as a control preparation (no bestatin) whilst the other iris from the same animal acted as a test preparation with bestatin added 5 min prior to neurokinin A application. These experiments were carried out in a separate series as this peptidase inhibitor has been shown to have effects per se in some other preparations (see Hall et al., 1990).

Z 1. Tissue preparation and recording arrangements

2.3. Expression of results and statistical analysis

Male New Zealand albino rabbits (2.5-2.7 kg) were killed by i.v. pentobarbitone sodium (Sagatal, 25-35 mg/kg) followed by exsanguination. The eyes were enucleated immediately after death and opened by an incision 2-3 mm dorsal to the limbus, followed by excision of the iris from the ciliary margin. The sphincter pupillae muscle was freed of dilator muscle and this preparation was suspended whole in 2 ml silanised glass organ baths in Krebs solution at 37 ° C. The preparations were attached to isometric Grass FT03B force-displacement transducers under an initial resting tension of 150 mg. Mechanical activity was recorded on Grass model 7E polygraphs. An equilibration period of 60 min was allowed before commencing regular dosing, after which time the maximal response to carbachol was established in individual preparations. The composition of the Krebs solution was (mM): Na ÷ 140, K ÷ 5.9, CI- 104.8, H , P O 4 1.2, HCO 3 24.9, Ca 2÷ 2.6, Mg 2÷ 1.15, SO52- -1.15, glucose 10. The Krebs solution was maintained at pH 7.4 by constant bubbling with 95% 02-5% CO 2. In all experiments, the Krebs solution contained

Responses are expressed as % maximal response to carbachol in individual preparations. Data from each experiment were combined and calculated as mean response _+ S.E.M. for each concentration, pD 2 ( - logl0 ECs0) estimates were determined from geometrically meaned EC50 estimates obtained in individual preparations, and relative potencies were calculated from the differences of these pD 2 estimates. For estimation of kinetics of offset of tachykinin responses, times to reach 50% of maximal response after washout, following cumulative dosing to maximal response, were averaged across preparations. Tests for significant differences were made using Student's t-test for independent or paired samples, and one-way analysis of variance, as appropriate.

2. 4. Source of agents used Agents were obtained as follows: carbamylcholine chloride, atropine sulphate, hexamethonium bromide, ibuprofen, phosphoramidon (Sigma, U.K.), mepyramine maleate, Sagatal (May and Baker, U.K.), cimeti-

dine (Smith, Kline and French, U.K.), guanethidine sulphate (CIBA, U.K.), enalaprilat (Merck, Sharp and Dohme, New Jersey, U.S.A.), DL-2-mercaptomethyl3-guanidinoethylthiopropanoic acid (mergetpa, Calibiochem, U.S.A.), substance P, bestatin, s u c c - [ A s p 6 , N Me-Phes]sP-(6-11) (senktide) (Peninsula Laboratories Europe), g-amino valeryl-[L-Prog,N-Me-Leut°]SP-(611) (GR73632) and [Lys3,GlyS-R-y-lactam,Leug]NKA(3-10) (GR64349) were kind gifts from Glaxo Group Research, Ware, U.K. All salts used were of analytical grade and were obtained from B.D.H., U.K. All peptides were dissolved in distilled water and deep frozen in small aliquots under N 2. Ibuprofen was dissolved in 5% Na2CO 3. All other agents were dissolved in distilled water.

3. R e s u l t s

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Log [Peptide] (M) Fig. 1. Log concentration-response curves to tachykinins in the rabbit iris sphincter pupillae preparation. The symbols denote ( o ) GR73632, (o) substance P, (zx) senktide and ( - ) GR64349. Responses shown were obtained in the absence of peptidase inhibitors and are the means+S.E.M, for 8-10 preparations, expressed as % maximal response to carbachol (CCh) in individual preparations. Experiments were carried out in the presence of hexamethonium (10 #M), atropine, mepyramine, cimetidine, guanethidine and ibuprofen (all 1 /~M).

3.1. Kinetics of responses Qualitatively, contractile responses to all agonists were similar, being rapid in onset (5-20 s) and reaching a peak response within 30 s: however, quantitative differences in response offset kinetics were apparent between the peptides. Maximal responses to GR64349 and GR73632 washed out relatively quickly (time to reach half-maximal responses were 1.1 + 0.28 and 1.5 +_0.32 min respectively, n = 8), those to senktide were somewhat more prolonged (time to reach half-maximal responses was 5.1 +_0.88 min, n = 8). In contrast, responses to substance P were still apparent for circa 20 min despite continual washing of the preparations (time to reach ha!f-maximal response was 35.3 ___5.4 min, n = 4). 3.2. Agonist relatiee potencies Log concentration-response curves for the four neurokinin receptor agonists are shown in fig. 1. The maximal response to these agonists was of simila~ magnitude to the raaximal response obtained with carbachol, and did not differ significantly between agonists (P > 0.05). The averaged log concentration-response curves were essentially parallel, though individual log concentration-response curves to GR64349 were generally biphasic in form. Agonist activities shown in table 1 are expressed as pD 2 values with relative potency estimates. The NK-1selective agonist GR73632, the NK-3-selective agonist senktide and the NK-2-selective agonist GR64349 had potencies of 933, 16.6 and 0.054 relative to substance P, respectively, in the absence of peptidase inhibitors. The presence of the peptidase inhibitors pbz,sphoramidon, enalaprilat and mergetpa (all 1 ~M) had no

significant effect on agonist p D 2 estimates (P > 0.05). The effect of peptidase inhibitors on full concentration-response curves to the four agonists are shown in fig. 2. Bestatin (100/.tM) was without effect on the log concentration-response curve to neurokinin A (data not shown).

4. D i s c u s s i o n

The data presented here with selective neurokinin receptor agonists strongly suggests the presence of NK-1, and most probably NK-3, receptors in the rabbit iris sphincter. Thus, the NK-l-selective agonist GR73632 (Hagan et al., 1989; in press) was very potent in contracting the iris (ECs0 = 0.32 pM) and had 933 times the potency of substance P. The absolute and relative potency of the NK-1 agonist in this preparation is even higher than that reported in other NK-1 preparations (Hagan et al., 1989; in press; Hall and Morton,

TABLE 1 Activities of neurokinin receptor agonists in the rabbit iris sphincter in the absence of peptidase inhibitors, pD 2 ( - l o g ECs0) estimates are averages for 8-10 individual preparations. Relative potencies were calculated in terms of differences in pD 2 estimates (substance P = 1.00) Experiments were carried out in the presence of hexamethonium (10 /.tM), atropine, mepyramine, cimetidine, guanethidine and ibuprofen (all 1 #M) Agonist

pD 2 (S.E.M.)

Relative potency

Substance P GR73632 Senktide GR64349

7.54 (0.28) 10.50 (0.24) 8.76 (0.27) 6.27 (0.18)

1.00 933 16.6 0.054

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Fig. -. Effect of peptidase inibitors on log concentration-response curves to tachykinins i,~ the rabbit iris sphincter. Responses to substance P (A). senktide (B), GR73632 (C) and GR64349 (D) were obtained in the absence (o) or the presence (©) of the peptidase inhibis.ors phosphoramidon, enalaprilat and mergetpa (all 1 gM). Values shown are the mean _+S.E.M. for 8-10 individual preparations. The presence of peptidase inhibitors was without significant effect on responses to tachykinins (P > 0.05).

1991). These results with GR73632 indicating the presence of NK-1 receptors in the rabbit iris support the findings from previous studies using the earlier NK-Iselective agonists substance P methyl ester (Cascieri et al., 1981; Watson et al., 1983) and [GIp6,L-Prog]SP-(6 11) (septide) (Piercey et al., 1985; Laufer et al., 1986) which both have high activity in this preparation (data not shown; Magbagbeola et al., 1988b). In this present study, the NK-3-selective agonist senktide (Wormser et al., 1986) was found to be 16.6 times more active than substance P (ECs0 = 1.7 nM). Senktide is more potent relative to substance P in other NK-3 receptor systems (Wormser et al., 1986; Hanani et al., 1988; Guard et al., 1990), but is devoid of activity in homogeneous NK-1 or NK-2 preparations (Laufer et al., 1986; Wormser et al., 1986; Hall and Morton, 1991). These present data with senktide therefore suggest the presence of NK-3 receptors in the rabbit iris sphincter. The agonist GR64349 which is very potent at NK-2 receptors (Hagan et al., 1989; in press) was active only at the highest concentrations tested (32 nM-0.32 /.tM) having only 0.054 the activity relative to substance P, thus discounting a major contribution from NK-2 receptors in the contractile response to tachykinins. The contractile response of the rabbit iris sphincter to this agonist at higher concentra-

tions may well reflect an interaction with NK-1 or NK-3 receptors (Hagan et al., 1989; Hall and Morton, 1991). Although these Jesuits clearly point to the presence of both NK-1 and NK-3 receptors in the rabbit iris sphincter preparation, recent work has highlighted the possible distorting influence of peptide degradation on agonist activities (Sekisawa et al., 1987; Stephens-Smith et al., 1988; Hall et al., 1990). Consequently, in the present study experiments were carried out to determine the effect of inhibitors of peptidases implicated in tachykinin metabolism in other preparations (see e.g. Littlewood et al., 1988), on agonist activity estimates. However, inhibitors of neutral endopeptidase, angiotensin-converting enzyme and certain carboxypeptidases were without significant effect on agonist potencies or slopes of the log concentration-response curves to tachykinins. Similarly, the aminopeptidase inhibitor bestatin was without significant effect on the activity of neurokinin A, a tachykinin known to be degraded by peptidases sensitive to this inhibitor in other systems (Nau et al., 1986). In this present study, in the absence of peptidase inhibitors, responses to the three synthetic analogues (GR73632, GR64349 and senktide) were relatively fast in offset whereas that to substance P was considerably more prolonged (see Results). Previous studies have reported similar results in the rabbit iris sphincter where responses to substance P, and also to physalaemin, were considerably more prolonged than those to other tachykinins including eledoisin, kassinin, neurokinin A and neurokinin B (Hosoki et al., 1985; 1987; Too et al., 1988; Magbagbeola et al., 1988a). Differential degradation by peptidases is unlikely to be a major contributing factor to the tachykinins response offset kinetics, since responses to the three synthetic analogues used in the present study, which have been proposed as being metabolically stable (Wormser et al., 1986; Hagan et al., 1989; in press), washed out faster than responses to substance P which is known to be susceptible to degradation by peptidases (Skidgel et al., 1984; Nau et al., 1986; Littlewood et al., 1988). It is suggested, therefore, that substance P (and physalaemin) have some property riot shared by other tachykinins which leads to the prolonged differer, ces in binding kinetics to receptor types ah'eady discussed, or due to an interaction with a site having unique properties. In conclusion, assuming a high degree of selectivity in the analogues used in this present study, these agonist data clearly point to the presence of both NK-1 and NK-3 receptor populations in the rabbit iris sphincter preparation. Further, the potencies of these novel neurokinin receptor agonists are not significantly influenced by peptidase activity. These results regarding mixed receptor populations should help explain

13

apparently anomalous results reported previously by other workers using agonists of lesser selectivity, and emphasise the necessity for the use of selective agents in such receptor classification studies. Confirmation of these agonist data, however, clearly requires the determination of the affinities of subtype-selective competitive neurokinin receptor antagonists which are now becoming available (Williams et al., 1988; Hagan et al., 1990; Rovero et al., 1990; Ward et al., 1990). Further analysis is especially important in view of the unusual, and unexplained characteristics; including offset kinetics from the receptor or in the mechanism mediating responses in this preparation to substance P and physalaemin uniquely. Studies relating to these points are currently in progress.

Acknowledgement We wish to thank Glaxo Group Research for the kind gifts of GR73632 and GR64349. We thank the Wellcome Trust for support to J.M.H.

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Williams, B.J., A.T. Curtis, A. Foster and R. Tridgett, 1988. Devel. opment of NK-2 selective antagonists, Peptides, 21, 189. Wormser, U., R. Laufer, Y. Hart, M. Chorev, C. Gilon and Z.C Selinger, 1986, Highly selective agonists for substance P receptoi subtypes, The EMBO J. 5, 2805.