European Journal of Pharmacology, 193 (1991) 231-238 6) 1991 Elsevier Science Publishers B.V. 0014-2999/91/$03.50 ADONIS 0014299991001858

231

EJP 51701

Bradykinin B 2 receptor evoked K + permeability increase mediates relaxation in the rat duodenum J u d i t h M. H a l l a n d I a n K . M . M o r t o n Pharmacology Group, Division of Biomedical Sciences, King's College London, Manresa Road, London S W3 6LX, U.K. Received 23 August 1990, revised MS received 2 November 1990, accepted 13 November 1990

We have investigated the receptors and associated coupling mechanisms that mediate the smooth muscle relaxant response to bradykinin (BK) in the rat duodenum in vitro. Relaxation in response to BK seems due to a direct action on the longitudinal smooth muscle since effects were demonstrable in the presence of ibuprofen, mepyramine, atropine, guanethidine (all 1 #M), hexamethonium (10/~M) and TTX (0.3/IM). Receptors involved are of the B2 subtype since agonists and antagonists active at B1 receptors were essentially inactive, and the B2 receptor antagonist Lys,Lys-[Hyp3,ThiS'8,D-PheT]BK was a potent competitive antagonist of BK-induced relaxation (pK B of 7.2_+ 0.1). The activity of both BK and the antagonist were unchanged by the presence of peptidase inhibitors including the carboxypeptidase inhibitor DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (mergetpa, 10 /IM), which prevents conversion of BK analogues to des-Arg9-Bl-active products. In high-K + solution, BK (0.1-10/~M) produced concentration-related increases in 86Rb efflux. Both this permeability increase in high-K + solution, and the relaxant responses in Krebs solution, were inhibited by low concentrations (10-100 nM) of apamin, as well as the B2 receptor antagonist Lys,Lys-[Hyp3,ThiS'8,D-PheV]BK (1 t~M). These results are compatable with the proposal that BK-evoked relaxation of the rat duodenum is mediated via a subset of B2 receptors for which the antagonist Lys,Lys-[Hyp3,ThiS'8,D-PheV]BK has a high affinity, and results from stabilisation of the smooth muscle membrane through the opening of apamin-sensitive 86Rb-permeable calcium-activated K + channels. Bradykinin; Bradykinin B2 receptors; Duodenum (rat); K + channels; Peptidase inhibitors; Lys,Lys-[Hyp3,ThiS'8,D-Phe7]BK

1. Introduction

Receptors for bradykinin (BK) were originally classified into two main subtypes: B 1 receptors where BK analogues lacking the carboxy terminal arginine residue (des-Arg9-BK analogues), are relatively active (Barab6 et al., 1977; Regoli and Barabr, 1980), and B2 receptors where analogues of this series are inactive. However, there is now evidence that the B 2 site does not represent a single receptor population, but rather that there is apparent heterogeneity in recognition properties within this main subtype (see Field et al., 1988; Plevin and Owen, 1988). Such conclusions have been drawn, in particular, from studies involving the use of BK-receptor antagonists which are active in a number of non-B 1 preparations. These putative B2 selective antagonists, which were originally developed by Vavrek and Stewart, 1985), are based on modifications of the linear BK

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

sequence by substitutions in positions 2, 3, 5 and 8 of the prototype [D-Phe7]BK structure. Several such antagonists appear to discriminate between the B2 receptors present in peripheral preparations such as the rat uterus, rabbit iris sphincter and rat duodenum where they have a relatively high apparent affinity (Griesbacher and Lembeck, 1987; Field et al., 1988; Griesbacher et al., 1989), and those present in the guinea-pig ileum, rat vas deferens and guinea-pig urinary bladder where they have a lower apparent affinity (Llona and Galleguilles, 1983; Vavrek and Stewart, 1985; Carter et al., 1986; Tousignant et al., 1987; Field et al., 1988; Maggi et al., 1989). Furthermore, analogues of this series are relatively inactive as antagonists of BK responses at the novel B 3 site recently described in the guinea-pig trachea (Farmer et al., 1989) and are also inactive in the guinea-pig taenia caeci (Field et al., 1988). The presence of both B 1 and B2 receptor populations has previously been described in the rat duodenum preparation, with the former claimed to mediate contraction, and the latter relaxation, in response to BK (Boschcov et al., 1984; Paiva et al., 1989). A previous

232 study (Griesbacher and Lembeck, 1987) reported competitive antagonism of the relaxant responses to BK in this preparation by Lys,Lys-[Hyp3,ThiS.S,D-PheT]BK, however, no attempt was made to determine the subtype of BK receptor involved. In the light of the proposed presence of a mixed B~ and B2 receptor population (Boschcov et al., 1984; Paiva et al., 1989), along with the recent evidence suggesting antagonism of B~ receptors by certain of the putative [D-PheT]BK substituted B2 receptor antagonists (Regoli et al., 1986a), we have carried out investigations to determine the subtype of BK-receptor involved in mediating relaxation in the rat duodenum. Further, because of the possibility that the B1 activity of B2 receptor antagonists is a consequence of their decarboxylation by kininase I to des-Arg 9 products (Regoli et al., 1986b; see Sheikh and Kaplan, 1986b), the influence of the carboxypeptides inhibitor mergetpa was investigated both on the activity of BK and on antagonist pK B estimates. The mechanism of the relaxant response of the rat duodenum to BK has previously not been investigated in any detail. However, studies from our own laboratory (Carter et al., 1986; Morton et al., 1987; Hall, 1990) and others (Gater et al., 1985; Den Hertog et al., 1988), have indicated that certain similar responses, the relaxant phase of the B2-mediated biphasic response to BK in the guinea-pig taenia caeci and relaxation of the guineapig ileum (see Hall and Bonta, 1973), involves the opening of apamin-sensitive calcium-activated K + channels. More recently, a similar mechanism has been proposed to account for the B2-mediated relaxation in response to BK in the guinea-pig urinary bladder (Maggi et al., 1989). We have therefore studied the mechanism of relaxation in response to B2 receptor stimulation in the rat duodenum using both conventional organ-bath experiments and measurement of 86Rb efflux as an indicator of K + channel permeability. Apamin was used in both types of experiment to test whether similar K + channels are involved in the Bz-mediated relaxation in the rat duodenum as for those suggested above to mediate relaxation in the guinea-pig caeci and ileum and the rat urinary bladder. Some of these results have been presented as a preliminary communication to the British Pharmacological Society (London) December 1987 (Field et al., 1988) and I U P H A R (Sydney) 1987 (Morton et al., 1987).

2. Materials and methods

2.1. Tissue preparation Male Sprague-Dawly rats (200-350 g) were killed by stunning and exsanguination. The duodenum was excised and attached pancreas and blood vessels were carefully removed.

2.2. Organ bath experiments The duodenum was divided into four sections of approximate length 10 mm, and preparations were attached with silk to Hall effect purpose-designed isotonic transducers (E. Dyett, University College, London) and placed under a 1 g load. Responses were recorded on JJ Instruments C452 potentiometric fiat bed recorders. All experiments were performed with four preparations set up in parallel, in Krebs solution at 37°C containing atropine, mepyramine, ibuprofen, guanethidine (all 1 /~M), hexamethonium (10 #M) and, where indicated, TTX (0.3 ~M). A 45 s contact time and 5 min dose cycle were used in all organ bath experiments.

2.2.1. Test for involvement of B l receptors The activities of kallidin (Lys-BK) and the Bl-selecrive agonist des-Argg-BK were determined along with BK in each preparation with agonists (0.1 nM-10 #M) applied in a fully randomised order. This allowed expression of agonist activity relative to BK. The activity of des-Argq-BK (1 ~M) was tested every 30 min in all experiments to identify possible time-dependent induction of B~ receptors (Marceau et al., 1984). Possible B1 receptor involvement was further tested using the selective B~-receptor antagonist des-Argg[LeuS]BK. Threepoint concentration-response curves to BK were obtained in the absence, and then after a 5 or 10 rain equilibration with des-Argg[LeuS]BK (1.0 ~M or 3.2 ~M). The effect of the carboxypeptidase inhibitor mergetpa was investigated on concentration-response curves to BK (see Sheikh and Kaplan, 1986b). Experiments were conducted concurrently on control (no mergetpa) or test (with mergetpa, 10 ktM) preparations, with a randomised order of dosing. In both sets of experiments, in addition to the mediator antagonists detailed above, the inhibitors of neutral endopeptidase (E.C.3.4.24.11, 'enkephalinase') a n d k i n i n a s e II (E.C.3.4.15.1, angiotensin-converting enzyme, ACE); phosphoramidon (1 ~M) and enalaprilat (1 /,M) respectively, were included in the Krebs solution.

2.2.2. Effect of apamin on agonist responses The effect of apamin was tested against relaxant responses to BK and certain of its analogues (kallidin, des-Arg9-BK, D-Arg-[ThiS'8,FdfV]BK) (see Materials). Submaximal responses were obtained in the absence, and then after a 1, 5 or 10 min equilibration, with apamin (10 or 100 nM). Reversibility of the effect of apamin after washout was established in each preparation. In these experiments, the Krebs solution in addition contained TTX (0.3/tM).

233

2.2.3. Bz receptor antagonist studies Initial screening of eleven putative B2 receptor antagonists had identified several as being of potential value in receptor classification studies (see Field et al., 1988). One such antagonist Lys,Lys-[Hyp3,ThiS'S,DPheV]BK was studied further in this present investigation. Here, three-point concentration-response curves were obtained for BK, and then suitably increased concentrations of BK were applied after a 5 min equilibration with antagonist (10 nM-10 #M) in order to estimate a range of dose ratios in the presence of the antagonist. Reversibility of responses after antagonist washout was established in each preparation, with up to three antagonist concentrations used in each individual preparation. In these experiments, the Krebs solution contained in addition to the antagonists stated above, the peptide inhibitors phosphoramidon (1 /~M) and captopril (1/~M) (see Sheikh and Kaplan, 1986a; Orawski and Simmons, 1989). In a separate series of experiments, similar to those described above, but designed to investigate the effect of carboxypeptidases, such as kininase I, on antagonist pKn estimates, the inhibitor mergetpa (10 /IM) (see Orawski and Simmons, 1989) was also included in the Krebs solution. 2.3. 86Rb-efflux experiments The duodenum was divided into four sections and the longitudinal smooth muscle removed as described for the guinea-pig ileum by Rang (1964). Single thickness longitudinal muscle strips were suspended in thermostatted perfusion chambers on holders made from stainless steel tubing (o.d. 0.9 mm). Preparations were attached with silk to Grass FT03B isometric force-displacement transducers to allow recording of tension on Grass 79C polygraphs. Preparations were superfused at 1 ml rain ~ for 30 min with Krebs solution, and thereafter with a high-K + medium aerated with 95%O2-5%CO 2 and maintained at 37 °C. Preparations were then incubated for 1 h in a high-K + medium containing a6Rb (specific activity c. 1 MBq mol i) by inserting 1 ml vials under each preparation within the perfusion chamber and aerating via the muscle holder tubing. After this time, superfusion was resumed and the preparations were washed for 30 min, the superfusate being discarded. Thereafter, the superfusate was collected every 3 rain with 9 min agonist application periods using a 15 min dose cycle. The protocol and medium used are based on those of Jenkinson and Morton (1967a,b) who used a depolarising medium to measure agonist-induced K+-permeability changes uncomplicated by membrane hyperpolarisation which otherwise tends to obscure increases in K + efflux (Jenkinson and Morton, 1967c). The effect of BK (0.1, 1.0 and 10/~M) alone or in the presence of either apamin (100 nM) or Lys,Lys-

[Hyp3,ThiS'8,D-Phe7]BK (1 /~M) was tested on rate of efflux of 86Rb. In each experiment, two of the four preparations acted as controls with BK alone, whilst one of the other preparations also received apamin in the perfusion medium from the time of washout, and another received Lys,Lys-[Hyp3ThiS'8,D-PheT]BK 3 min before each dose of BK. In all experiments, the perfusing medium contained atropine, mepyramine, ibuprofen, guanethidine, phosphoramidon, captopril (all at 1 /~M) and hexamethonium (10/~M). Tissues were solubilised in NCS tissue solubiliser for up to 12 h at 37°C, and were counted with all samples for ,{-radioactivity by CErenkov radiation scintillation spectrometry.

2.4. Solutions and drugs 2.4.1. Solutions The composition of the Krebs solution was (mM): Na + 140, K + 5.9, CI- 104.8, H2PO 4 1.2, H C O f 24.9, Ca 2+ 2.6~ Mg 2+ 1.15, SOd - 1.15, glucose 10. The composition of the high-K + solution was (mM): K + 235, Na + 5, Mg 2+ 1.15, Ca 2+ 5, CI- 17, S O t - 109.5, HCO 3 16, Rb + 7.5, phosphate buffer 0.4, glucose 10. This solution was also used as 'load solution', the amount of 'cold' Rb adjusted to 7.5 mM according to final concentration of radioactive 86Rb in individual experiments. 2.4. 2. Materials S6Rb (specific activity c. 6 MBq mol -~) and NCS tissue solubiliser were obtained from Amersham International, U.K. Salts used were of analytical grade and obtained from B.D.H., U.K. Other agents were obtained as follows; bradykinin, kallidin, phosphoramidon, des-Argg-BK and des-Arg 9[LeuS]BK (Peninsula Laboratories Europe), atropine sulphate, ibuprofen and hexamethonium bromide (Sigma, U.K.), captopril (Squibb, U.S.A.), enalaprilat (Merck Sharp and Dohme, New Jersey), guanethidine sulphate (CIBA, U.K.), mepyramine maleate (May and Baker, U.K.) and mergetpa (D,L-2-mercaptomethyl-3guanidinoethylthiopropanoic acid) (Calbiochem, U.S.A.). Lys,Lys-[Hyp3,ThiS'8,D-PheV]BK ( B 4 3 1 0 / B4311) and D-Arg-[ThiS"8,FdfV]BK (B4360) ( F d f = pfluoro-D-phenylalanine) were kind gifts from Dr. R. Vavrek and Prof. J.M. Stewart, University of Colarado. 2.5. Statistical analysis Throughout estimates are shown as means_+ S.E. Tests for significance were made using Student's t-test for paired or two independent samples, as appropriate. Rate coefficients for 86Rb efflux were calculated in terms of fractional release (% min -~) of total tissue content with time as described previously (Jenkinson

234

and Morton, 1967a). Drug effects where quantified for significance tests in terms of % peak effect during BK application over control rate for the three previous control periods. For graphical representation, rate coefficients are shown as means + S.E. of absolute rate coefficients for each collection period. The p K B estimates and their S.E. were obtained from individual dose-ratio estimates (x) by direct calculation from the Gaddum-Schild equation, p K B = log]0 ( x - 1) - logl0 [At, assuming a Normal distribution for log ( x - 1), where [At is the applied antagonist concentration. Tests for unity slope were made using linear regression analysis. Statistical significance is shown as * P < 0.05 or • * P < 0.02.

3. Results

3.1. Organ bath experiments 3.1.1. Involvement of

B1

receptors

No evidence for a major contribution from B]-receptors to relaxation was found. Thus, the activity of the B t receptor agonist des-Arg9-BK was found to be c. 0.001 as compared to BK (BK = 1.00), though in occasional individual preparations this agonist had some activity (c. 0.1 relative to BK). Kallidin (Lys-BK) was approximately equiactive with BK. The p D 2 ( - l o g E C s 0 ) for BK in the absence of mergetpa was c. 10. The sensitivity of the duodenum to des-Arg9-BK did not change over the duration of the experiments (up to 6 h, data not shown). The selective B t receptor antagonist des-Argg-[LeuS]BK (1.0-3.2 /~M) was inactive against responses to BK, though the antagonist was occasionally seen to relax individual preparations in its own right as has been previously reported (Paiva et al., 1989). In all experiments, relaxant responses to BK and analogues were predominant, though at higher concentrations, a secondary delayed contractile response was seen. Figure 1 shows the effect of the carboxypeptidase inhibitor mergetpa on the log concentration-response curve for relaxation with BK. The peptidase inhibitor was without significant effect on responses to BK, and had no very obvious effects on washout kinetics of responses to BK even though inhibition of some peptidases had previously been shown to prolong peptide action in other tissues (Hall et al., 1990).

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Log [Bradykinin] (M) Fig. 1. Log concentration-relaxation curves to bradykinin in the absence (O), or presence (0), of the carboxypeptidase inhibitor DL-2mercaptomethyl-3-guanidinoethylthiopropanoic acid (mergetpa, 10 ~M). Experiments were carried out in Krebs solution containing atropine, mepyramine, ibuprofen, guanethidine (all 1 ~aM) and hexamethonium (10 ~M), along with the neutral endopeptidase inhibitor phosphoramidon and the kininase II inhibitor enalaprilat (1 #M) at 3 7 o c . Each point represents the mean+_S.E, for six preparations expressed as % maximal response to bradykinin in individual preparations. Mergetpa was without significant effect on responses to bradykinin (P > 0.05).

3.1.3. Be-receptor antagonist studies p K B estimates of 7.2 (+0.1) and 7.4 (+0.1) were obtained for the antagonist Lys,Lys-[Hyp3,ThiS"8,DPheT]BK against relaxant responses to BK from experi-

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BK (1 nM I

BK (1 riM)

BK (1 nM)

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APAMIN (100 nM)

3.1.2. Effect of apamin on agonist responses Figure 2 shows the effect of apamin (100 nM) on relaxant responses to BK and D-Arg-[ThiS'8,FdfV]BK. Relaxant responses were abolished by apamin and this effect was readily reversible on washout. In a number of preparations, the presence of apamin increased the spontaneous mechanical activity of the duodenum (data not shown).

Fig. 2. Abolition by apamin (100 riM) of relaxant responses of the rat duodenum to (a) bradykinin (BK, 1 nM) and (b) D-Arg[Thi 5'8, FdfT]BK (B4360, 1 /tM). Apamin was applied as indicated by the bars. Note the ready reversibility of the apamin effect. Relaxant responses were obtained using isotonic recording conditions in Krebs solution at 37 °C. The Krebs solution contained atropine, mepyramine, ibuprofen, guanethidine (all 1 /LM), TTX (0.3 #M) and hexamethonium (10 ~M). The figure shows a representative tracing from 10 experiments.

235

ments. Linear regression analysis showed that the slopes of these Schild plots were close to, and not significantly different from 1.0 (P > 0.05), thus suggesting a competitive interaction.

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3.2. 86Rb-efflux experiments

0.0 / //~' -7.5 -7.0

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Log [Lys,Lys-[Hyp3,ThiS,B.D-Phe7] -BK] (M)

Fig. 3. Schild plot analysis of antagonism of the relaxant responses of the rat duodenum to bradykinin by the Brreceptor antagonist Lys,Lys[Hyp3,ThiS"8,D-PheT]BK. Experiments carried out in the absence (e), or the presence (ll L of the carboxypeptidase inhibitor D,L-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (mergetpa, 10 /~M). The Krebs solution in both cases contained atropine, mepyramine, ibuprofen, guanethidine (all 1 ktM) and hexamethonium (10 /~M), along with the neutral endopeptidase inhibitor phosphoramidon, and the kininase II inhibitor captopril (both at 1 #M). For experiments with and without mergetpa, each point is the mean+S.E, for five determinations. The slopes of the fines shown, fitted by linear regression analysis (b = 1.09 and 0.99 respectively) do not differ significantly from unity (P > 0.05). Estimates of p K B calculated from the Gaddum-Schild equation were 7.42 ( + 0.12) for experiments carried out in the presence of mergetpa and 7.24 (+0.10) for experiments carried out in the absence of mergetpa, p K a estimates under the two conditions do not differ significantly (P > 0.05).

The effect of BK on the rate coefficient for 86Rb efflux in the depolarised rat duodenum is shown in fig. 4. A concentration-related increase in efflux is seen, which is attenuated in the presence of apamin (100 nM), and the BK receptor antagonist Lys,Lys-[Hyp3,ThiS'8,DPheV]BK (1 #M). These inhibitory effects of apamin and Lys,Lys-[Hyp3,ThiS"8,D-Phe7]BK were especially marked against responses to 1 /~M BK, and the appropriate plots are shown in fig. 5, where actual rate coefficients and the time course of the response can be observed. It may be noted that, somewhat unexpectedly, the duodenum exhibited spontaneous activity in the 1'

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ments carried out in the absence or the presence of mergetpa (10 #M), respectively. Figure 3 shows antagonist activity of Lys,Lys-[Hyp3,ThiS'8,D-PheT]BK displayed as Schild plots from both series of experi-

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Fig. 4. Bradykinin-evoked efflux of 86Rb from rat duodenum longitudinal muscle strips superfused at 37°C with a medium containing high-K + (235 mM) throughout. Every strip was exposed to three different bradykinin (BK) concentrations, each over three consecutive 3 min collection periods, and peak increases in rate coefficient over control rates are shown as means + S.E. for 7-14 preparations, Within experiments matched tissues were tested in the presence of apamin (I, 100 nM) throughout, or Lys,Lys[Hyp3,ThiS'sD-PheVlBK (A, 1/~M) for 3 rain prior to BK, or acted as controls (O). Significant difference at P < 0.02 level is shown as * * for treated strips compared to controls. The high-K + solution contained atropine, mepyramine, ibuprofen, guanethidine, phosphoramidon, captopril (all 1 jaM) and hexamethonium (10 ttM).

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Fig. 5. Bradykinin-evoked efflux of 86Rb from rat duodenum longitudinal muscle strips. Rate coefficients from seven experiments are shown as mean + S.E. and the application of bradykinin (BK, l ~tM), indicated by a horizontal bar, is over three consecutive 3 min collection periods. Rate coefficients were calculated at the mid-period. Panels (a) control preparations, (b) preparations in the presence of apamin (100 nM) and (c) preparations in the presence of Lys,Lys[Hypa,ThiS'8,D-PheT]BK (1 #M). Significant increase from control for a given time period is shown * for P < 0.05. The high-K + solution contained atropine, mepyramine, ibuprofen, guanethidine, phosphoramidon, captopril (all 1/LM) and hexamethonium (10 ~M).

236 high-K + medium, though the concurrent mechanical trace did not show relaxation of the duodenum in response to BK under these conditions (data not shown). The presence per se of a p a m i n or Lys,Lys[Hyp3,ThiS'8,D-PheV]BK had no obvious effect on the rate coefficient for 86Rb efflux.

4. Discussion

4.1. The involvement of relaxation

B2

receptors in smooth muscle

This study investigated receptors and mechanisms of relaxation in the rat duodenum preparation in response to BK. In a number of intestinal preparations, cyclooxygenase products (Walker and Wilson, 1979), acetylcholine (Goldstein et al., 1983; Yau et al., 1986) and catecholamines (Tfirker et al., 1964) have been implicated in the actions of BK. However, since all experiments in the present study were performed in the presence of antagonists or inhibitors to these and other mediators, it is likely that the relaxant response described here represents a predominantly direct action of BK through interaction with receptors present on the longitudinal smooth muscle. It should be noted that additional interactions, including that of BK with sensory nerves to release mediators such as calcitonin gene-related peptide or adenosine trisphosphate, both of which relax this preparation (Ferrero et al., 1980; Maggi et al., 1986) may also contribute to relaxation. Since relaxation still occurred in the presence of T'I'X, however, it seems unlikely that there was any major component from such a mechanism in the present experiments. It is clear that the predominant BK receptor population mediating relaxation of the rat duodenum, under the conditions of this study, is not of the B~ subtype, since des-Argg-BK had very low relaxant activity, and des-Arg9-[LeuS]BK was inactive as an antagonist. Furthermore, the log concentration-response curve to BK was unaffected by the presence of the carboxypeptidase inhibitor mergetpa which has been shown to inhibit breakdown by kininase I of BK to the Bl-selective agonist des-Argg-BK (Regoli et al., 1986b; Orawski and Simmons, 1989). These findings with des-Argg-BK are in contrast to those reported by others (Boschcov et al., 1984; Paiva et al., 1989) who found a predominant contractile response to this agonist in the rat duodenum. A possible reason for this discrepancy with the present data may lie in the fact that these authors conducted all their experiments in a solution containing low calcium, which might be expected to effect calcium homeostasis within the cells (see discussion of calcium-activated K + channels below). An interaction of BK with the recently proposed B3 receptor is also unlikely since antagonists

based on the [D-PheT]BK substituted series are very active in the duodenum (see Field et al., 1988) but have low antagonist activity in B3 preparations (Farmer et al., 1989). The involvement of B2 receptors mediating responses to BK is confirmed by the high activity of the BKantagonist Lys,Lys-[Hyp3,ThiS~8,D-PheV]BK. Schild plot analysis of experiments carried out in the presence of blockers of neutral endopeptidase and kininase II suggested a competitive interaction with B2 receptors for which the antagonist has a relatively high affinity (pK = 7.2), which was not significantly different when determined in the additional presence of the carboxypeptidase inhibitor mergetpa (pK B = 7.4). Furthermore, preliminary studies indicate no change in the pK B of this antagonist when tested in the continuous presence of the B 1 receptor antagonist des-Arg9-[LeuS]BK (1/~M) (data not shown). An identical pK B estimate was reported by others for Lys,Lys-[Hyp3,ThiS'8,D-PheV]BK in the rat duodenum preparation in the absence of any peptidase inhibitors and under somewhat different conditions (Griesbacher et al., 1989). These results allow speculation as to possible B2 receptor heterogeneity between peripheral preparations. Thus, similar high p K B estimates have been reported for this particular antagonist in other B2 preparations including the rabbit iris sphincter and rat uterus (Griesbacher and Lembeck, 1987; Field et al., 1988; Plevin and Owen, 1988; Griesbacher et al., 1989). In contrast, we found the antagonist to be relatively low in activity in several other preparations including the ileum and the rat vas deferens and inactive in the guinea-pig taenia caeci (Field et al., 1988). These observations suggest the presence of different subtype(s) of B 2 receptor.

4.2. Mechanism of B2-mediated relaxation Considering the mechanism of B2-mediated relaxation of the rat duodenum, BK was seen to increase efflux of 86Rb from depolarised longitudinal smooth muscle preparations in a concentration-related manner. The antagonist Lys,Lys-[Hyp3,ThiS'8,D-PheV]BK inhibited these increases in efflux, thus showing that the B2-mediated relaxation in normal polarised conditions can be related to B2-mediated increases in 86Rb efflux (as measured under depolarised conditions). These resuits suggest that the opening of a subset of K + channels, and subsequent membrane stabilisation or hyperpolarisation, can most readily account for the smooth muscle relaxation by BK. The concentrations of BK required to demonstrate significant opening of the subset of 86Rb-passing calcium activated K + channels in the high-K + medium were higher than those necessary to relax preparations in normal Krebs solution. This

237

was also the case in experiments, using essentially the same medium, demonstrating opening of the similar, and now well documented, apamin-sensitive channels by noradrenaline (Jenkinson and Morton, 1967a,b) and bradykinin (Gater et al., 1985) in the guinea-pig taenia caeci. Such differences in concentrations required might reflect some voltage sensitivity of these channels and altered calcium homeostasis in the depolarised cells. Probably more importantly, in normal medium, these highly excitable tissues would be stabilised by the opening of very few channels, whereas in tracer experiments maximal additional channel opening is necessary given the high basal permeability of K + channels as a whole. It is noteworthy that Liebmann et al. (1987) reported an increase in cAMP levels with BK in the rat duodenum preparation, which in principle could also account for the relaxation. However, there can be no doubt that an increase in calcium-activated K+-channel permeability is the major event initiating relaxation in response to BK, as described below. Thus, the mechanical response in Krebs solution, as well as the BK-evoked 86Rb efflux in high-K + medium, were attenuated by low concentrations of apamin, which is known to selectively block a subset of calcium-activated K + channels in several preparations (Banks et al., 1979; Gater et al., 1985; Hall, 1990). A similar mechanism involving the opening of apamin-sensitive K + channels has also been proposed to account for the relaxant response t o B 2 receptor activation in other smooth muscle preparations; for example, in the guinea-pig caeci (Carter et al., 1986; Morton et al., 1987; Den Hertog et al., 1988), guinea-pig ileum (Carter et al., 1987) and rat urinary bladder (Maggi et al., 1989). Furthermore, it may be noted that in the guinea-pig taenia caeci an evident apamin-sensitive increase in 86Rb efflux though calcium-activated K ÷ channels has also been reported for BK (Gater et al., 1985; Hall, 1990), and may be related to calciummobilisation through increased phosphatidylinositol turnover by BK demonstrated both in the guinea-pig taenia caeci and ileum (Hall, 1990). However, though the mechanism of relaxation in these latter two preparations seems similar to that described in this present study in the rat duodenum, the antagonist Lys,Lys[Hyp3,ThiS'8,D-PheV]BK has a lower apparent affinity for the Bz receptor populations in the guinea-pig ileum and taenia caeci (see Field et al., 1988). In conclusion, the relaxant response of the rat duodenum is mediated by a B2 receptor subtype for which the antagonist Lys,Lys-[Hyp3,ThiS"8,D-PheT]BK has a high affinity. Activation of these receptors results in the opening of apamin-sensitive calcium-activated 86Rb-permeable K + channels. The opening of these channels by BK under polarising conditions would result in membrane stabilisation and could therefore adequately account for smooth muscle relaxation.

Acknowledgements We acknowledge an MRC Studentship to J.M.H. and thank Debra Mitchell for excellent technical help with the antagonist experiments with mergetpa. We thank Prof. John Stewart and Dr. Ray Vavrek for the kind gift of B4310, B4311 and B4360.

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