Circular Muscle Of Guinea-pig Colon Research Articles

Differences in electromechanical coupling between bradykinin and the nonpeptide kinin B2 receptor agonist, FR 190997, in the circular muscle of guinea-pig colon.

We have compared the effect of bradykinin (BK) and the nonpeptide kinin B2 receptor agonist, FR 190997, in producing changes in membrane potential and tension in the circular muscle of guinea-pig colon by the sucrose gap technique. In the presence of atropine (1 microM), S-ketoprofen (3 microM) and apamin (0.1 microM), BK (1 microM for 20 s) induced a transient depolarization of the membrane with superimposed action potentials (spikes) and transient contraction. Nifedipine (1 microM) eliminated the spikes and markedly inhibited the BK-induced contractions. FR 190997 (3-10 microM for 20 s) induced a slowly developing sustained small depolarization associated with a slowly developing and sustained contraction but, contrary to BK, FR 190997 was unable to trigger spikes. Nifedipine had no effect on depolarization and contraction induced by FR 190997. In the presence of 1 microM nifedipine, the combined application of a blocker of receptor-operated cation channels, SKF 96365 (50 microM for 30 min), and of an inhibitor of sarcoplasmic reticulum calcium pump, cyclopiazonic acid (CPA 10 microM for 30 min), reduced the BK-induced depolarization and contraction by about 45%-60%. The same treatment induced about 40% reduction of the sustained contraction induced by FR 190997, whereas the concomitant depolarization was not significantly affected. The tyrosine kinase inhibitor genistein (40 microM for 20 min) had no effect on the BK- and FR 190997-induced depolarization and contraction in the presence of nifedipine. In radioligand binding experiments performed in membranes of colonic smooth muscle cells, both agonists displaced the [3H]BK specific binding with a pIC50 of 9.6 and 8.5 for BK and FR 190997, respectively. These findings indicate a substantial qualitative difference in mechanisms of excitation contraction coupling activated by BK and FR 190997 via B2 receptors in guinea-pig colon.

Antimuscarinic, calcium channel blocker and tachykinin NK2 receptor antagonist actions of otilonium bromide in the circular muscle of guinea-pig colon.

We have analyzed, by the sucrose gap method, the action of otilonium bromide, a quaternary ammonium derivative in use for the symptomatic therapy of irritable bowel syndrome, on the electrical and mechanical responses initiated by different stimuli in the circular muscle of the guinea-pig proximal colon. Otilonium bromide produced a concentration-dependent inhibition of membrane depolarization (IC50 4.1 microM), action potentials (APs) and contraction (IC50 3.7 microM) produced by the muscarinic receptor agonist, methacholine. It also produced a concentration-dependent inhibition of APs and accompanying contraction (IC50 31 microM) produced by KCl (30 mM), and had a biphasic effect on the cholinergic excitatory junction potential (e.j.p.) produced by single pulse electrical field stimulation: at low concentrations (0.1-0.3 microM) otilonium bromide enhanced the e.j.p. and, at higher concentrations (IC50 22 microM and 16 microM toward depolarization and contraction), produced a concentration-dependent inhibition. Otilonium bromide eliminated the APs superimposed on the depolarization induced by the tachykinin NK1 receptor agonist, [Sar9]substance P-sulphone and suppressed the corresponding contraction (IC50 43 microM) but had little effect on the sustained membrane depolarization induced by this agonist. On the other hand, otilonium bromide produced a similar inhibitory effect on both membrane depolarization and contraction (IC50 38 microM and 45 microM, respectively) induced by the tachykinin NK2 receptor agonist [betaAla8]neurokinin A (4-10). When tested in the presence of nifedipine (1 microM), otilonium bromide had no effect on the membrane depolarization induced by [Sar9]substance P-sulphone but inhibited in a concentration-dependent manner the depolarization induced by [betaAla8]neurokinin A (4-10) (IC50 41 microM). In contrast, the blocker of receptor-operated cation channels, SKF 96365, inhibited with similar potency the depolarization induced by both [Sar9]substance P-sulphone and [betaAla8]neurokinin A (4-10) (IC50 60 microM and 54 microM, respectively). In radioligand binding experiments otilonium bromide produced a concentration-dependent inhibition of the binding of both an agonist ([125I]neurokinin A, Ki 7.2 microM) and an antagonist ([3H]SR 48968, Ki 2.2 microM) to membranes of Chinese hamster ovary cells transfected with the human tachykinin NK2 receptor. In conclusion, the present findings demonstrate that, in the microM range of concentrations, otilonium bromide acts as a muscarinic and tachykinin NK2 receptor antagonist and as a calcium channel blocker. The latter property is likely to account for its ability to suppress contraction initiated by the tachykinin NK1 receptor agonist. Therefore multiple mechanisms of action account for the ability of otilonium bromide to reduce stimulated motility of intestinal smooth muscle.

Evidence for the involvement of multiple mechanisms in the excitatory action of bradykinin in the circular muscle of guinea-pig colon.

We have investigated, by using the sucrose gap technique, the mechanisms of the excitatory action of bradykinin in the circular muscle of the guinea-pig proximal colon. In the presence of atropine (1 microM) and S-ketoprofen (3 microM), the application of bradykinin (1 microM for 20 s) produced complex changes in membrane potential and muscle tension. The prevailing response was a small hyperpolarization followed by a slowly developing depolarization and a tonic contraction. The selective B2 receptor antagonist, HOE 140 (0.3 microM) blocked the responses to bradykinin (1 microM) while tetrodotoxin (0.3 microM) had no affect. The selective B1 receptor agonist, [des-Arg9]bradykinin (1 microM) did not affect the electrical or mechanical activities of the circular muscle. Apamin (0.1 microM) blocked the transient hyperpolarization and potentiated the bradykinin-induced depolarization and contraction. In the presence of apamin, nifedipine (1 microM) blocked spikes (when present) and the phasic contraction while leaving the tonic contraction unaffected. The excitatory action of bradykinin was further investigated in the presence of atropine (1 microM), S-ketoprofen (3 microM), apamin (0.1 microM) and nifedipine (1 microM). The depolarization but not the contraction induced by bradykinin was reduced by about 30% in low-Na+ (25 mM) but not in low Cl- (9.7 mM) Krebs solution. The depolarization and contraction evoked by bradykinin were reduced (by about 30 and 75%, respectively) in Ca2+-free (2 min) Krebs solution. The blocker of the sarcoplasmic reticulum Ca2+ pump, cyclopiazonic acid (CPA, 10 microM) reduced the nifedipine-resistant depolarization and contraction induced by bradykinin by about 40 and 60%, respectively. The inhibitor of receptor-operated cation channels, SKF 96365 (50 microM) reduced the nifedipine-resistant bradykinin-induced depolarization and contraction by about 40 and 30%, respectively, whereas the inhibitor of Ca2+-dependent chloride channels, niflumic acid (100 microM) was without effect. The inhibitory effect of SKF 96365 (50 microM) and CPA (10 microM) was additive: in the presence of both drugs the bradykinin-induced depolarization and contraction were reduced by about 70-80%. The protein kinase C inhibitor, GF 109203x (10 microM) did not affect the nifedipine-resistant bradykinin-induced depolarization and contraction. At a concentration of 30 microM, GF 109203x reduced the bradykinin-induced contraction by about 50% while leaving the bradykinin-induced depolarization unaffected. The KCl (40 mM)-induced contraction was significantly reduced (by about 30%) by GF 109203x (30 microM). The present findings indicate that, in the presence of apamin and nifedipine, the bradykinin-induced contraction of circular muscle of the guinea-pig colon is due to the influx of extracellular Ca2+ via non-selective cation channels and, in part, to the release of Ca2+ from a loosely bound internal store. Intracellular Ca2+ facilitates the bradykinin-induced depolarization, a response which does not involve a protein kinase C-dependent mechanism.

Effect of cyclopiazonic acid on contractions produced by tachykinin NK1 and NK2 receptor agonists in the circular muscle of guinea-pig colon.

1 This study aimed to assess the effect of cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic reticulum calcium (Ca) pump, against contractile responses produced by selective tachykinin NK1 and NK2 receptor agonists, [Sar9]substance P (SP) sulfone and [beta Ala8]neurokinin A (NKA) (4-10), respectively, on the circular muscle of guinea-pig colon. All experiments were performed in the presence of atropine (1 microM) and indomethacin (10 microM). 2 In organ bath experiments, a submaximally equieffective concentration of the two agonists (10 nM) was selected: [Sar9]SP sulfone (10 nM) produced a biphasic contraction, the two amplitudes averaging 75 +/- 2 and 43 +/- 3% of the maximal response to KCl (80 mM) at 1 and 15 min from application of the agonist, respectively. CPA (3 microM for 60 min) slightly reduced the phasic response to [Sar9]SP sulfone (16 +/- 4% inhibition) and markedly suppressed the tonic component (89 +/- 3% inhibition). 3 The contraction produced by [beta Ala8]NKA (4-10) (10 nM) was more sustained than that induced by the NK1 receptor agonist: it averaged 69 +/- 5 and 73 +/- 4% of the response to KCl at 1 and 15 min from application of the agonist, respectively. CPA slightly and evenly depressed the response to [beta Ala8]NKA (4-10) (18 +/- 7 and 21 +/- 5% inhibition at 1 and 15 min). 4 In the presence of tachykinin NK1 and NK2 receptor antagonists (SR 140333 and MEN 10627, respectively, 1 microM each) and of L-nitroarginine (100 microM), KCl (40 mM) produced a distinct phasic and tonic contraction which was suppressed by 1 mM nifedipine. CPA (3 microM) did not affect the phasic contraction to KCl but abolished the tonic component of the response. 5 In the presence of 1 microM nifedipine, the response to [beta Ala8]NKA (4-10) was slightly depressed (32 +/- 6% inhibition) in its early component only, while the response to [Sar9]SP sulfone was abolished. CPA produced a slight inhibition (15 +/- 9 and 33 +/- 10% at 1 and 15 min, respectively) of the nifedipine-resistant response to [beta Ala8]NKA (4-10), an effect similar to that observed in the absence of nifedipine. Therefore, a large part of the response to [beta Ala8]NKA (4-10) persisted in the presence of both CPA and nifedipine. 6 In the sucrose gap, a prolonged superfusion with [Sar9]SP sulfone (0.1 microM for 5 min) produced sustained depolarization with superimposed spikes and contraction. CPA (3 microM) produced transient depolarization and contraction. In the presence of CPA, the initial responses (depolarization, spikes and contraction) to [Sar9]SP sulfone were unaffected but the sustained component of contraction was absent; the latter effect was accompanied by a suppression of spikes while the sustained depolarization was present. 7 We conclude that, during sustained depolarization produced by the NK1 receptor agonist, blockade of the sarcoplasmic reticulum Ca pump by CPA produces a faster Ca-dependent inactivation of Ca channels, thereby eliminating spikes and abolishing the tonic component of contraction. Ca mobilization/reuptake from a CPA-sensitive store seems to be of minor importance for regulating the NK2 receptor-mediated contractile responses.

Sequential activation of the triple excitatory transmission to the circular muscle of guinea-pig colon

The aim of this study was to resolve the temporal relationships of the triple excitation of the circular muscle of guinea-pig colon that occurs in response to activation of the intrinsic excitatory nerves by using atropine and tachykinin NK 1 and NK 2 receptor selective antagonists to define the relative contribution of the transmitters involved. In organ bath experiments, performed in the presence of blockers of inhibitory innervation, a train of electrical pulses at 5 Hz for 300 s produced a sustained contraction of the circular muscle of guinea-pig colon: the sequential addition of atropine (1 μM), of the tachykinin NK 1 receptor antagonist, SR 140333 (0.3 μM) and of the tachykinin NK 2 receptor antagonist, MEN 11420 (1 μM) produced a cumulative inhibitory effect and progressively delayed the onset of the contractile response to nerve stimulation. In the presence of peptidase inhibitors, atropine was less effective in inhibiting the contractile response for prolonged periods of stimulation: however, the pattern of inhibition of the evoked response produced by the sequential addition of blocker drugs was not substantially affected. The selective tachykinin NK 3 receptor agonist, senktide, produced a concentration-dependent contraction of guinea-pig colon. The sequential addition of atropine (1 μM), SR 140333 (0.3 μM) and MEN 11420 (1 μM) reproduced the effect of the same drugs on the response to electrical nerve stimulation. The peptide blocker of N-type voltage-dependent calcium channels, ω-conotoxin (0.1 μM) produced a partial inhibitory effect of the response to senktide. The ω-conotoxin-resistant response to 1 μM senktide was inhibited and delayed by the progressive application of atropine, SR 140333 and MEN 11420, similar to the effect observed in the absence of ω-conotoxin. In sucrose gap, single-pulse electrical field stimulation produced a fast excitatory junction potential which was largely (>90%) inhibited by atropine; application of a low concentration of the potassium channel blocker, 4-aminopyridine (30 μM), markedly enhanced the atropine-resistant excitatory junction potential which was abolished by the NK 1 receptor antagonist, GR 82334. We conclude that, during prolonged electrical or chemical stimulation of excitatory motorneurons, there is a sequential, time-dependent activation of the three excitatory mechanisms in the circular muscle of guinea-pig colon: the pattern of activation is relatively independent of the intensity of stimulation and/or the mechanisms of secretion of released transmitters. Postjunctional factors predominate in determining the relative contribution of the three transmitters, acetylcholine, substance P and neurokinin A, in producing excitation of the circular muscle.

CGRP potentiates excitatory transmission to the circular muscle of guinea-pig colon

We aimed to assess whether calcitonin gene-related peptide (CGRP) can modulate the release of tachykinins which are the main nonadrenergic noncholinergic (NANC) excitatory transmitters to the circular muscle of the guinea-pig proximal colon. In organ bath experiments, electrical field stimulation (EFS) in the presence of atropine (1 μM) and guanethidine (3 μM) evoked twitch phasic NANC contractions which were abolished by the combined administration of tachykinin NK 1 and NK 2 receptor antagonists. Human αCGRP (CGRP, 1–100 nM) produced a concentration-dependent potentiation of the amplitude of the NANC contractions induced by EFS while salmon calcitonin (up to 1 μM) had no effect. The potentiating effect of CGRP was unaffected by in vitro capsaicin pretreatment (10 μM for 15 min), peptidase inhibitors (captopril, bestatin and thiorphan, 1 μM each), apamin (0.3 μM) plus l-nitroarginine ( l-NOARG, 100 μM) and by the CGRP 1 receptor antagonist, the C-terminal fragment CGRP(8–37) (1 μM). The NK 2 receptor antagonist MEN 10627 which, when administered alone, had only a partial inhibitory effect on the amplitude of NANC twitches, concentration-dependently (10 nM–1 μM) inhibited the potentiating effect of CGRP. CGRP (1–100 nM) produced a concentration-dependent potentiation of the atropine-sensitive cholinergic contractions evoked by EFS in the presence of guanethidine and of tachykinin NK 1 and NK 2 receptor antagonists. Similar to the effect of CGRP, application of capsaicin (0.1–1 μM) potentiated the amplitude of the NANC contraction to EFS, an effect undergoing complete desensitization upon a second application of the drug. CGRP (0.1 μM) did not affect the contractile action of a submaximally effective concentration of neurokinin A (2 nM) while it inhibited that induced by substance P (2 nM). In sucrose gap, single pulse EFS in the presence of atropine (1 μM) and guanethidine (3 μM) induced an inhibitory junction potential (i.j.p.) and a small excitatory junction potential (e.j.p.). CGRP (0.1 μM) produced membrane hyperpolarization and relaxation without affecting i.j.p. amplitude but concomitantly increased the e.j.p. amplitude to induce a contraction in correspondence to each electrical pulse. In the presence of the NK 1 receptor antagonist, GR 82334 (3 μM), the membrane hyperpolarization and relaxation produced by CGRP and the EFS-evoked i.j.p. were unaffected, while the potentiating effect of CGRP on the EFS-evoked NANC e.j.p. and the corresponding contraction were abolished. We conclude that, in addition to the previously characterized direct smooth muscle relaxant action via CGRP 1 receptors (Maggi et al. Regulatory Peptides 61, 27–36, 1996), CGRP also induces a remarkable potentiation of excitatory neurotransmission to the circular muscle of the guinea-pig colon via CGRP 2 receptors. The latter effect, documented in this study, is evidenced on both the atropine-sensitive and the atropine-resistant (tachykinin-mediated) components of excitatory transmission: this effect does not involve mediator(s) release from capsaicin-sensitive primary afferent nerves, nor inhibition of peptide degradation or modulation of NANC inhibitory transmission.

Tachykinin receptors and intestinal motility

Substance P (SP) and neurokinin A (NKA) are synthesized by enteric cholinergic motorneurons that project to the longitudinal and circular muscle of the mammalian intestine. Thus, acetylcholine, SP, and NKA are the excitatory neuromuscular transmitters in the intestine. Tachykinin NK1 and NK2 receptors are expressed by smooth muscle cells in most regions of the intestine: the corelease of SP and NKA from nerves thus realizes paradigms of tachykininergic cotransmission. Examples have been found in which a cooperative model can be applied to account for the action of SP-NKA acting at NK1 and NK2 receptors (e.g., circular muscle of guinea-pig duodenum), as well as examples in which the message produced by activation of the two receptors diverges sharply in producing responses that have a markedly different time course and use different effector systems (e.g., circular muscle of guinea-pig colon). NK3 receptors are expressed on both excitatory and inhibitory motor neurons: indirect contractions (via release of acetylcholine and tachykinins) and relaxations (via release of nitric oxide) can be evoked in the gut by selective stimulation of NK3 receptors. Although a role of NK3 receptors in certain enteric reflexes has been evidenced, the importance of this system in mediating hexamethonium-resistant enteric transmission appears less important than previously speculated.

Calcitonin gene-related peptide (CGRP) in the circular muscle of guinea-pig colon: role as inhibitory transmitter and mechanisms of relaxation

In the presence of 1 μM tetrodotoxin (TTX), human α calcitonin gene-related peptide (CGRP) produced a concentration-dependent relaxation (EC 50 1.1 nM; E max 86% of the relaxation to 1 μM isoprenaline) of mucosa-free circular muscle strips from the guinea-pig proximal colon. In the presence of TTX, the C-terminal fragment CGRP(8–37) produced a concentration (0.3–3 μM)-dependent rightward shift of the curve to CGRP. The TTX-resistant, receptor-mediated, CGRP-induced relaxation was unaffected by apamin (0.3 μM) and l-nitroarginine ( l-NOARG, 100 μM), alone or in combination, as well as by glibenclamide (3 μM) or ( S)-ketoprofen (10 μM). Tetraethylammonium (TEA, 1–10 mM) and cyclopiazonic acid (CPA, 3–10 μM) produced a concentration-dependent partial inhibition of the relaxant response to CGRP. The inhibitory effect of TEA on the maximal relaxation produced by CGRP was prevented by nifedipine (1 μM) which did not affect the CGRP-relaxation of its own. In the presence of atropine (1 μM), guanethidine (3 μM), SR 140,333 (0.3 μM), MEN 10,627 (1 μM), apamin (0.3 μM) and l-NOARG (100 μM), the application of 1 μM capsaicin produced a transient relaxation of the strips. This response was not reproduced upon a second application of capsaicin, 60 min later, indicating complete desensitization. CGRP(8–37) (0.3–1.0 μM) produced a partial inhibitory effect (about 50% inhibition) of the relaxant response to capsaicin. In the presence of atropine (1 μM), guanethidine (3 μM), SR 140,333 (0.3 μM), MEN 10,627 (1 μM), apamin (0.3 μM), l-NOARG (100 μM) and after capsaicin in vitro pretreatment (10 μM for 15 min), electrical field stimulation (EFS, 10 Hz for 5 s) produced a transient relaxation which was unchanged by CGRP(8–37) (1 μM) while being abolished by TTX. In sucrose gap, brief superfusion with 0.3 μM CGRP produced a TTX (1 μM)- resistant membrane hyperpolarization and relaxation: the hyperpolarization produced by CGRP was inhibited by about 50% by either TEA (10 mM) or CPA (10 μM), while being unaffected by glibenclamide (3 μM). The combined application of TEA and CPA was not more effective (65% inhibition) in inhibiting the CGRP-induced hyperpolarization than each drug alone. We conclude that CGRP produces a direct relaxation of the circular muscle of the guinea-pig proximal colon by activating receptors sensitive to blockade by CGRP(8–37). Activation of Ca-dependent potassium channels and Ca release/reuptake from internal store(s) appear both to be involved in the action of CGRP. Endogenous CGRP mediates part of the relaxant response evoked by stimulation of capsaicin-sensitive primary afferent nerves in the circular muscle of guinea-pig colon, while it is not involved in the apamin and l-NOARG-resistant nonadrenergic noncholinergic (NANC) relaxation produced by electrical field stimulation of intrinsic inhibitory nerves.

Neuronal tachykinin NK2 receptors mediate release of non-adrenergic non-cholinergic inhibitory transmitters in the circular muscle of guinea-pig colon.

The aims of this study were: (i) verify the usefulness of the recently described non-peptide antagonist, SR 142801, for blocking tachykinin NK3 receptors in the circular muscle of the guinea-pig colon and (ii) after occlusion of NK3 receptors by SR 142801, test the hypothesis that tachykinins may activate non-adrenergic non-cholinergic inhibitory neurons via non-NK3 receptors. In sucrose gap, we found that SR 142801 (0.1 microM) time-dependently inhibited the senktide-induced atropine (1 microM)-sensitive depolarization, action potentials and contractions of circular muscle of guinea-pig colon without affecting the cholinergic excitatory junction potential and contraction produced by single pulse electrical field stimulation. Likewise, SR 142801 (0.1 microM) time-dependently inhibited the senktide-induced non-adrenergic non-cholinergic hyperpolarization and relaxation of the circular muscle, without affecting the non-adrenergic non-cholinergic inhibitory junction potentials and relaxation produced by single pulse electrical field stimulation. Therefore, SR 142801 is a suitable tool to occlude neuronal NK3 receptors in guinea-pig colon. In the presence of SR 142801 (0.1 microM), atropine (1 microM), guanethidine (3 microM), indomethacin (3 microM) and nifedipine (1 microM) superfusion with neurokinin A (0.3 microM) produced depolarization on which a series of inhibitory junction potentials were superimposed. The incidence, number and amplitude of the inhibitory junction potentials evoked by neurokinin A was partly reduced by pretreatment with either apamin (0.1 microM) or L-nitroarginine (30 microM) and was totally blocked by pretreatment with apamin plus L-nitroarginine or by tetrodotoxin (1 microM). None of these treatments affected the depolarization and contraction produced by neurokinin A. The NK1 receptor selective antagonist, GR 82,334 (3 microM), did not affect the responses to neurokinin A, which were abolished by the NK2 receptor-selective antagonist GR 94,800 (0.1 microM). Substance P (0.3 microM) produced a large depolarization of the membrane but was poorly effective in producing superimposed inhibitory junction potentials. The NK1 receptor-selective agonist [Sar9]substance P sulfone (0.3 microM) produced large depolarization without inducing superimposed inhibitory junction potentials, while the NK2 receptor-selective synthetic agonist [beta-Ala8]neurokinin A(4-10) (0.3 microM) produced depolarization and superimposed inhibitory junction potentials. We conclude that neurokinin A, in addition to direct excitation and contraction of circular muscle activates, via neuronal NK2 receptors, inhibitory non-adrenergic non-cholinergic motorneurons. Thus, neuronal NK2 receptors should be considered as targets for endogenous tachykinins in enteric circuitries leading to descending relaxation in guinea-pig colon.

Different Ca 2+ influx pathways mediate tachykinin receptor-induced contraction in circular muscle of guinea-pig colon

We used an electrophysiological approach (single sucrose gap) to compare the mechanism of action of selective tachykinin NK 1 and NK 2 receptor agonists ([Sar 9]substance P sulfone and [βAla 8]neurokinin A-(4–10), respectively) in producing contraction of the circular muscle of the guinea-pig proximal colon. [Sar 9]Substance P sulfone produced a marked depolarization, action potentials and increase in membrane conductance. On the other hand, [βAla 8]neurokinin A-(4–10) produced less depolarization of the cell membrane and did not change membrane resistance. Nifedipine (1 μM) greatly reduced (80% inhibition) the contraction due to [Sar 9]substance P sulfone while that due to [βAla 8]neurokinin A-(4–10) was slightly affected (13% inhibition). Action potentials induced by either agonist were suppressed by nifedipine, while depolarization was reduced only to a minor extent. When tested in a Ca 2+-free medium, the contraction produced by either agonist was greatly reduced (84–89%) as compared to the control. In organ bath experiments [Sar 9]substance P sulfone and [βAla 8]neurokinin A-(4–10) produced concentration-dependent contraction of the circular muscle of the colon (EC 50 8 and 12 nM, respectively). Nifedipine (1 μM) markedly suppressed the response to [Sar 9]substance P sulfone while that to [βAla 8]neurokinin A-(4–10) was only slightly depressed. These findings demonstrate that NK 1 receptor-mediated contraction is strictly linked to membrane depolarization and action potentials generation through nifedipine-sensitive Ca 2+ channels (electromechanical coupling) while the NK 2 receptor-mediated contraction is substantially unrelated to depolarization and, while being largely dependent upon extracellular Ca 2+, is nifedipine-resistant, possibly linked to the opening of non-selective (Ca 2+-permeable) receptor-gated cation channels (pharmacomechanical coupling).