Functional role of M2 and M3 muscarinic receptors in the urinary bladder of rats in vitro and in vivo.

1. Urinary bladder smooth muscle is enriched with muscarinic receptors, the majority of which are of the M2 subtype whereas the remaining minority belong to the M3 subtype. The objective of the present study was to assess the functional role of M2 and M3 receptors in the urinary bladder of rat in vitro and in vivo by use of key discriminatory antagonists. 2. In the isolated bladder of rat, (+)-cis-dioxolane produced concentration-dependent contractions (pEC50 = 6.3) which were unaffected by tetrodotoxin (0.1 microM). These contractions were antagonized by muscarinic antagonists with the following rank order of affinity (pA2) estimates: atropine (9.1) > 4-diphenyl acetoxy-methyl piperidine methiodide (4-DAMP) (8.9) > darifenacin (8.5) > para fluoro hexahydrosiladifenidol (p-F-HHSiD) (7.4) > pirenzepine (6.8) > methoctramine (5.9). These pA2 estimates correlated most favourably (r = 0.99, P < 0.001) with the binding affinity (pKi) estimates of these compounds at human recombinant muscarinic m3 receptors expressed in Chinese hamster ovary cells, suggesting that the receptor mediating the direct contractile responses to (+)-cis-dioxolane equates with the pharmacologically defined M3 receptor. 3. As M2 receptors in smooth muscle are negatively coupled to adenylyl cyclase, we sought to determine whether a functional role of M2 receptors could be unmasked under conditions of elevated adenylyl cyclase activity (i.e., isoprenaline-induced relaxation of KCl pre-contracted tissues). Muscarinic M3 receptors were preferentially alkylated by exposing tissues to 4-DAMP mustard (40 nM, 1 h) in the presence of methoctramine (0.3 microM) to protect M2 receptors. Under these conditions, (+)-cis-dioxolane produced concentration-dependent reversal (re-contraction) of isoprenaline-induced relaxation (pEC50 = 5.8) but had marginal effects on pinacidil-induced, adenosine 3':5'-cyclic monophosphate (cyclic AMP)-independent, relaxation. The re-contractions were antagonized by methoctramine and darifenacin, yielding pA2 estimates of 6.8 and 7.6, respectively. These values are intermediate between those expected for these compounds at M2 and M3 receptors and were consistent with the involvement of both of these subtypes. 4. In urethane-anaesthetized rats, the cholinergic component (approximately 55%) of volume-induced bladder contractions was inhibited by muscarinic antagonists with the following rank order of potency (ID35%inh, nmol kg-1, i.v.): 4-DAMP (8.1) > atropine (20.7) > methoctramine (119.9) > darifenacin (283.3) > pirenzepine (369.1) > p-F-HHSiD (1053.8). These potency estimates correlated most favourably (r = 0.89, P = 0.04) with the pKi estimates of these compounds at human recombinant muscarinic m2 receptors. This is consistent with a major contribution of M2 receptors in the generation of volume-induced bladder contractions, although the modest potency of darifenacin does not exclude a role of M3 receptors. Pretreatment with propranolol (1 mg kg-1, i.v.) increased the ID35%inh of methoctramine significantly from 95.9 to 404.5 nmol kg-1 but had no significant effects on the inhibitory responses to darifenacin. These data suggest an obligatory role of beta-adrenoceptors in M2 receptor-mediated bladder contractions in vivo. 5. The findings of the present study suggest that both M2 and M3 receptors can cause contraction of the rat bladder in vitro and may also mediate reflex bladder contractions in vivo. It is proposed that muscarinic M3 receptor activation primarily causes direct contraction of the detrusor whereas M2 receptor activation can contract the bladder indirectly by reversing sympathetically (i.e. beta-adrenoceptor)-mediated relaxation. This dual mechanism may allow the parasympathetic nervous system, which is activated during voiding, to cause more efficient and complete emptying of the bladder.

5-HT4 receptor mediated stimulation of gastric emptying in rats.

It is well documented that certain substituted benzamides, such as cisapride, and benzimidazolones, such as BIMU 8, enhance gastric emptying in rats. As these compounds possess 5-HT3 antagonistic and 5-HT4 agonistic properties, the precise mechanisms (5-HT3 or 5-HT4) underlying their gastroprokinetic effects is still unclear. In the present study, we used SC 49518 (a benzamide and selective 5-HT4 receptor agonist) and two selective 5-HT4 receptor antagonists (RS 23597-190 and SB 204070) to elucidate the role of 5-HT4 receptors in gastroprokinesis. SC 49518 (1-316 micrograms/kg; ip) produced significant and dose-dependent stimulation of gastric emptying in rats (ED50 = 2.3 micrograms/kg; ip). SC 49518 also produced dose-dependent inhibition of bradycardia induced by 2-methyl 5-HT (von Bezold-Jarisch reflex) but with a 156 fold lower potency (ID50 = 0.36 mg/kg; ip). The gastroprokinetic effects of SC 49518 (3-316 micrograms/kg; ip) were significantly antagonized by the selective 5-HT4 receptor antagonist RS 23597-190 (0.1 mg/kg/min; iv). SB 204070 (0.003-1 mg/kg; ip), another selective 5-HT4 receptor antagonist, produced dose-dependent inhibition of the gastroprokinetic effects of SC 49518 (10 micrograms/kg; ip), the inhibition attaining statistical significance at the dose of 0.1 mg/kg; ip. RS 23597-190 had no effects on gastric emptying per se whereas SB 204070 significantly increased gastric emptying by itself at 1 mg/kg; ip but not at 0.1 mg/kg; ip. These findings show, for the first time, that SC 49518, a selective 5-HT4 receptor agonist, produces potent stimulation of gastric emptying in rats via a mechanism involving activation of 5-HT4 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for the involvement of 5-hydroxytryptamine 4 receptors in 5-hydroxytryptophan-induced diarrhea in mice.

The objective of this study was to characterize the receptor(s) to 5-HT mediating 5-HTP-induced diarrhea in mice. The severity of diarrhea in mice was assessed using an arbitary scoring scale ranging from 0 (normal stools) to 3 (watery diarrhea). Administration of 5-HTP (1-30 mg/kg i.p.) produced a dose-dependent increase in diarrhea score (ED50, 1.47 mg/kg i.p.). 5-HTP (10 mg/kg i.p.)-induced diarrhea was unaffected by atropine (3 mg/kg i.p.) but was completely abolished by the aromatic L-amino acid decarboxylase inhibitor benserazide (10 mg/kg i.p.). Pretreatment (5 min before 5-HTP) with DAU 6285, a marginally selective 5-HT4 receptor antagonist, significantly inhibited 5-HTP-induced diarrhea (ID50, 0.58 mg/kg i.p.). Pretreatment (5 min before 5-HTP) with GR 113808 or SB 204070, two highly selective 5-HT4 antagonists, significantly inhibited 5-HTP-induced diarrhea with ID50 estimates of 0.31 and 0.003 mg/kg i.p., respectively. The maximal inhibition produced by DAU 6285, GR 113808 and SB 204070 was 63%, 68% and 36%, respectively. Neither GR 113808 (1 and 3 mg/kg i.p.) nor SB 204070 (0.1 and 1 mg/kg i.p.) had any effect on 16,16-dimethyl prostaglandin E2 (30 micrograms/kg i.p.)-induced diarrhea in mice. DAU 6285 significantly inhibited 16,16-dimethyl prostaglandin E2-induced diarrhea at the highest dose (3 mg/kg i.p.). Pretreatment (30 min before 5-HTP) with methysergide (0.1-3 mg/kg i.p.), metergoline (0.01-0.1 mg/kg i.p.), ketanserin (0.01-1 mg/kg i.p.), YM 060 (0.01-0.1 mg/kg i.p.) or ondansetron (0.01-3 mg/kg i.p.) had no significant effects on 5-HTP-induced diarrhea.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological evaluation of 1229U91, a novel high-affinity and selective neuropeptide Y-Y1 receptor antagonist.

The physiological role of neuropeptide Y (NPY), peptide YY (PYY) and their receptors (Y1 and Y2) has been difficult to elucidate mainly due to the lack of selective and high-affinity antagonists. Recently, Burroughs Wellcome disclosed a series of cyclic peptides, including the compound 1229U91, which were reported to be selective NPY receptor antagonists (PCT Publication No. WO 94/00486). The objective of this study was to evaluate the pharmacological properties of 1229U91. In radioligand binding studies, 1229U91 displaced specifically bound [125I]PYY from SK-N-MC cells (Y1 receptors) and SK-N-BE(2) cells (Y2 receptors) yielding pKi +/- S.E.M. estimates of 10.9 +/- 0.2 and 7.9 +/- 0.2, respectively. In the isolated perfused kidney of rat (Y1 receptor assay), NPY (10-1000 ng, bolus injection) evoked concentration-dependent increases in perfusion pressure (EC50 = 54.5 ng). In this assay, 1229U91 (1, 10 and 100 nM) produced concentration-dependent dextral displacement of the concentration-effect curve to NPY. The antagonism was surmountable at 1 nM 1229U91 (apparent pA2 estimate +/- S.E.M. = 9.3 +/- 0.4). At concentrations of 10 and 100 nM, 1229U91 produced significant depression of the maximum response to NPY (36 and 67%, respectively). In the vas deferens of rat (Y2 receptor assay), 1229U91 (3 microM) had no effect on NPY-induced inhibition of electrically evoked twitch response. In pithed rats, 1229U91 (0.3, 1 and 3 micrograms/kg/min i.v.) produced dose-dependent dextral displacement of the pressor dose-response curve to NPY yielding dose-ratio estimates of 2.4, 25.4 and 57.5, respectively. 1229U91 (3 micrograms/kg/min i.v.) had no effect on the pressor responses to norepinephrine or angiotensin II.(ABSTRACT TRUNCATED AT 250 WORDS)