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We examined the potential stimulatory effects of U46619 (a prostanoid TP receptor agonist) and five prostanoids on the contractile activities of urinary bladder smooth muscle (UBSM), focusing on the role of the TP receptor and its associated CaChanges in the basal tone and spontaneous contractile activity (amplitude and frequency) of isolated guinea pig UBSM were measured isotonically. The presence of TP receptors in UBSM was examined by RT-qPCR and immunofluorescence.U46619, prostaglandin (PG) EProstanoids can enhance UBSM contractile activities and thus may be endogenous candidates for induction of detrusor overactivity. The TP receptor and TP-receptor-activated VDCCs/SOCCs are key molecules responsible for these effects.

Aims β-Adrenoceptors (β-ADRs) mediating the relaxation of rat superior mesenteric arteries (SMAs) were pharmacologically identified, and the effects of chemical sympathetic denervation on β-ADR-mediated relaxation were examined. Main methods The tension changes of endothelium-denuded SMAs were isometrically recorded and the mRNA of endothelium-denuded SMA β-ADR was detected using RT-PCR. Key findings In endothelium-denuded SMAs contracted with ≥10−7 M phenylephrine (an α1-ADR agonist), isoprenaline (a β-ADR agonist)-induced relaxation was competitively inhibited by 3 × 10−9–10−8 M propranolol (a β1,2-ADR antagonist), but not further affected by ≥10−8 M propranolol. Although isoprenaline-induced relaxation was not affected by ICI-118,551 (10−9–10−8 M; a β2-ADR antagonist), it was competitively inhibited by atenolol (10−7–3 × 10−7 M; a β1-ADR antagonist) in the presence of ICI-118,551. In the presence of 10−7 M propranolol, isoprenaline- and CGP-12177A (a β3-ADR partial agonist)-induced relaxation was competitively inhibited by high concentrations of bupranolol (a β1,2,3-ADR antagonist), with pA2 values of 6.49 and 5.76, respectively. We detected the mRNA of β1- and β3-ADRs in endothelium-denuded SMAs. Treatment with 6-hydroxydopamine (a catecholaminergic neurotoxin) reduced maximal isoprenaline-induced relaxation in the presence and absence of 10−7 M propranolol, but not CGP-12177A-induced relaxation. Significance Isoprenaline-induced relaxation of rat SMAs is mediated by β1- and β3-ADRs. β-ADR-mediated relaxation of rat SMAs is shown to be attenuated by chemical sympathetic denervation. The differences in the effects of bupranolol and chemical sympathetic denervation on the responses to isoprenaline and CGP-12177A in rat SMAs might be explained by the possible presence of multiple β3-ADRs with different pharmacological properties.

Aims We examined the potential stimulatory effects of U46619 (a prostanoid TP receptor agonist) and five prostanoids on the contractile activities of urinary bladder smooth muscle (UBSM), focusing on the role of the TP receptor and its associated Ca2+ influx routes to understand the roles of prostanoids in the regulation of UB contractile activity. Main methods Changes in the basal tone and spontaneous contractile activity (amplitude and frequency) of isolated guinea pig UBSM were measured isotonically. The presence of TP receptors in UBSM was examined by RT-qPCR and immunofluorescence. Key findings U46619, prostaglandin (PG) E2, PGF2α, and PGA2 enhanced UBSM basal tone and spontaneous contractile activities, which were measured as amplitudes and frequencies. The enhancing effects of U46619 were completely suppressed by SQ 29,548 (a TP receptor antagonist), which also partially suppressed the stimulating effects of other prostanoids. The expression of TP receptors in UBSMs was verified at the mRNA and protein level. The enhancing effects of U46619 completely disappeared in Ca2+-free solution. U46619-enhanced basal tone was completely suppressed by verapamil, an inhibitor of voltage-dependent Ca2+ channels (VDCCs), and verapamil strongly decreased the spontaneous contraction frequency. The spontaneous contractions remaining in the presence of verapamil were strongly suppressed by SKF-96365 (an inhibitor of receptor-operated Ca2+ channels (ROCCs)/store-operated Ca2+ channels (SOCCs)), but not by LOE-908 (an inhibitor of ROCCs). Significance Prostanoids can enhance UBSM contractile activities and thus may be endogenous candidates for induction of detrusor overactivity. The TP receptor and TP-receptor-activated VDCCs/SOCCs are key molecules responsible for these effects.

Alpha1-adrenergic receptors (alpha1-ARs) are classified into three subtypes: alpha1A-AR, alpha1B-AR, and alpha1D-AR. Triple disruption of alpha1A-AR, alpha1B-AR, and alpha1D-AR genes results in hypotension and produces no contractile response of the thoracic aorta to noradrenalin. Presently, we characterized vascular contractility against other vasoconstrictors, such as potassium, prostaglandin F2alpha (PGF(2alpha)) and 5-hydroxytryptamine (5-HT), in alpha1A-AR, alpha1B-AR, and alpha1D-AR triple knockout (alpha1-AR triple KO) mice.The contractile responses to the stimulation with vasoconstrictors were studied using isolated thoracic aorta.As a result, the phasic and tonic contraction induced by a high concentration of potassium (20 mM) was enhanced in the isolated thoracic aorta of alpha1-AR triple KO mice compared with that of wild-type (WT) mice. In addition, vascular responses to PGF(2alpha) and 5-HT were also enhanced in the isolated thoracic aorta of alpha1-AR triple KO mice compared with WT mice. Similar to in vitro findings with isolated thoracic aorta, in vivo pressor responses to PGF(2alpha) were enhanced in alpha1-AR triple KO mice. Real-time reverse transcription-polymerase chain reaction analysis and western blot analysis indicate that gene expression of the 5-hydroxytryptamine 2A (5-HT(2A)) receptor was up-regulated in the thoracic aorta of alpha1-AR triple KO mice while the prostaglandin F2alpha receptor (FP) was unchanged.These results suggest that loss of alpha1-ARs can lead to enhancement of vascular responsiveness to the vasoconstrictors and may imply that alpha1-ARs and the subsequent signaling regulate the vascular responsiveness to other stimulations such as depolarization, 5-HT and PGF(2alpha).

This study evaluates ocular (iris, ciliary body and ciliary process) and nonocular (atria and lung) beta-adrenoceptors in rabbit to characterize the plasma membrane beta-adrenoceptors and binding affinities of beta-adrenoceptor antagonists.The tissue segment binding method with a hydrophilic radioligand (-)-4-[3-t-butylamino-2-hydroxypropoxy]-[5,7-(3)H]benzimidazol-2-one ([(3)H]-CGP12177) was employed.Specific and saturable binding of [(3)H]-CGP12177 to intact tissue segments was detected by using (+/-)-propranolol to define nonspecific binding, showing a single population of plasma membrane binding sites with high affinity. Competition experiments with selective beta(1)- and beta(2)-adrenoceptor antagonists revealed a single population of beta(2)-adrenoceptors in ocular tissues and of beta(1)-adrenoceptors in atria, but mixed populations of beta(1)- and beta(2)-adrenoceptors in 70% and 30%, respectively, in lung. A competition curve for timolol was biphasic in lung and its binding affinity for beta(2)-adrenoceptors was approximately 158-fold higher than for beta(1)-adrenoceptors, indicating the beta(2)-selectivity of timolol. In contrast, competition curves for stereoisomers of befunolol, carteolol, and propranolol were monophasic in all tissues. The (-)-enantiomers of these antagonists were more potent than corresponding (+)-enantiomers in displacing from [(3)H]-CGP12177 binding, and the isomeric potency ratios of befunolol and carteolol were less than those of propranolol.This study with tissue segment binding method suggests that the binding affinity of (-)-enantiomers of beta-adrenoceptor antagonists for plasma membrane beta-adrenoceptors (beta(1)-adrenoceptors of atria, beta(2)-adrenoceptors of ocular tissues, and mixed beta(1)-/beta(2)-adrenoceptors of lung) is higher than that of corresponding (+)-enantiomers and their stereoselectivity is different between beta-adrenoceptor antagonists.

Recently, tissue segment binding method with a hydrophilic radioligand [(3)H]-CGP12177 was developed to detect plasma membrane beta-adrenoceptors in rat heart (Horinouchi et al., 2006). In the present study, propranolol (40 mg kg(-1) day(-1)), atenolol (40 mg kg(-1) day(-1)) and bevantolol (200 mg kg(-1) day(-1)) were administered to rats for 6 weeks, and the changes of plasma membrane beta-adrenoceptors and their mRNA expression in rat ventricle were examined. Chronic administration of propranolol increased the beta(1)-adrenoceptors but decreased the beta(2)-adrenoceptors without changing total amount of plasma membrane beta-adrenoceptors. Atenolol increased both plasma membrane beta(1)- and beta(2)-adrenoceptors, whereas bevantolol had no effect on the beta-adrenoceptor density and their subtype proportions. In contrast, the density of beta-adrenoceptors detected in conventional homogenate binding study was extremely low (approximately 60% of plasma membrane beta-adrenoceptors detected with the tissue segment binding method) and the effects of chronic administration of beta-adrenoceptor antagonists were not necessarily in accord with those at the plasma membrane beta-adrenoceptors. The mRNA levels of beta(1)- and beta(2)-adrenoceptors were not altered by propranolol treatment, while beta(1)-adrenoceptor mRNA significantly decreased after administration of atenolol or bevantolol without changing the level of beta(2)-adrenoceptor mRNA. The present binding study with intact tissue segments clearly shows that the plasma membrane beta(1)- and beta(2)-adrenoceptors of rat heart, in contrast to the homogenate binding sites and the mRNA levels, are differently affected by chronic treatment with three beta-adrenoceptor antagonists; up- and down-regulations of beta(1)- and beta(2)-adrenoceptors, respectively, by propranolol, and up-regulation of both the subtypes by atenolol, but no significant change in both the subtypes by bevantolol.

Summary In isolated mouse left atria, acetylcholine (ACh) produced a biphasic inotropic response; a transient decrease in developed tension was followed by an increase. Both negative and positive responses were concentration dependent and were inhibited by atropine. The negative and positive inotropic responses were also observed with a nonselective muscarinic stimulant, oxotremorine-M, but not with an M1-receptor selective stimulant, McN-A343. Pirenzepine, an M1-receptor antagonist, inhibited both negative and positive inotropic responses at high concentrations. Gallamine, an M2receptor antagonist, inhibited the negative response. Hexahydro-siladifenidol hydrochloride, p-fluoro analog (p-F-HHSiD), an M3-receptor antagonist, inhibited the positive response with no effect on the negative phase. In pertussis toxin (PTX) treated preparations, negative inotropic response to ACh was not observed. These results suggest that the negative and positive inotropic responses to acetylcholine in mouse atria are mediated by M2 and M3 receptors, respectively. The negative phase, but not the positive phase, was mediated by a PTX-sensitive G protein.

The effects of hirsutine, an indole alkaloid from Uncaria rhynchophylla MIQ. JACKSON with antihypertensive, negative chronotropic and antiarrhythmic activity, and its C3 structural epimer, dihydrocorynantheine, on membrane potentials of rabbit sino-atrial node and guinea-pig right ventricle and left atrium were studied with microelectrode techniques. In sino-atrial node preparations, hirsutine and dihydrocorynantheine (0.1 microM to 10 microM) concentration-dependently increased cycle length, decreased slope of the pacemaker depolarization (phase 4 depolarization), decreased maximum rate of rise and prolonged action potential duration. In atrial and ventricular preparations, both compounds (0.1 microM to 30 microM) concentration-dependently decreased maximum rate of rise and prolonged action potential duration. These results indicate that hirsutine and dihydrocorynantheine have direct effects on the action potential of cardiac muscle through inhibition of multiple ion channels, which may explain their negative chronotropic and antiarrhythmic activity.

The present study was undertaken to know whether the formation of inositol 1,4,5-trisphosphate (IP 3 ) is increased by quick stretch, a dynamic mechanical stimulus in porcine coronary artery in order to inquiry the possibility that IP 3 could mediate Ca 2+ release in the stretch-induced contraction. Quick stretching of a helical strip of porcine coronary artery at a rate of 10 cm/sec, the amount of stretch equivalent to 140% of the initial muscle length (= 100%), and the stimulus period of 30 sec with 20-min intervals, produced delayed contraction. Quick stretching increased the content of IP 3 about three-fold over the control basal level, which always preceded the contraction. A putative phospholipase C inhibitor, 2-nitro-4-carboxyphenyl-N, N-diphenylcarbamate (NCDC), abolished the increase in the formation of IP 3 and partially inhibited the stretch-induced contraction. The results suggest that quick stretching increases the formation of IP 3 through a possible mechanism for activation of phospholipase C, which may lead to release of Ca 2+ into myoplasm and to further activation of the contractile elements.

This study evaluates beta-adrenoceptors in rat atria and ventricle using the tissue segment binding method and compares the results with those obtained using conventional homogenate binding assays. In studies with tissue segment binding, the hydrophilic radioligand [(3)H]-CGP12177 selectively bound to plasma membrane beta-adrenoceptors, and the B(max) levels were significantly higher than those obtained with homogenate binding. However, both binding approaches revealed similar proportions of beta(1)- and beta(2)-adrenoceptors. The regional distribution of plasma membrane beta(1)- and beta(2)-adrenoceptors in rat hearts were also determined using tissue segment binding. Abundance of beta-adrenoceptors and proportion of beta(1)-adrenoceptors were higher in atria than in ventricle, but there was no significant difference between right and left atria or within ventricle (right and left ventricle free walls, apex, and interventricular septum). To establish the ability of the tissue segment binding method to study beta-adrenoceptor regulation such as the internalization of receptors, the effect of prolonged exposure of rat ventricle to (-)-isoprenaline was also investigated by using tissue segments and homogenate binding. Incubation with (-)-isoprenaline for 1 h in vitro caused a concentration-dependent decrease in the density of beta-adrenoceptors, predominantly beta(2)-adrenoceptors, when assessed with tissue segment binding method. In contrast, the subtype-specific change after treatment with (-)-isoprenaline was not detected using homogenate binding. In summary, the tissue segment binding method with [(3)H]-CGP12177 enables a more precise quantitation of plasma membrane beta(1)- and beta(2)-adrenoceptors in rat hearts and is suitable for studying their regulation.

NIP-142 was shown to prolong atrial effective refractory period and to terminate atrial fibrillation and flutter in in vivo canine models. To obtain information on its antiarrhythmic action, we examined the effect of NIP-142 on cloned human cardiac K + channel Kv1.5 (hKv1.5) currents stably expressed in a human cell line using whole-cell voltage clamp methods. NIP-142 inhibited the hKv1.5 current in a concentration-dependent and voltage-independent manner. The inhibition was larger at the end of depolarizing pulse than at the outward current peak. The IC 50 for inhibition of the steady-state phase was 4.75 μM. A cross-over phenomenon was observed when current traces in the absence and presence of NIP-142 were superimposed. Inhibition of hKv1.5 current by NIP-142 was frequency-independent; changing the depolarizing pulse frequencies (0.1, 0.2, 1 Hz) and little effect on the degree of inhibition. NIP-142 decreased the maximal peak amplitude of kHv1.5 current at the first command pulse after 3 min rest in the presence of the drug. These results suggest that NIP-142 has inhibitory effects on the hKv1.5 current through interaction with both open and closed states of the channel, which may underlie its antiarrhythmic activity in the atria.

Efonidipine is a dihydropyridine Ca2+ antagonist with inhibitory effects on both L-type and T-type Ca2+ channels and potent bradycardiac activity especially in patients with high heart rate. In the present study, we examined the frequency dependence of efonidipine action on the T-type Ca2+ channel in isolated guinea-pig ventricular myocytes. The potency of efonidipine to inhibit the T-type Ca2+ current was higher under higher stimulation frequencies. The IC50 values were 1.3 x 10(-8), 2.0 x 10(-6) and 6.3 x 10(-6) M under stimulation frequencies of 1, 0.2 and 0.05 Hz, respectively. The reduction of T-type Ca2+ current amplitude was not accompanied by change in the time course of current decay. Efonidipine (10 microM) inhibited T-type Ca2+ current elicited by depolarization from holding potentials ranging from -90 to -30 mV by about 30%; the voltage-dependence of steady-state inactivation was not changed by the drug. Efonidipine slowed the recovery from inactivation following an inactivating prepulse. In conclusion, efonidipine was shown to have frequency-dependent inhibitory effects on the T-type Ca2+ channel, which could be explained by slow dissociation of the drug from the inactivated state of the channel.

The effects of efonidipine, a 1,4-dihydropyridine phosphonate, and structurally related compounds on rabbit sino-atrial node action potential were examined with microelectrodes. 3NIC5NZ has a phosphonate moiety identical to that of efonidipine at the C5 position of the dihydropyridine ring and a side chain identical to nicardipine at C3, while 3NZ5NIC has C5 and C3 side chains identical to nicardipine and efonidipine, respectively. All four compounds decreased the slope and prolonged the early and late phases of pacemaker depolarization. The selectivity for the late phase against the early phase was in the order of efonidipine > 3NIC5NZ >> nicardipine > 3NZ5NIC. Thus, the phosphonate moiety at C5 position of the may be important for the characteristic prolongation of the late phase pacemaker depolarization by efonidipine.

Vasorelaxant effects of ATP-sensitive potassium (K ATP ) channel openers were examined on the tonic phase of vascular contraction induced by norepinephrine (NE) in guinea pig and rat aorta. K ATP channel openers, NIP-121 and cromakalim, produced glibenclamide-sensitive and concentration-dependent relaxations in guinea pig and rat aorta preconstricted with NE. However, the vascular relaxations induced by both K ATP channel openers were less pronounced in guinea pig aorta than in rat aorta. d - cis -Diltiazem, at the concentration up to 10 −5 M, did not appreciably inhibit the NE-induced contraction of guinea pig aorta, whereas the compound almost completely inhibited the NE-induced contraction of rat aorta at the same concentration. By contrast, sodium nitroprusside relaxed the NE-induced contractions in both guinea pig and rat aorta with similar potencies. These findings suggest that vasorelaxant effects of K ATP channel openers on the NE-induced sustained contraction in guinea pig aorta is not attributable to the subsequent inhibition of Ca 2+ influx through L-type voltage-gated Ca 2+ channels. Lower sensitivity of guinea pig aortic smooth muscle to K ATP channel openers is most likely due to the low dependence of NE-induced contraction on the Ca 2+ influx in this vascular smooth muscle.

Effects of zaprinast, an inhibitor of guanosine 3', 5'-cyclic monophosphate (cGMP)-specific phosphodiesterase, and methylene blue, an inhibitor of soluble guanylate cyclase, on the negative chronotropic response to CD-832, a novel dihydropyridine derivative with a nitrate moiety, and nifedipine were examined with isolated guinea-pig right atria in the presence and absence of isoproterenol. CD-832 and nifedipine produced concentration-dependent negative chronotropic effects both in the absence and presence of isoproterenol. In the absence of isoproterenol, the concentration-response curves for CD-832 and nifedipine were neither potentiated by zaprinast nor inhibited by methylene blue. In the presence of isoproterenol (10[-8] M), zaprinast produced a three-fold leftward shift of the concentration-response curve for CD-832, while methylene blue produced a three-fold rightward shift. The concentration-response curve for nifedipine was not affected by these agents. SIN-1, a nitric oxide (NO) donor, had no chronotropic effect in the absence of isoproterenol, but had a concentration-dependent negative chronotropic effect in the presence of isoproterenol: the beating rate decreased to values close to that in the absence of isoproterenol. These findings suggest that NO-cGMP mediated pathway is involved in the negative chronotropic actions of CD-832 under beta-adrenergic stimulation.