Inotropic response of rabbit ventricular myocytes to endothelin-1: difference from isolated papillary muscles.

Endothelin-1 (ET-1) increased cell shortening and Ca2+ transients over the concentration of 3 x 10(-11) M to 10(-9) M with EC50 of 8.3 x 10(-11) M in rabbit single ventricular myocytes. Thus ET-1 was approximately 60 times more potent in single myocytes than in papillary muscles (EC50 = 5.1 x 10(-9) M) of the same species. In single myocytes, ET-1 at 10(-8) M elicited an inhibitory response that counteracted the facilitatory response: the concentration-response curve (CRC) for ET-1 was bell shaped. The ET(A)-receptor antagonist BQ-485 shifted CRC for ET-1 to the right in parallel; however, the facilitatory response to 10(-8) M ET-1 was markedly enhanced by BQ-485 and also by the ET(B) antagonist BQ-788. The ET(A)/ET(B) antagonist TAK-044 abolished the ET-1-induced response. These findings indicate that the response to ET-1 of single myocytes is different from that of papillary muscles in concentration dependence, characteristics of the response, and susceptibility to ET-receptor antagonists. Anomalous pharmacological characteristics of ET-1-induced response in rabbit papillary muscles may be due to integrated regulatory mechanisms that may involve also various types of noncardiac cell in ventricular myocardium.

Pharmacological characteristics of inhibitory action of the selective alpha1-antagonist JTH-601 on the positive inotropic effect mediated by alpha1-adrenoceptors in isolated rabbit papillary muscle.

Influence of JTH-601 [N-(3-hydroxy-6-methoxy-2,4,5-trimethylbenzyl)-N-methyl-2-(4-hydroxy-2-isopropyl-5-methylphenoxy)ethylamine hemifumarate], a selective alpha1-adrenoceptor antagonist, on alpha1-mediated positive inotropic effect (PIE) was studied in isolated rabbit papillary muscle (1 Hz at 37 degrees C). JTH-601 (0.1-10 microM) shifted the concentration-response curve (CRC) for PIE of phenylephrine mediated by alpha1-adrenoceptor (with timolol at 1 microM) to the right and downward. In the presence of 100 nM WB 4101, an alpha1A antagonist, the shift to the right disappeared and JTH-601 (1-3 microM) shifted CRC for phenylephrine downward. The antagonistic action of JTH-601 was unchanged by 100 nM (+)-niguldipine, another alpha1A antagonist. Following pretreatment with 10 microM chloroethylclonidine, an alpha1B antagonist, the shift of CRC for phenylephrine to the right disappeared and JTH-601 (3-10 microM) shifted CRC downward. Antagonistic action of JTH-601 (3 microM) was unaltered by 100 nM BMY 7378, an alpha1D antagonist. JTH-601 (10 microM) had no effect on beta-mediated PIE of isoproterenol. These results indicate that JTH-601 exerts an inhibitory action on alpha1-mediated PIE through antagonism of alpha1A- and/or alpha1B-adrenoceptors in rabbit ventricular myocardium. As an alpha1 antagonist, JTH-601 is much less potent in rabbit ventricular muscle than in smooth muscle.

Role of Na+/Ca2+ exchange in endothelin-1-induced increases in Ca2+ transient and contractility in rabbit ventricular myocytes: pharmacological analysis with KB-R7943.

1. The effects of endothelin-1 (ET-1) on intracellular Ca2+ ion level and cell contraction were simultaneously investigated in rabbit ventricular cardiac myocytes loaded with indo-1/A1. The role of Na+/Ca2+ exchange in ET-1-induced positive inotropic effect (PIE) was examined by using KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate), a selective inhibitor of reverse mode Na+/Ca2+ exchange. 2. ET-1 at 0.3 pM - 1 nM increased cell contraction and Ca2+ transient (CaT) with EC50 values of 2.9 pM and 1.2 pM, respectively, and the increase in amplitude of CaT was much smaller relative to the PIE: ET-1 at 1 nM increased peak cell shortening by 237%, while it increased peak CaT by 167%. For a given level of PIE, ET-1-induced increase in CaT was much smaller than that induced by elevation of [Ca2+]o and by isoprenaline. Therefore, ET-1 shifted the relationship between peak CaT and cell shortening to the left relative to the relationship for increase in [Ca2+]o, an indication that ET-1 increased myofibrillar Ca2+ sensitivity. 3. KB-R7943 at 0.1 microM and higher inhibited contraction and CaT induced by 0.1 nM ET-1 and at 0.3 microM it abolished the increase in CaT while inhibiting the PIE by 48.1%. Over concentration range of 0.1-0.3 microM, KB-R7943 neither inhibited baseline contraction and CaT nor the isoprenaline-induced response, although at 1 microM and higher it had a significant inhibitory action on these responses. 4. These results indicate that in rabbit ventricular myocytes both increases in CaT and myofibrillar Ca2+ sensitivity contribute to the ET-induced PIE, and the activation of reverse mode Na+/Ca2+ exchange may play a crucial role in increase in CaT induced by ET-1 in rabbit ventricular cardiac myocytes.

Differential regulation of intracellular Ca2+ signalling induced by high K+ and endothelin-1 in single smooth muscle cells of intact canine basilar artery: detection by means of confocal laser microscopy.

Changes in intracellular calcium concentration ([Ca2+]i) in smooth muscle cells play the key role in regulation of vascular smooth muscle tone and pathogenesis of cerebral vasospasm. In this study, we adopted the confocal laser microscopy to detect the fluorescence signals arising from the individual smooth muscle cells of canine basilar artery. Ring preparations were made, loaded with fluo-3 and changes in fluorescence induced by high K+ and endothelin-1 (ET-1) were measured by confocal laser microscopy. In some unstimulated smooth muscle cells Ca2+ waves arising from discrete region of the cell propagated to the whole cell with a velocity of approximately 10 microm/s. High K+ (80 mmol/L) induced a rapid rise in [Ca2+]i, the peak level being consistently reached approximately 10 s after stimulation. In contrast, the time to peak level of [Ca2+]i induced by ET-1 (0.3 micromol/L) varied widely between 13 and 26 s among individual cells, an indication that the extent of nonuniform coordination of increases in [Ca2+]i in individual cells may be partly responsible for the different time courses of tension development of vascular smooth muscle in response to the vasoactive stimulants. The increase in [Ca2+]i induced by ET-1 was transient but a pronounced and sustained contraction developed further in response to ET-1. Thus ET-1 has a biological property as a potential candidate to elicit cerebral vasospasm. Confocal laser microscopy could be a useful tool to measure the changes in [Ca2+]i in individual smooth muscle cells of cerebral artery.

Endothelin: receptor subtypes, signal transduction, regulation of Ca2+ transients and contractility in rabbit ventricular myocardium.

Endothelin (ET) isopeptides, ET-1, ET-2 and ET-3, elicit a positive inotropic effect (PIE) in association with a negative lusitropic effect, essentially with identical efficacies and potencies in the isolated rabbit papillary muscle, but with different concentration-dependent properties. Pharmacological analysis indicates that the PIE of ET-1 is mediated by an ETA2 subtype that is less sensitive to BQ-123 and FR139317, whereas the PIE of ET-3 is mediated by an ETA1 subtype that is highly sensitive to these ETA antagonists. ETs increased the amplitude of intracellular Ca2+ transient (CaT) in indo-1 loaded rabbit ventricular myocytes, but the increase was much smaller than that produced by elevation of [Ca2+]o or isoproterenol for a given extent of PIE, an indication of increased myofibrillar Ca2+ sensitivity. ETs stimulate phosphoinositide (PI) hydrolysis, which leads to production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Evidence for the role of IP3-induced Ca2+ release in cardiac E-C coupling is tenuous. Generation of IP3 induced by ET-1 was transient and returned to the baseline level when the PIE reached an elevated steady level. Protein kinase C (PKC) that is activated by DAG and also via other pathways triggered by ETs stimulates Na+-H+ exchanger to lead to an increased [Na+]i and alkalinization. The former may contribute to an increase in the amplitude of CaT through Na+-Ca2+ exchanger, and the latter, to an increase in myofibrillar Ca2+ sensitivity. A number of PKC inhibitors, such as staurosporine, H-7, calphostin C and chelerythrine, consistently and selectively inhibited the PIE of ET-3 without affecting the PIE of isoproterenol and Bay k 8644. The maximum inhibition was 20-30% of the total response. A Na+-H+ exchange inhibitor, [5-(N-ethyl-N-isopropyl) amiloride (EIPA)] or a Ca2+ antagonist, verapamil, could not completely inhibit the PIE of ET-3, but the combination of both inhibitors totally abolished the PIE of ET-3. These findings indicate that activation of PKC and subsequent activation of Na+-H+ exchanger and/or L-type Ca2+ channels may play a crucial role in the cardiac action of ET isopeptides in the rabbit ventricular myocardium.

Pharmacological characteristics of endothelin receptors in the rabbit ventricular myocardium: the nonselective endothelin receptor antagonist PD 145065 antagonizes the positive inotropic effect of endothelin-3 but not of endothelin-1.

Endothelin-3 (ET-3) elicited a concentration-dependent positive inotropic effect on rabbit papillary muscle, the maximal response being approximately 65% of the maximal response to isoproterenol. ET-1 induced a positive inotropic effect over the concentration range below 10(-9) M, at which ET-3 did not produce a positive inotropic effect, but the maximal response to ET-1 was equivalent to or slightly lower than that of ET-3. The nonselective ET receptor antagonist PD 145065 effectively antagonized the positive inotropic effect of ET-3 in a concentration-dependent manner and abolished it at 10(-5) M. PD 145065 decreased the positive inotropic effect induced by ET1 at lower concentrations (< 10(-9) M) but it did not affect the main portion of the concentration-response curve for the positive inotropic effect, i.e., the effect induced by high concentrations (> 10(-9) M) of ET-1. PD 145065 antagonized also the positive inotropic effect of sarafotoxin S6c. PD 145065 inhibited the specific binding of [125I]ET-1 and of [125I]ET-3 with a high- and a low-affinity site for competition. ETB selective ligands, RES-701-1 and sarafotoxin S6c, displaced [125Iuc]ET-3 with high affinity but they scarcely affected the [125I]ET-1 binding. These findings indicate that different subtypes of the ET receptor are responsible for the induction of the positive inotropic effect of ET-3 and ET-1. ET receptors involved in the production of the positive inotropic effect in the rabbit ventricular myocardium have pharmacological characteristics that are different from those of conventional ET receptors originally classified based on the pharmacological findings in noncardiac tissues. The positive inotropic effect of ET-3 in the rabbit ventricular muscle may be mediated predominantly by ETA1 receptors that are susceptible to PD 145065 as well as BQ-123 and FR139317, and partially mediated by ETB receptors that are inhibitable with RES-701-1. ETA2 receptors that are resistant to ETA selective as well as nonselective antagonists may mainly be responsible for the positive inotropic effect of ET-1 in the rabbit ventricular muscle.

The positive inotropic effect and the hydrolysis of phosphoinositide induced by endothelin-3 in rabbit ventricular myocardium: inhibition by a selective antagonist of ET(A) receptors, FR139317.

Endothelin-3 (ET-3), an isopeptide of ET, had a concentration-dependent positive inotropic effect (PIE) on rabbit papillary muscle. The maximal inotropic response to ET-3 was 65% of the maximal response to isoproterenol. ET-1 elicited a PIE below 10(-9) M, namely, over a concentration range at which ET-3 did not elicit a PIE. The selective ET(A) antagonist FR139317 effectively antagonized the PIE of ET-3. FR139317 abolished the PIE induced by ET-1 (<10(-9)M) but did not inhibit the PIE induced by high concentrations of ET-1. FR139317 also antagonized the PIE of sarafotoxin S6c. ET-3 caused a time- and concentration-dependent increase in [3H]inositol phosphates (inositol monophosphate, inositol bisphosphate and inositol trisphosphate). FR139317 at 10(-5) M decreased the ET-3-induced increase in inositol phosphates by about 60%, whereas it attenuated the increase in [3H]IP1 induced by ET-1 (3 x 10(-8)M) by only 20%. Thus, in the presence of FR139317, the PIEs of ET-3 and ET-1 were partially dissociated from the PI hydrolysis that was induced by these isopeptides. FR139317 inhibited the specific binding of [125I]ET-1 and of [125I]ET-3, and it was apparent that FR139317 had a high-affinity and a low-affinity site for competing for specific binding with each ligand. These findings indicate that different subtypes of the ET receptor are involved in the induction of the PIEs of ET-3 and ET-1. The PIE of ET-3 may be mediated predominantly by ET(A1) receptors that are susceptible to FR139317 and BQ-123 and partially by ET(B) receptor that are inhibited by RES-701-1. Both ET(A1) and ET(A2) receptors may be responsible for the PIE of ET-1, depending on the concentration in rabbit ventricular myocardium.

Methoxamine-induced inhibition of the positive inotropic effect of endothelin via alpha1-adrenoceptors in the rabbit heart.

The influence of methoxamine on the positive inotropic effect of endothelin was assessed in the isolated rabbit ventricular myocardium. Methoxamine by itself elicited a positive inotropic effect and it simultaneously inhibited the positive inotropic effects of endothelin-1 and endothelin-3 without affecting the acceleration of the hydrolysis of phosphoinositide that was induced by the endothelin isopeptides. By contrast, the positive inotropic effects induced by elevation of concentration of external Ca2+ ions, by Bay k 8644 (methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5- carboxylate), by dihydroouabain and by forskolin were unaffected by methoxamine. The inhibitory action of methoxamine was abolished by alpha1-adrenoceptor antagonists, such as prazosin, WB 4101 (2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane hydrochloride) and (+/-)-tamsulosin; and it was inhibited to a lesser extent by chlorethylclonidine. In addition, methoxamine did not modify the specific binding of [125I]endothelin-3 to ventricular membrane fraction. These results indicate that methoxamine antagonizes the positive inotropic effect of endothelin isopeptides at the level of the signal-transduction process, subsequent to acceleration of the hydrolysis of phosphoinositide, via activation of alpha1-adrenoceptors in the rabbit ventricular myocardium.

The G-protein G(o) in mammalian cardiac muscle: localization and coupling to A1 adenosine receptors.

To investigate the functional relevance of the G-protein G(o) to adenosine-induced effects in the heart, we studied the localization of G(o) and its interaction with A1 adenosine receptors. Concentrations of G(o) in various mammalian hearts differed markedly between the atrial and the ventricular muscle, as well as among species. In most species examined, the concentration of G(o) was much higher in the atrium than in the ventricle. The highest levels of G(o) in atria and ventricles were found in the ferret heart. An immunohistochemical study of the ferret heart with G(o) alpha-specific antibodies showed that G(o) was localized throughout the membranes of cardiac myocytes, including the intercalated disks. In addition, G(o) was densely distributed in the nerve fibers and Purkinje fibers. Analyses of G(o) alpha subtypes showed that bovine atrium mainly contained G(o)A alpha, while bovine ventricle contained only G(o)B alpha. By contrast, ferret ventricle contained both subtypes of G(o) alpha. To study the coupling of G(o) to A1 adenosine receptors, receptors in ventricular membranes of ferrets, which had been pretreated with pertussis toxin, or purified receptors were reconstituted with purified G(o)A and G(o)B. The reconstitution experiments indicated that both subtypes of G(o) coupled with A1 adenosine receptors. These results suggest that the effect of adenosine is mediated by both G(o)A and G(o)B in membranes of ferret cardiac myocytes.

Effects of a novel cardiotonic agent (+-)-6-[3-(3,4-dimethoxybenzylamino)-2-hydroxypropoxy]-2(1H)-quinolino ne (OPC-18790) on contractile force, cyclic AMP level, and aequorin light transients in dog ventricular myocardium.

We studied the effects of a novel cardiotonic agent OPC-18790 [(+-)-6-[3-(3,4-dimethoxybenzylamino)-2-hydroxypropoxy]-2(1H)- quinolinone] on isometric contractions, intracellular aequorin light transients, and cyclic AMP levels in isolated dog ventricular trabeculae. The positive inotropic effect (PIE) of OPC-18790 (1-30 microM) was consistently associated with an abbreviation of contractions and an increase in the amplitude of aequorin light transients. The maximum responses of Ca2+ transients and force to OPC-18790 were approximately 40% of the isoproterenol-induced maximum. Carbachol (3 microM) markedly attenuated the increases in force, light transients, and cyclic AMP accumulation induced by OPC-18790. These results indicate that OPC-18790 is a cardiotonic agent with moderate effectiveness, and that the PIE of OPC-18790 may be produced mainly by an increase in intracellular Ca2+ transients induced by cyclic AMP accumulation. For a given increase in amplitude of Ca2+ transients, OPC-18790 produced a more pronounced increase in force of contraction (FOC) than did isoproterenol, suggesting that OPC-18790 does not produce as great a decrease in Ca2+ sensitivity of contractile proteins as does isoproterenol. These observations indicate that among cardiotonic agents acting through cyclic AMP pathway, regulation of contractility produced by the selective cyclic AMP phosphodiesterase III (PDE-III) inhibitor OPC-18790 is qualitatively different from the regulation induced by isoproterenol that acts on cyclic AMP generation in intact myocardial cells.

Pronounced direct inhibitory action mediated by adenosine A1 receptor and pertussis toxin-sensitive G protein on the ferret ventricular contraction.

An adenosine A1 receptor agonist R-N6-phenylisopropyladenosine (R-PIA) elicited a pronounced negative inotropic effect with the EC50 value of 0.69 mumol/l in the presence of a beta-adrenoceptor blocking agent bupranolol (0.3 mumol/l) in the isolated ferret papillary muscle. The negative inotropic effect of R-PIA was not associated with changes in cyclic AMP level. Adenosine and other A1 receptor agonists also elicited a negative inotropic effect. DPCPX (1,3-dipropyl-8-cyclopentyl xanthine) antagonized the negative inotropic effect of R-PIA in a competitive manner (pA2 value = 8.4). The inhibitory action of R-PIA was markedly attenuated in the ventricular muscle preparation isolated from ferrets pretreated with pertussis toxin that caused ADP-ribosylation of 39 kDa proteins in the membrane fraction. In the membrane fraction derived from the ferret ventricle, [3H]-DPCPX bound to a single binding site in a saturable and reversible manner with high affinity (Kd value = 1.21 +/- 0.41 nmol/l; Bmax = 12.8 +/- 3.02 fmol/mg protein; n = 7). The binding characteristics of [3H]-DPCPX in the rat ventricle (Kd value = 1.51 +/- 0.09 nmol/l; Bmax = 12.7 +/- 1.47 fmol/mg protein; n = 5) were similar to those in the ferret. On the other hand, the content of G(o), a major pertussis toxin-sensitive G protein in the ferret heart, was much higher in the ferret than in the rat ventricle. The present results indicate that adenosine receptors may play an important role in the inhibitory regulation of ventricular contractility in the ferret in contrast to other mammalian species. The signal transduction process subsequent to agonist binding to A1 receptors including the pertussis toxin-sensitive G protein and ion channels may be responsible for the unique inhibitory action of adenosine in this species.

Inotropic effects of staurosporine, NA 0345 and H-7, protein kinase C inhibitors, on rabbit ventricular myocardium: selective inhibition of the positive inotropic effect mediated by alpha 1-adrenoceptors.

The influence of protein kinase C (PKC) inhibitors, staurosporine, NA 0345 and H-7, on the alpha 1- and beta-adrenoceptor-mediated positive inotropic effect (PIE) was studied in rabbit ventricular myocardium. Staurosporine (1-10 nM), NA 0345 (10-100 nM) and H-7 (1-10 microM) selectively attenuated the PIE mediated by alpha 1-adrenoceptors at concentrations that did not affect the beta-mediated PIE and basal force of contraction. Staurosporine at higher concentrations (> 10 nM) decreased the basal force, while NA 0345 and H-7 did not. In membrane fractions derived from rabbit ventricular muscle, neither staurosporine, NA 0345 nor H-7 modified the specific [3H]prazosin binding at the concentrations that elicited the functional modulation. Accumulation of [3H]inositol monophosphate (IP1) induced by alpha 1-adrenoceptor stimulation was not affected by the PKC inhibitors. Phorbol 12,13-dibutyrate (PDBu), a PKC activator, also selectively attenuated the alpha 1-mediated PIE, but in association with the inhibition of the alpha 1-mediated IP1 accumulation. Staurosporine (1 nM), but not H-7, antagonized the PDBu-induced inhibitory action on the alpha 1-mediated PIE. These findings indicate that staurosporine, NA 0345 and H-7 produce a selective inhibition of the alpha 1-mediated PIE, probably through inhibition of the alpha 1-adrenoceptor-mediated activation of PKC. On the contrary, externally administered phorbol ester may act by uncoupling of alpha 1-adrenoceptors to activation of phospholipase C through a pathway different from endogenous diacylglycerol to lead to a selective inhibition of the alpha 1-mediated PIE.

Selective inhibition by phorbol 12,13-dibutyrate of the alpha 1-receptor-mediated positive inotropic effect.

Influence of the protein kinase C activator phorbol 12,13-dibutyrate on the alpha 1- and beta-adrenoceptor-mediated positive inotropic effect was studied in the rabbit ventricular myocardium. Phorbol 12,13-dibutyrate (10(-8)-10(-6) M) inhibited the positive inotropic effect mediated by alpha 1-adrenoceptors in a concentration-dependent manner, while the positive inotropy mediated by beta-adrenoceptors was not affected by phorbol 12,13-dibutyrate up to 3 x 10(-7) M. Phorbol 12,13-dibutyrate at 10(-6) M decreased the beta-mediated effect, but the extent of inhibition was less than that of alpha 1-mediated effect produced by 10(-8) M phorbol 12,13-dibutyrate. Thus, the inhibition induced by phorbol 12,13-dibutyrate was 100-fold more selective for alpha 1- than for beta-mediated inotropy. Phorbol 12,13-dibutyrate at 10(-7) M increased the basal force of contraction in some preparations, but decreased it at 3 x 10(-7) M and higher in a concentration-dependent manner. In membrane fractions derived from the rabbit ventricular muscle, phorbol 12,13-dibutyrate did not affect the specific binding of [3H]prazosin. A nonhydrolyzable GTP analogue GTP gamma S shifted the epinephrine-induced displacement curve of [3H]prazosin to the right, but phorbol 12,13-dibutyrate did not affect the curve. Accumulation of [3H]inositol monophosphate induced by alpha 1 stimulation was inhibited by phorbol 12,13-dibutyrate. These findings indicate that phorbol 12,13-dibutyrate may induce the selective uncoupling of the myocardial alpha 1-receptor stimulation to activation of phospholipase C, and inhibit selectively the alpha 1-mediated positive inotropy.

On the mechanism of action of the beta-1 partial agonist denopamine in regulation of myocardial contractility: effects on myocardial alpha adrenoceptors and intracellular Ca++ transients.

Experiments were carried out to study the effects of denopamine on myocardial alpha-1 adrenoceptors in the rabbit and on intracellular Ca++ transients in the dog ventricular muscle. Denopamine displaced the specific binding of the alpha-1 receptor antagonist [3H]prazosin with high and low affinities in the membrane fraction derived from the rabbit ventricle. The positive inotropic effect (PIE) of denopamine, however, was not affected by prazosin. A beta receptor antagonist bupranolol antagonized the PIE of denopamine in a concentration-dependent manner. In the rabbit denopamine acted as a beta receptor partial agonist with an intrinsic activity of 0.8 and shifted the concentration-response curve for isoproterenol to the right (Kp = 1.5 microM). In addition, denopamine attenuated the maximal inotropic response to phenylephrine mediated by alpha-1 receptors, but did not affect the pD2 value for phenylephrine. In isolated dog right ventricular muscle, the bell-shaped concentration-response relationship for the inotropic effect of denopamine was associated with coinciding increase and decrease in the amplitude of aequorin light transients. The relationship between the increase in peak Ca++ transients and developed tension in response to denopamine was the same as the relation during administration of isoproterenol. The present results indicate that denopamine binds myocardial alpha-1 adrenoceptors with high affinity and may thereby inhibit the maximal response of phenylephrine mediated by alpha-1 receptors. The PIE of denopamine is mediated exclusively by beta receptors. The change in Ca++ sensitivity caused by denopamine may not be different from that induced by a beta receptor full agonist isoproterenol.

Effect of (+)-niguldipine on myocardial alpha 1-adrenoceptors in the rabbit.

The influence of the alpha 1A-adrenoceptor subtype-selective antagonist (+)-niguldipine on the alpha 1-mediated positive inotropic effect was assessed in the isolated rabbit ventricular myocardium. (+)-Niguldipine displaced the specific binding of [3H]prazosin to a membrane fraction derived from rabbit ventricular muscle with high (Ki = 64.6 pmol/l; RH = 23%) and low (Ki = 7.08 nmol/l) affinity. (+)-Niguldipine displaced specific [3H]CGP-121177 binding only at very high concentrations (Ki = 118 nmol/l). (+)-Niguldipine at 0.1 pmol/l and higher shifted the concentration-response curve for the alpha 1-mediated positive inotropic effect downwards, but at higher concentrations (up to 10 and 100 nmol/l) it did not cause a further shift of the curve. (+)-Niguldipine (1-100 nmol/l) did not affect the beta-mediated positive inotropic effect and the basal force of concentration. (-)-Niguldipine also showed a selective inhibitory action on the alpha 1-adrenoceptor-mediated positive inotropic effect, but its affinity and potency were approximately 1-2 log units lower than those of (+)-niguldipine. The present results indicate that the alpha 1A-adrenoceptor subtype is involved in the alpha 1-mediated positive inotropic effect. (+)-Niguldipine (or (-)-niguldipine with lower affinity) is able to antagonize selectively the cardiac alpha 1A-adrenoceptor-mediated positive inotropic effect. The magnitude of the alpha 1A-mediated inotropic effect, however, may be much less than that mediated by the alpha 1B-subtype in the rabbit ventricular myocardium.

Effects of vasopressin on phosphoinositide hydrolysis and myocardial contractility.

The effects of vasopressin on phosphoinositide hydrolysis, ventricular contractility and adrenoceptor-mediated inotropy were studied in the rabbit. Vasopressin caused an accumulation of [3H]inositol monophosphate in ventricular slices prelabelled with myo-[3H]inositol, whereas it elicited a small but definite negative inotropic effect. In addition, vasopressin attenuated the positive inotropic effect elicited via alpha 1- and endothelin receptors without affecting the beta-adrenoceptor-mediated effect. The nonpeptide-selective V1 receptor antagonist OPC 21268 (a quinolinone derivative) inhibited the vasopressin-induced accumulation of [3H]inositol monophosphate. The present results indicate that vasopressin stimulates phosphoinositide hydrolysis via V1 receptors, but inhibits force and the alpha 1-mediated positive inotropic effect in the rabbit ventricular myocardium.

Role of alpha 1A adrenoceptor subtype in production of the positive inotropic effect mediated via myocardial alpha 1 adrenoceptors in the rabbit papillary muscle: influence of selective alpha 1A subtype antagonists WB 4101 and 5-methylurapidil.

In order to elucidate the contribution of alpha 1A subtype to the positive inotropic effect mediated by myocardial alpha 1 adrenoceptors, the influence of the alpha 1A selective antagonists WB 4101 and 5-methylurapidil on the alpha 1-mediated positive inotropic effect (induced by phenylephrine in the presence of a beta adrenoceptor blocking agent bupranolol) was assessed in the isolated rabbit papillary muscle. WB 4101 (10(-9)-10(-7) mol/l) shifted the concentration-response curve of the alpha 1-mediated positive inotropic effect to the right in parallel, but the slope of Schild plot did not meet the competitive antagonism: WB 4101 shifted the curve by log one unit at 10(-9) mol/l, whereas it did not cause further shift at higher concentrations of 10(-8) and 10(-7) mol/l. WB 4101 did not affect the beta adrenoceptor-mediated positive inotropic effect. 5-Methylurapidil (10(-9) to 10(-7) mol/l) shifted the curve of alpha 1-mediated positive inotropic effect to the right and downwards in a concentration-dependent manner; the slope of Schild plot calculated at the level of 20% of the maximum response to phenylephrine was close to unity. 5-Methylurapidil at 3 x 10(-7) mol/l abolished the alpha 1-mediated positive inotropic effect. In addition, 5-methylurapidil inhibited the beta adrenoceptor-mediated positive inotropic effect in the same concentration range as it antagonized the alpha 1-mediated positive inotropic effect, indicating that 5-methylurapidil is not selective for myocardial alpha 1 adrenoceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological characteristics of adenosine-induced inhibition of dog ventricular contractility: dependence on the pre-existing level of beta-adrenoceptor activation.

Experiments were carried out to characterize the adenosine-induced negative inotropic effect in relation to the extent of beta-adrenoceptor activation in the isolated dog left ventricular myocardium. Adenosine and R-N6-phenylisopropyladenosine inhibited the positive inotropic effect of isoprenaline (10(-7) mol/l and lower) about 20% of its maximal response, which was antagonized by an A1 adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine in a concentration-dependent manner. The negative inotropic effect of adenosine disappeared and that of R-N6-phenylisopropyl-adenosine decreased when the isoprenaline concentration was elevated to the level higher than 10(-7) mol/l. Adenosine deaminase (1.5 U/ml) that abolished the negative inotropic effect of adenosine enhanced the effect of R-N6-phenylisopropyladenosine, indicating that endogenous adenosine released by high isoprenaline concentration (10(-6) mol/l) modulates the interaction. The maximal response to adenosine and R-N6-phenylisopropyladenosine determined in the presence of 10(-7) mol/l isoprenaline was 50% of that of carbachol which elicited the maximal inhibition even in the presence of 10(-6) mol/l isoprenaline. The negative inotropic effects of R-N6-phenylisopropyladenosine and carbachol were additive to the maximal response equivalent to that of carbachol. The difference in the efficiency between the adenosine and muscarinic receptor agonists may be partly ascribed to the difference in densities of the respective receptors in the dog ventricular myocardium. The negative inotropic effect of R-N6-phenylisopropyladenosine in the presence of isoprenaline was associated with decrease in cyclic AMP levels elevated previously by isoprenaline.(ABSTRACT TRUNCATED AT 250 WORDS)

Potent inhibitory action of chlorethylclonidine on the positive inotropic effect and phosphoinositide hydrolysis mediated via myocardial alpha 1-adrenoceptors in the rabbit ventricular myocardium.

The influence of the alpha 1b-adrenoceptor-selective antagonist chlorethylclonidine on the alpha 1-adrenergic positive inotropic effect and the phosphoinositide hydrolysis induced by phenylephrine was investigated in the rabbit ventricular myocardium. Pretreatment of membrane fractions derived from the rabbit ventricular muscle with 10(-5) mol/l chlorethylclonidine decreased the specific binding of [3H]prazosin (at a saturating concentration of 10(-9) mol/l) from the control value of 11.27 +/- 0.48 to 4.18 +/- 1.87 fmol/mg protein. The inhibition by adrenaline of the binding of [3H]prazosin (slope factor and affinity) was not affected by chlorethylclonidine. The positive inotropic effect of phenylephrine (in the presence of 3 x 10(-7) mol/l bupranolol) was inhibited by chlorethylclonidine in a concentration-dependent manner (10(-7)-10(-5) mol/l) and abolished by 10(-5) mol/l chlorethylclonidine. The concentration of chlorethylclonidine to inhibit the phenylephrine-induced maximum response to 50% was 2.4 x 10(-6) mol/l. The accumulation of [3H]inositol monophosphate and [3H]inositol trisphosphate induced by 10(-5) mol/l phenylephrine was inhibited by chlorethylclonidine in the same concentration range. These findings indicate that the myocardial alpha 1-adrenoceptors mediating a positive inotropic effect in the rabbit ventricular myocardium may belong to the chlorethylclonidine-sensitive alpha 1b-subtype, and that the subcellular mechanism of action involve phosphoinositide hydrolysis.

Different mechanisms involved in the positive inotropic effects of benzimidazole derivative UD-CG 115 BS (pimobendan) and its demethylated metabolite UD-CG 212 Cl in canine ventricular myocardium.

UD-CG 115 BS produced a positive inotropic effect in a concentration-dependent manner (EC50 = 9.2 x 10(-5) M, efficacy = 0.65) in isolated canine ventricular muscle. UD-CG 212 Cl also elicited a positive inotropic effect (EC50 = 1.9 x 10(-7) M, efficacy = 0.23); its potency was higher, but its efficacy was much less than that of UD-CG 115 BS. Although the effect of UD-CG 115 BS was not altered by a beta-adrenoceptor antagonist, bupranolol (3 x 10(-7) M), the response to UD-CG 212 Cl in high concentrations became transient in the presence of bupranolol: After reaching a peak, the force decreased gradually to the control level at greater than or equal to 10(-4) M. Both UD-CG 115 BS and UD-CG 212 Cl elevated the cyclic AMP level, but to a much smaller extent than other newly developed cardiotonic agents such as amrinone, milrinone, enoximone, and piroximone. Carbachol (3 x 10(-6) M) abolished the accumulation of cyclic AMP produced by these agents while it suppressed the maximum contractile response to UD-CG 115 BS by only 30%. The positive inotropic effect of UD-CG 212 Cl was converted to a negative effect by carbachol. Both UD-CG 115 BS and UD-CG 212 Cl produced a leftward shift in the concentration-response curve for the positive inotropic effect of isoproterenol. These results suggest that an elevation of cyclic AMP levels owing to cyclic AMP phosphodiesterase inhibition may be predominantly responsible for the positive inotropic effect of UD-CG 212 Cl but that a cyclic AMP-independent mechanism may contribute significantly to the positive inotropic effect of UD-CG 115 BS. UD-CG 212 Cl (greater than 3 x 10(-6) M) elicits a negative inotropic effect that is unmasked by beta-adrenoceptor blockade.