Ikuo Norota

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.

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

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/AM. 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. 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. KB‐R7943 at 0.1 μM and higher inhibited contraction and CaT induced by 0.1 nM ET‐1 and at 0.3 μM it abolished the increase in CaT while inhibiting the PIE by 48.1%. Over concentration range of 0.1–0.3 μM, KB‐R7943 neither inhibited baseline contraction and CaT nor the isoprenaline‐induced response, although at 1 μM and higher it had a significant inhibitory action on these responses. 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.

Signal Transduction and Ca2+ Signaling in Contractile Regulation Induced by Crosstalk Between Endothelin-1 and Norepinephrine in Dog Ventricular Myocardium

Abstract— In certain cardiovascular disorders, such as congestive heart failure and ischemic heart disease, several endogenous regulators, including norepinephrine (NE) and endothelin-1 (ET-1), are released from various types of cell. Because plasma levels of these regulators are elevated, it seems likely that cardiac contraction might be regulated by crosstalk among these endogenous regulators. We studied the regulation of cardiac contractile function by crosstalk between ET-1 and NE and its relationship to Ca2+ signaling in canine ventricular myocardium. ET-1 alone did not affect the contractile function. However, in the presence of NE at subthreshold concentrations (0.1 to 1 nmol/L), ET-1 had a positive inotropic effect (PIE). In the presence of NE at higher concentrations (100 to 1000 nmol/L), ET-1 had a negative inotropic effect. ET-1 had a biphasic inotropic effect in the presence of NE at an intermediate concentration (10 nmol/L). The PIE of ET-1 was associated with an increase in myofilament sensitivity to Ca2+ ions and a small increase in Ca2+ transients, which required the simultaneous activation of protein kinase A (PKA) and PKC. ET-1 elicited translocation of PKC&egr; from cytosolic to membranous fraction, which was inhibited by the PKC inhibitor GF 109203X. Whereas the Na+-H+ exchange inhibitor Hoe 642 suppressed partially the PIE of ET-1, detectable alteration of pHi did not occur during application of ET-1 and NE. The negative inotropic effect of ET-1 was associated with a pronounced decrease in Ca2+ transients, which was mediated by pertussis toxin-sensitive G proteins, activation of protein kinase G, and phosphatases. When the inhibitory pathway was suppressed, ET-1 had a PIE even in the absence of NE. Our results indicate that the myocardial contractility is regulated either positively or negatively by crosstalk between ET-1 and NE through different signaling pathways whose activation depends on the concentration of NE in the dog.

Potent inhibitory action of chlorethylclonidine on the positive inotropic effect and phosphoinositide hydrolysis mediated via myocardial alpha1-adrenoceptors in the rabbit ventricular myocardium

SummaryThe influence of the alphalb-adrenoceptor-selective antagonist chlorethylclonidine on the alpha1-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 × 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 × 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 alpha1-adrenoceptors mediating a positive inotropic effect in the rabbit ventricular myocardium may belong to the chlorethylclonidine-sensitive alpha1b-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 × 10 -5 M, efficacy = 0.65) in isolated canine ventricular muscle. UD-CG 212 Cl also elicited a positive inotropic effect (EC50 – 1.9 × 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 β-adrenoceptor antagonist, bupranolol (3 × 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 ≥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 × 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 carhachol. 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 he predominantly responsible for the positive inotropie 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 (>3 × 10 -6 M) elicits a negative inotropic effect that is unmasked by β-adrenoceptor blockade.

Negative inotropic effects of angiotensin II, endothelin-1 and phenylephrine in indo-1 loaded adult mouse ventricular myocytes

Angiotensin II (Ang II). endothelin-1 (ET-1) and phenylephrine are receptor agonists that share the signal transduction acting through acceleration of phosphoinositide hydrolysis in the heart. Because the regulation of myocardial contractility induced by these receptor agonists shows a wide range of species-dependent variation among experimental animals, we carried out experiments to elucidate the mechanism of contractile regulation induced by these agents in mice which are employed currently more as transgenic models. Effects of Ang II, ET-1 and phenylephrine on cell shortening and Ca2+ transients were investigated in single ventricular myocytes loaded with indo-1/AM. Ang II (10(-8), 10(-7) M), ET-1 (10(-10), 10(-9) M) and phenylephrine (10(-6), 10(-5) M in the presence of the beta-adrenoceptor antagonist timolol) decreased the cell shortening [Ang II: 58.4+/-9.03 (n = 8), 50.3+/-11.90% (n = 6); ET-1: 48.4+/-8.27, 31.2+/-6.45% (n = 5); phenylephrine: 45.7+/-11.60, 28.7+/-5.89% (n = 5)]. By contrast, the amplitude of Ca2+ transients was not significantly influenced by these agonists. The selective protein kinase C inhibitor chelerythrine at 10(-6) M significantly inhibited the decrease in cell shortening induced by these receptor agonists. These results indicate that Ang II, ET-1 and phenylephrine elicit a negative inotropic effect with insignificant alteration of Ca2+ transients, which may be mainly mediated by activation of protein kinase C in mouse ventricular cardiomyocytes.

Role of alpha1A adrenoceptor subtype in production of the positive inotropic effect mediated via myocardial alpha, adrenoceptors in the rabbit papillary muscle: influence of selective alpha1A subtype antagonists WB 4101 and 5-methylurapidil

SummaryIn order to elucidate the contribution of alpha1A subtype to the positive inotropic effect mediated by myocardial alpha, adrenoceptors, the influence of the alpha1A selective antagonists WB 4101 and 5-methylurapidil on the alpha,-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 (10−9-10−7mol/l) shifted the concentration-response curve of the alpha,-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/1, 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 alpha1-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 × 10−7 mol/1 abolished the alpha1-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 alpha1-mediated positive inotropic effect, indicating that 5-methylurapidil is not selective for myocardial alpha, adrenoceptors. In the membrane fraction derived from the rabbit ventricular muscle, 5-methylurapidil displaced the specific binding of [3H]CGP-12177 with high affinity, whereas WB 4101 did not affect the [3H]CGP-12177 binding in the concentration range that it antagonized the alpha,-mediated positive inotropic effect. The present results indicate that alpha1A adrenoceptor subtype plays a role in production of the positive inotropic effect mediated by myocardial alpha, adrenoceptors, but the extent is less than that mediated by alpha1B subtype in the rabbit ventricular myocardium.

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

Summary: 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 μM) 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 ∼40% of the isopro-terenol-induced maximum. Carbachol (3 μM) 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.

Differential action of a protein tyrosine kinase inhibitor, genistein, on the positive inotropic effect of endothelin-1 and norepinephrine in canine ventricular myocardium.

1 Experiments were carried out in isolated canine ventricular trabeculae and acetoxymethylester of indo‐1‐loaded single myocytes to elucidate the role of protein tyrosine kinase (PTK) in the inotropic effect of endothelin‐1 (ET‐1) induced by crosstalk with norepinephrine (NE). The PTK inhibitor genistein was used as a pharmacological tool. 2 Genistein but not daidzein inhibited the positive inotropic effect and the increase in Ca2+ transients induced by ET‐1 by crosstalk with NE at low concentrations. 3 Genistein and daidzein antagonized the negative inotropic effect and the decrease in Ca2+ transients induced by ET‐1 by crosstalk with NE at high concentrations, but genistein did not affect the antiadrenergic effect of carbachol. 4 Genistein but not daidzein enhanced the positive inotropic effect and the increase in Ca2+ transients induced by NE via β‐adrenoceptors, while the enhancing effect of genistein was abolished by the protein tyrosine phosphatase inhibitor vanadate. 5 These findings indicate that genistein (1) induces a positive inotropic effect in association with an increase in Ca2+ transients, (2) inhibits the positive inotropic effect of ET‐1 induced by crosstalk with NE, and (3) enhances the positive inotropic effect of NE induced via β‐adrenoceptors by inhibition of PTK. In addition, genistein inhibits the negative inotropic effect of ET‐1 induced by crosstalk with NE through a PTK‐unrelated mechanism. PTK may play a crucial role in the receptor‐mediated regulation of cardiac contractile function in canine ventricular myocardium.

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

SummaryAn adenosine A1 receptor agonist R-N6-phenylisopropyladenosine (R-PIA) elicited a pronounced negative inotropic effect with the EC50 value of 0.69 μmol/1 in the presence of a β-adrenoceptor blocking agent bupranolol (0.3 μmol/1) 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 Go, 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.