Hong-Xin Wang

Effects of κ-opioid receptor stimulation in the heart and the involvement of protein kinase C

The role of protein kinase C (PKC) in mediating the action of κ‐receptor stimulation on intracellular Ca2+ and cyclic AMP production was determined by studying the effects of trans‐(±)‐3,4‐dichloro‐N‐methyl‐N‐(2‐[1‐pyrrolidinyl] cyclohexyl) benzeneacetamide methanesulphonate (U50,488H), a selective κ‐receptor agonist, and phorbol 12‐myristate 13‐acetate (PMA), a PKC agonist, on the electrically‐induced [Ca2+]i transient and forskolin‐stimulated cyclic AMP accumulation in the presence and absence of a PKC antagonist, staurosporine or chelerythrine, in the single rat ventricular myocyte. U50,488H at 2.5–40 μM decreased both the electrically‐induced [Ca2+]i transient and forskolin‐stimulated cyclic AMP accumulation dose‐dependently, effects which PMA mimicked. The effects of the κ‐agonist, that were blocked by a selective κ‐antagonist, nor‐binaltorphimine, were significantly antagonized by the PKC antagonists, staurosporine and/or chelerythrine. The results indicate that PKC mediates the actions of κ‐receptor stimulation. To determine whether the action of PKC was at the sarcoplasmic reticulum (SR) or not, the [Ca2+]i transient induced by caffeine, that depletes the SR of Ca2+, was used as an indicator of Ca2+ content in the SR. The caffeine‐induced [Ca2+]i transient was significantly reduced by U50,488H at 20 μM. This effect of U50,488H on caffeine‐induced [Ca2+]i transient was significantly attenuated by 1 μM chelerythrine, indicating that the action of PKC involves mobilization of Ca2+ from the SR. When the increase in IP3 production in response to κ‐receptor stimulation with U50,488H in the ventricular myocyte was determined, the effect of U50,488H was the same in the presence and absence of staurosporine, suggesting that the effect of PKC activation subsequent to κ‐receptor stimulation does not involve IP3. The observations suggest that PKC may act directly at the SR. In conclusion, the present study has provided evidence for the first time that PKC may be involved in the action of κ‐receptor stimulation on Ca2+ in the SR and cyclic AMP production, both of which play an essential role in Ca2+ homeostasis in the heart.

Inhibition of [3H]‐U69593 binding and the cardiac effects of U50,488H by calcium channel blockers in the rat heart

The calcium channel blockers (CCBs), nifedipine, nicardipine, diltiazem and verapamil, were used to displace the binding of [3H]‐U69593 ((5a,7a,8b)‐(+)‐N‐methyl‐N‐(7‐[1‐pyrrolidinyl]‐1‐oxaspiro[4,5]dec‐8‐yl)‐benzeneacetamide), a specific κ‐opioid agonist, in the rat cardiac sarcolemma. The CCBs competed with the binding of [3H]‐U69593 (4 nm) in a dose‐dependent manner. The displacing potency of verapamil was 55 times greater than that of nifedipine. The effects of two CCBs, verapamil and nifedipine, on the arrhythmogenic action of κ‐receptor stimulation by a specific κ‐receptor agonist, U50,488H (trans‐(±‐3,4‐dichloro‐N‐methyl‐N‐(2‐[1‐pyrrolidinyl] cyclohexyl) benzeacetamide methanesulphonate), were also studied in the rat isolated perfused heart. U50,488H 80–800 nmol dose‐dependently induced arrhythmias, which were completely abolished by a selective κ‐receptor antagonist, nor‐BNI (nor‐binaltorphimine,17,17′‐(dicyclopropylmethyl)‐6,6′,7,7′‐6,6′‐imino‐7,7′‐binorphinan‐3,4′,14, 14′‐tetrol), at 100 nmol. The arrhythmogenic effect was also attenuated by both verapamil and nifedipine in a dose‐dependent manner. The ED50 values for verapamil and nifedipine were 2.75 and 63.7 nmol, respectively. The antiarrhythmic potencies of these two CCBs were correlated to their displacing potencies and inversely related to their well known potencies in inhibiting transmembrane Ca2+ influx in the cardiac muscle. Measurement of [Ca2+]i in the absence of free extracellular Ca2+ by a spectrofluorometric method, with fura‐2 as Ca2+ indicator, showed that U50,488H 5×10−5 m slowly increased [Ca2+]i in single ventricular myocytes and this effect was abolished by pretreatment with nor‐BNI (5 μm), or ryanodine (5 μm). Verapamil 1 and 10 μm abolished the effect of U50,488H in 37.5% (3 out of 8) and 100% (12 out of 12) of the cells studied, respectively. On the other hand, nifedipine 10 and 100 μm had no effect at all. Neither verapamil nor nifedipine exerted any significant effect on the caffeine‐induced Ca2+ transient. The observations suggest that CCBs may inhibit the actions of κ‐receptor stimulation at the level of the κ‐receptor.

Tetrandrine inhibits electrically induced [Ca2+]i transient in the isolated single rat cardiomyocyte

The effect of tetrandrine on the electrically induced elevation of cytosolic Ca2+ concentration, [Ca2+]i, in the single isolated rat cardiomyocyte was studied with a fluorometric ratio method using fura-2 acetomethylester (fura-2/AM) was Ca2+ indicator. Tetrandrine (3-100 microM) concentration and time dependently inhibited the amplitude of the [Ca2+]i transient without any significant effect on the resting level of [Ca2+]i. At high concentrations (60-100 microM), tetrandrine also prolonged the time to reach the peak (t1.0) and the time to decline the 20% of the peak level (t0.2) of the electrically induced [Ca2+]i transient. The effect of tetrandrine was fast in onset and fully reversible upon washout. Tetrandrine (10 microM) partially inhibited the elevation of [Ca2+]i in response to KCl-induced depolarization. Verapamil and diltiazem mimicked the effects of tetrandrine given at low concentrations, but not at high concentrations. At high concentrations, tetrandrine reduced the magnitude of the caffeine-induced [Ca2+]i transient. Tetrandrine (100 microM) administered after thapsigargin, which itself decreased the amplitude and prolonged the duration of the electrically induced [Ca2+]i transient, further decreased the amplitude of the [Ca2+]i elevation. After ryanodine, which itself decreased the amplitude of the [Ca2+]i transient, 100 microM tetrandrine not only further reduced the amplitude, but also prolonged the duration of the electrically induced [Ca2+]i transient. These results provide evidence that in addition to its inhibitory effect on Ca2+ influx at the sarcolemma at the therapeutically relevant concentrations, tetrandrine at high concentrations may inhibit Ca2+ uptake into the sarcoplasmic reticulum.

Reduced responsiveness of [Ca2+]i to adenosine A1- and A2-receptor stimulation in the isoproterenol-stimulated ventricular myocytes of spontaneously hypertensive rats.

To determine the modulatory action of adenosine-receptor stimulation on [Ca2+]i responses to beta-adrenoceptor stimulation in the heart of the spontaneously hypertensive rat (SHR), the electrically induced [Ca2+]i transient in response to isoproterenol (ISO) in single ventricular myocytes pretreated with adenosine agonists in SHRs and its normotensive control Wistar-Kyoto (WKY) rats was measured with a spectrofluorometric method by using fura-2/AM as the calcium indicator. In both types of rat, ISO at 0.001-1 microM augmented the electrically induced [Ca2+]i transient, and the effect was blocked by a beta-adrenoceptor blocker, propranolol. In SHRs that did not exhibit cardiac hypertrophy, the resting level of [Ca2+]i and the amplitude of the electrically induced [Ca2+]i transient were the same as those in WKY rats, whereas the augmentation of the electrically induced [Ca2+]i transient in response to ISO was significantly lower than that in WKY rats. In WKY rats, the effects of ISO on the electrically induced [Ca2+]i transient were inhibited by the adenosine A1-receptor agonist, R(-)-N6-(2-phenylisopropyl)adenosine (R-PIA) at 0.01-10 microM. In contrast, the effects of ISO were further enhanced by the adenosine A2-receptor agonist, N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl)]adenosine (DPMA) at 1-10 microM. In SHRs, the inhibitory effect of R-PIA was significantly reduced, whereas the excitatory effect of DPMA was absent. The effects of both adenosine-receptor agonists in both types of rat were abolished by the respective adenosine-receptor antagonists, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and 3,7-dimethyl-1-propargylxanthine (DMPX). The results indicate that the modulatory actions of adenosine-receptor stimulation on [Ca2+]i response to beta-adrenoceptor stimulation in the hearts of SHRs are reduced, which is independent of cardiac hypertrophy.

Reduced inhibitory actions of adenosine A1 and kappa 1-opioid receptor agonists on beta-adrenoceptors in spontaneously hypertensive rat heart.

1. The modulatory actions of both adenosine A1 and K1‐opioid receptor agonists on β‐adrenoceptor stimulation in the heart of both spontaneously hypertensive rats (SHR) and nor‐motensive Wistar‐Kyoto (WKY) rats were compared.

High carbachol increases the electrically induced [Ca2+]i transient in the single isolated ventricular myocyte of rats

In order to investigate the mechanisms responsible for the inotropic effects of muscarinic acetylcholine receptor stimulation by high concentrations of muscarinic receptor agonists, we studied the effects of carbachol at 30-300 microM on the electrically induced [Ca2+]i transient of rat isolated ventricular myocytes. Carbachol at this dose range increased the amplitude and duration of the electrically induced [Ca2+]i transient time and dose dependently. It also increased the resting fluorescence ratio and time to 80% decline of amplitude from the peak. At 100-300 microM the increase in [Ca2+]i transient was followed by a cluster of Ca2+ oscillations in 50-83% of the cells studied. The effects were blocked by atropine, but not pertussis toxin. Depletion of Ca2+ from sarcoplasmic reticulum by ryanodine, which itself reduced the amplitude of the [Ca2+]i transient and increase resting fluorescence, abolished the effect of carbachol on the [Ca2+]i transient without affecting its effect on resting fluorescence ratio. The caffeine-induced [Ca2+]i transient was unaffected by prior addition of carbachol in a Ca2+ free and low Na+ solution. Inhibition of Ca2+ by the L-type Ca2+ channel blocker, verapamil, which itself reduced the amplitude of the [Ca2+]i transient without affecting the resting fluorescence ratio, attenuated the augmentation of the amplitude of the [Ca2+]i transient elicited by carbachol. Ni2+, a non-specific Ca2+ channel blocker and an inhibitor of Na(+)-Ca2+ exchange, abolished the effects of carbachol on both [Ca2+]i transient and resting fluorescence ratio. Low external Na+, which increased the resting fluorescence ratio due to its inhibitory effect on Na(+)-Ca2+ exchange, also abolished the effects of carbachol. The results indicate that the inotropic effect of muscarinic acetylcholine receptor stimulation by high concentrations of a muscarinic receptor agonist may be due to an increase in the electrically induced [Ca2+]i transient in ventricular myocytes via a process which is not pertussis toxin sensitive. The increase in the electrically induced [Ca2+]i transient may result from increases in Na2(+)-Ca2+ exchange and influx of Ca2+ via voltage-gated Ca2+ channels, and mobilization of Ca2+ from the intracellular store. The mobilization of Ca2+ from the intracellular store is a secondary event. The study has provided for the first time that muscarinic acetylcholine receptor stimulation by high concentrations of carbachol increases Ca2+ influx via the Ca2+ channel and mobilization of Ca2+ from its intracellular store. The study has also demonstrated for the first time the occurrence of Ca2+ oscillations induced by high concentrations of carbachol.

DIFFERENT MECHANISMS FOR CA2+I OSCILLATIONS INDUCED BY CARBACHOL AND HIGH CONCENTRATIONS OF CA2+O IN THE RAT VENTRICULAR MYOCYTE

1. The purpose of the present study was to explore the different mechanisms of [Ca2+]i oscillations induced by high concentrations of either carbachol (CCh) or extracellular Ca2+ ([Ca2+]o). First, we compared the oscillations induced by CCh at concentrations of 100–300 μmol/L and [Ca2+]o (5 mmol/L) in the single rat ventricular myocyte. Second, we studied CCh‐ and [Ca2+]o‐induced [Ca2+]i oscillations following either interference with the production of inositol trisphosphate (IP3), reductions in cytosolic Ca2+ ([Ca2+]i), inhibition of Ca2+ influx and Na+–Ca2+ exchange or depletion of Ca2+ from its intracellular store.

ELECTRICALLY INDUCED INTRACELLULAR Ca2+ TRANSIENT IN SINGLE VENTRICULAR MYOCYTES: A USEFUL PARAMETER FOR THE STUDY OF CARDIAC DRUGS

1. Fluorescent Ca2+ indicators, such as fura‐2/AM and calcium green‐1, have become one of the most popular tools for measuring intracellular calcium ([Ca2+]i).