Wei-Min Zhang

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.

Decreased affinity of k-receptor binding during reperfusion following ischaemic preconditioning in the rat heart

The effects of ischaemic preconditioning with three cycles of ischaemia of 3 min and reperfusion of 5 min each cycle on ventricular fibrillation threshold (VFT) and ventricular fibrillation (VF), and binding properties of tritiated U69,593, a selective kappa opioid-receptor (k-receptor) agonist, during subsequent ischaemia and/or reperfusion were studied in the rat heart. It was found that ischaemic preconditioning significantly enhanced the VFT values during ischaemic and reperfusion. VF during the subsequent reperfusion period was also significantly reduced. The Kd of the [3H]U69,593 binding sites in the sarcolemma of the heart at 5 min of reperfusion was significantly increased following ischaemic preconditioning. The Bmax was, however, not altered after the preconditioning. The study provides evidence for the first time suggesting that the cardioprotective effects of ischaemic preconditioning may be related to a reduction in affinity of the K-receptor binding.

k-opioid receptor agonist inhibits the cholera toxin sensitive G-protein in the heart

Summary: To explore the signaling mechanisms of the negative modulation of &bgr;‐adrenoceptors by &kgr;‐opioid receptors (&kgr;‐OR) in the heart, the possibility of the interaction at the level of G protein and receptor was determined. Cholera toxin, an activator of the stimulatory G protein (Gs), elevated electrically induced intracellular Ca2+ ([Ca2+]i) transients and induced ribosylation of the &agr;‐subunit of Gs (Gs&agr;) in rat ventricular myocytes. The effects were significantly attenuated by U50,488H, a specific agonist of &kgr;‐OR, and were abolished by nor‐binaltorphimine, a selective &kgr;‐OR antagonist. The content of Gs&agr;, however, was not affected by U50,488H. Receptor binding experiments showed that neither Bmax nor Kd of the binding of [3H]CGP‐12177, a &bgr;‐adrenoceptor antagonist, was affected by U50,488H. The current study provides the first evidence that &kgr;‐OR stimulation inhibits the ribosylation of the &agr;‐subunit of the Gs protein, thus inhibiting the action of cholera toxin on the protein.

PRO- AND ANTI-ARRHYTHMIC EFFECTS OF A KAPPA OPIOID RECEPTOR AGONIST : A MODEL FOR THE BIPHASIC ACTION OF A LOCAL HORMONE IN THE HEART

1. The effects of κ opioid receptor stimulation on cardiac rhythm and the underlying signal pathways were investigated in the rat.

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.