Molecular cloning and expression of inducible nitric oxide synthase in chick embryonic ventricular myocytes

In order to determine the effect of kappa-opioid receptor agonist on the beta1-adrenoceptor stimulation in the heart, the effects of norepinephrine (NE), a beta1-adrenoceptor agonist, on contraction and electrically induced intracellular calcium ([Ca2+]i) transient in the single rat ventricular myocyte pretreated with a kappa-opioid receptor agonist, trans-(+/-)-3, 4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl)-benzeneacetamide (U50,488H), at 0.01-1 microM were studied with a video edge tracker method and a spectrofluorometric method using fura-2 as calcium indicator, respectively. NE at 0.01-10 microM augmented both twitch amplitude and electrically induced [Ca2+]i transient dose-dependently, which were abolished by propranolol at 1 microM, a beta-adrenoceptor antagonist. The effects of NE on both contraction and [Ca2+]i transient were attenuated in a dose-dependent manner by U50,488H at 0.01-1 microM, which itself had no effect at all. The maximum response ( Emax) was decreased, while the concentration that produces 50% of the maximum response (EC50) was enhanced, by U50, 488H. The inhibitory effects of U50,488H on beta-adrenoceptor stimulation were completely blocked by pretreatment with norbinaltorphimine, a specific kappa-opioid receptor antagonist at 1 microM, or preincubation with pertussis toxin (PTX) at 200 ng/ml for 6 h. On the other hand, the inhibition on NE-induced augmentation in electrically induced [Ca2+]i transient by U50,488H was not affected by pretreatment with U73122, a specific inhibitor of phospholipase C (PLC), at 10 microM for 30 min. U50,488H attenuated the augmentation of the electrically stimulated [Ca2+]i transient induced by forskolin at 0.1 and 0.5 microM. It did not, however, affect the augmentation of the electrically induced [Ca2+]i transient by N6, 2'-O-dibutyryl adenosine cyclic monophosphate (DB-cAMP). The results suggest that kappa-opioid receptor stimulation by U50,488H at 10(-6 )M or lower may inhibit the effects of beta-adrenoceptor stimulation by acting at a PTX-sensitive G-protein and AC, but not via the phosphoinositol pathway.