Mechanism of Ca2+ release and entry during contraction elicited by norepinephrine in rat resistance arteries.

The intracellular Ca2+ stores and the mechanisms of Ca2+ entry produced by norepinephrine (NE) were investigated in small mesenteric resistance arteries of the rat. In Ca2+-free medium, NE (10 μM) elicited a transient increase in both intracellular free Ca2+concentration ([Ca2+]i) and tension that were both drastically reduced by caffeine and only partially reduced by the two sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) blockers thapsigargin and cyclopiazonic acid, despite the presence of SERCA2a and SERCA2b isoforms in the medial smooth muscle layer of the artery. After depletion of intracellular Ca2+ stores with 10 μM NE, addition of exogenous CaCl2 (2.5 mM) produced large and sustained increases in both [Ca2+]iand contraction of the arteries provided that the agonist was continuously present. In these conditions, the responses to CaCl2 were inhibited by the voltage-dependent Ca2+ entry blocker nitrendipine (1 μM), the putative inhibitor of receptor-operated Ca2+ entry SKF-96365 (30 μM), and NiCl2 (1 mM). The inhibition produced by SKF-96365 and NiCl2 was greater than that of nitrendipine. Also, the responses to CaCl2 were greatly reduced or abolished in the presence of the Na+/Ca2+exchanger inhibitors 1,3-dimethyl-2-thiourea, 3',4'-dichlorobenzamil, MgCl2, and amiloride or after omission of NaCl in the medium. Also, protein kinase C inhibitors, calphostin C and staurosporine, and tyrosine kinase inhibitors, genistein and tyrphostin 23, both reduced the responses to CaCl2. The inhibitory effect of protein kinase C inhibitor and tyrosine kinase were additive. These results suggest that NE releases Ca2+ from intracellular stores that are caffeine sensitive and partially sensitive to SERCA inhibitors. They indicate that in addition to Ca2+ influx via nitrendipine-sensitive and SKF-96365-sensitive channels, Na+/Ca2+exchanger participates in the CaCl2-induced contraction produced in NE-exposed vessels. The pathway leading to Ca2+ entry probably involves tyrosine kinase and protein kinase C. All the above mechanisms require ongoing receptor stimulation.