Mechanism of carteolol-induced cytosolic Ca2+ mobilization in cultured vascular smooth muscle cells

Abstract: An increase in cytosolic calcium concentration triggers intracellular signal transduction in vascular cells, which then regulates the vascular contraction. In the present study, the regulatory mechanism of carteolol on the intracellular free Ca2+ ([Ca2+] i ) mobilization was investigated in cultured A7r5 vascular smooth muscle cells. The A7r5 cells were cultured and loaded with fura-2-AM, which was used as a Ca2+ sensitive fluorescent probe. In both the presence and absence of external Ca2+, carteolol increased [Ca2+] i with a dose-dependent manner in A7r5 cells at concentrations between 608µM and 6.08µM. In a Ca2+-containing buffer, carteolol-induced [Ca2+] i showed an initial peak followed by a secondary and persistent plateau. Pretreatment of the cells with La3+, the plasma membrane Ca2+ pump inhibitor, and nifedipine, a L-type Ca2+ channel inhibitor, both partially restrained the carteolol-induced initial peak in [Ca2+] i by 92% and 86%, respectively. Pretreatment of the cells with adrenoceptor antagonists, prazosin inhibited the [Ca2+] i response by 80%, and propranolol enhanced the response by 61%. In the Ca2+−-free buffer, pretreatment of the cells with carteolol inhibited the endoplasmic reticulum Ca2+ pump inhibitor of thapsigargin-induced [Ca2+] i increase by 97%. Pretreatment of the cells with thapsigargin also inhibited the carteolol-induced [Ca2+] i rise by 98%. The internal Ca2+ release induced by the carteolol was partially inhibited by U73122 (phospholipase C inhibitor) and aristolochic acid, quinacrine (phospholipase A2 inhibitors). After incubation of carteolol in the Ca2+-free buffer, the addition of CaCl2 increased the Ca2+ influx, implying that the release of Ca2+ from internal stores further induced capacitative Ca2+ entry. These results suggest that carteolol-induced [Ca2+] i increase is mediated by the initial influx via the α1-adrenoceptor, L-type Ca2+ channel, nonselective calcium entry channels and release of Ca2+ from an intracellular store, which is mainly in the endoplasmic reticulum followed by capacitative Ca2+ entry but decrease via the β2-adrenoceptor. The intracellular Ca2+ release was also modulated by phospholipase A2, C-coupled events. [Copyright &y& Elsevier]