SUBCELLULAR CA2+-GRADIENTS IN A7R5 VASCULAR SMOOTH-MUSCLE

The free calcium concentrations in nucleus ([Ca2+](n)) and in cytoplasm ([Ca2+]c) of cultured A7r5 smooth muscle were estimated by confocal laser microscopy using the Ca2+-indicator Indo-1. Upon stimulation with 5 mu M vasopressin (AVP) a cytosolic Ca2+ gradient was observed whereby the highest increase was observed in the subplasmalemmal region. The maximal nuclear Ca2+ concentration ([Ca2+](n)) attained a lower level than that in the cytoplasm ([Ca2+](c) > [Ca2+](n)). After the initial rise, a second sustained change of the Ca2+ level was found and the initial gradient ([Ca2+](c) > [Ca2+](n)) was preserved. In Ca2+-free solution containing 2 mM EGTA the maximal [Ca2+]c value after AVP stimulation was significantly lower than in the Ca2+-containing solution, but it remained higher than [Ca2+](n) which was the same in both conditions. The initial Ca2+ rise was followed by a monoexponential decline. When the influx of Ca2+ through voltage-sensitive Ca2+ channels was blocked, the maximal and steady state values of [Ca2+](c) but not of [Ca2+](n) were lower as compared to the values in non-treated cells. Preincubation with 10 mu M verapamil and 2 mM Ni2+ resulted in initial [Ca2+](c) and [Ca2+](n) rises which were not significantly different from the levels found in the absence of Ni2+, but the sustained phase was absent in both compartments. The differential effect with [CH2+](c) > [Ca2+](n) was not observed if 1 nM AVP was applied instead of 5 mu M AVP. The results indicate that cytosolic and nuclear Ca2+ stores behave differently with respect to their dependence on the agonist concentration and also with respect to the effect of Ca2+-entry mechanisms.