Regulation of Ca2+ entry in rat aortic smooth muscle cells in primary culture

We characterized Ca2+ entry in rat aortic smooth muscle cells (SMCs) maintained in primary culture by measuring uptake of 45Ca2+ or Mn2+ from a normal balanced salt solution and the extracellular Ca(2+)-induced increase in the intracellular Ca2+ concentration ([Ca2+]i) in a medium [high pH (pH 8.8)/high Mg2+ (20 mM) medium containing a sarcoplasmic reticulum Ca(2+)-ATPase inhibitor, thapsigargin] that inhibits removal of Ca2+ from the cytoplasm. Such measurements in the presence or absence of a dihydropyridine (DHP) calcium channel antagonist (PN200-110) or hyperpolarizing agent (valinomycin) revealed that DHP-sensitive voltage-gated Ca2+ channels (VGCCs) are activated in these SMCs under resting conditions and that DHP-sensitive Ca2+ entry occurs mostly via these VGCCs. We found that receptor stimulation by endothelin-1 in these SMCs resulted in activation of neither DHP-sensitive nor -insensitive Ca2+ entry, but rather resulted in marked suppression of the former. Utilizing the DHP-sensitive extracellular Ca(2+)-induced increase in [Ca2+]i as a monitor of activity of the DHP-sensitive VGCCs, we investigated the effects of protein kinase activators and phosphatase inhibitors on the regulation of these VGCCs. We found that the DHP-sensitive VGCCs were inhibited by endothelin-1 through the activation of protein kinase C. We also found that they were inhibited by 8Br-cGMP, okadaic acid, and calyculin A.