REGULATION OF CA2-MUSCLE CELLS IN PRIMARY CULTURE( ENTRY IN RAT AORTIC SMOOTH)

We characterized Ca2+ entry in rat aortic smooth muscle cells (SMCs) m aintained in primary culture by measuring uptake of Ca-45(2+) or Mn2from a normal balanced salt solution and the extracellular Ca2+-induce d increase in the intracellular Ca2+ concentration ([Ca2+](i)) in a me dium [high pH (pH 8.8)/high Mg2+ (20 mM) medium containing a sarcoplas mic reticulum Ca2+-ATPase inhibitor, thapsigargin] that inhibits remov al of Ca2+ from the cytoplasm. Such measurements in the presence or ab sence of a dihydropyridine (DHP) calcium channel antagonist (PN200-110 ) or hyperpolarizing agent (valinomycin) revealed that DHP-sensitive v oltage-gated Ca2+ channels (VGCCs) are activated in these SMCs under r esting conditions and that DHP-sensitive Ca2+ entry occurs mostly via these VGCCs. We found that receptor stimulation by endothelin-1 in the se SMCs resulted in activation of neither DHP-sensitive nor -insensiti ve Ca2+ entry, but rather resulted in marked suppression of the former . Utilizing the DHP-sensitive extracellular Ca2+-induced increase in [ Ca2+](i) as a monitor of activity of the DHP-sensitive VGCCs, we inves tigated the effects of protein kinase activators and phosphatase inhib itors on the regulation of these VGCCs. We found that the DHP-sensitiv e VGCCs were inhibited by endothelin-1 through the activation of prote in kinase C. We also found that they were inhibited by 8Br-cGMP, okada ic acid, and calyculin A.