The major action of forskolin, the diterpine activator of adenylate cyclase, in primary (unpassaged) rat aortic smooth muscle cells is to reduce vasopressin-stimulated Ca2+ concentrations. In repetitively passaged cells, however, forskolin by itself increased Ca2+ levels by apparently stimulating Ca2+ uptake into the cell and had much smaller effects on inhibiting vasopressin-stimulated Ca2+ elevations. Both primary and passaged smooth muscle cells contained adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. Guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase was greatly reduced or absent in passaged smooth muscle cells. The introduction of purified cGMP-dependent protein kinase into the cytoplasm of passaged cells prevented forskolin from elevating intracellular Ca2+ and restored the capacity of forskolin to reduce vasopressin-stimulated Ca2+ mobilization. Similar effects were observed for isoproterenol in passaged smooth muscle cells. When introduced into cells, the active catalytic subunit of the cAMP-dependent protein kinase did not lead to reductions in Ca2+ levels. These results suggest that cAMP elevations lead to profound changes in Ca2+ metabolism through activation of both cAMP- and cGMP-dependent protein kinases. Activation of cGMP-dependent protein kinase by cAMP leads to the reduction in intracellular Ca2+, whereas activation of cAMP-dependent protein kinase may only mediate the uptake of Ca2+ from extracellular sources.
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