Effects of 8-bromo-cGMP on Ca2+ levels in vascular smooth muscle cells: possible regulation of Ca2+-ATPase by cGMP-dependent protein kinase.

Abstract

The effects of 8-bromo-cGMP on intracellular calcium concentrations in cultured rat aortic smooth muscle cells were studied. Both angiotensin II and depolarizing concentrations of K+ stimulated Ca2+ accumulation in the cytoplasm. The increase in Ca2+ due to angiotensin II was associated with an increase in inositol phosphates, while that due to K+ was not. Preincubation of cells with 8-bromo-cGMP (100 microM) caused an inhibition of peak Ca2+ accumulation to either angiotensin II or K+. To probe the mechanism of action of cGMP in vascular smooth muscle, the effects of cGMP-dependent protein kinase on Ca2+-ATPase from the cultured cell particulate material were investigated. Ca2+-activated ATPase was stimulated approximately equal to 2-fold by exogenous calmodulin and up to 4-fold by low concentrations of purified cGMP-dependent protein kinase. The inclusion of both calmodulin and cGMP-dependent protein kinase resulted in an additive stimulation of Ca2+-ATPase. Stimulation of Ca2+-ATPase activity was observed at all Ca2+ concentrations tested (0.01-1.0 microM). cAMP-dependent protein kinase catalytic subunit and protein kinase C were either ineffective or less effective than cGMP-dependent protein kinase in stimulating the Ca2+-ATPase from rat aortic smooth muscle cells. These results suggest a possible mechanism of action for cGMP in mediating decreases in cytosolic Ca2+ through activation of a Ca2+-ATPase and the subsequent removal of Ca2+ from the cell.