Sarcolemmal (Ca2(+)+Mg2+)-ATPase of vascular smooth muscle and the effects of protein kinases thereupon.
To elucidate the regulation mechanisms for sarcolemmal Ca2(+)-pumping ATPase of vascular smooth muscle, the preparation of the membrane fraction of porcine aorta with which the enzyme activity could be analyzed was attempted. A Ca2(+)-activated, Mg2(+)-dependent ATPase [Ca2(+)+Mg2+)-ATPase) activity with high affinity for Ca2+ (Km = 79 +/- 18 nM) was found in a sarcolemma-enriched fraction obtained from digitonin-treated microsomes that possessed the essential properties of plasma membrane (PM) Ca2(+)-pumping ATPases, as determined for the erythrocyte and cardiac muscle enzymes. The activity was stimulated by calmodulin and inhibited by low concentrations of vanadate. Saponin had a stimulatory effect on it. The existence of the PM enzyme in the membrane fraction was substantiated by the Ca2(+)-dependent, hydroxylamine sensitive phosphorylation of a 130K protein, which could be selectively enhanced by LaCl3. The enzyme activity was potentiated by either cGMP or a purified G-kinase. Purified protein kinase C potentiated the enzyme activity. However, none of these agents stimulated the activity of the enzyme purified from microsomes by calmodulin affinity chromatography. The results suggest that the sarcolemmal Ca2(+)-pumping ATPase of vascular smooth muscle is regulated by these protein kinases not through phosphorylation of the enzyme itself but through phosphorylation of membrane components(s) other than the enzyme. Phosphatidylinositol phosphate was found to stimulate the enzyme, suggesting its role in mediation of the stimulatory effects of the protein kinases.
Imai
S
S
Department of Pharmacology, Niigata University School of Medicine.
Yoshida
Y
Y
Sun
H T
HT
D000818:Animals; D001011:Aorta / Q000187:drug effects / Q000201:enzymology; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology; D006152:Cyclic GMP / Q000494:pharmacology; D004912:Erythrocytes / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology*; D010716:Phosphatidylinositols / Q000494:pharmacology*; D011494:Protein Kinases / Q000494:pharmacology*; D012503:Saponins / Q000494:pharmacology; D012508:Sarcolemma / Q000187:drug effects / Q000201:enzymology*; D013552:Swine; D014638:Vanadates / Q000494:pharmacology
D002147:Calmodulin; D010716:Phosphatidylinositols; D012503:Saponins; D014638:Vanadates; D011494:Protein Kinases; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D006152:Cyclic GMP