Effects of cAMP- and cGMP-dependent protein kinases, and calmodulin on Ca²⁺ uptake by highly purified sarcolemmal vesicles of vascular smooth muscle

Sarcolemmal fractions of vascular smooth muscles were prepared from porcine thoracic aortae by differential and sucrose density gradient centrifugation. In these fractions, there was a high activity of 5′-nucleotidase, a putative marker enzyme of plasma membrane, and a low activity of rotenone insensitive NADH-cytochrome c reductase a marker of sarcoplasmic reticulum. In these fractions, the Ca²⁺ uptake was ATP-dependent. A low concentration of saponin which inhibited Ca²⁺ uptake by the plasma membrane but not by the sarcoplasmic reticulum, inhibited 65% of the Ca²⁺ uptake of this fraction. The Ca²⁺ uptake of this fraction was enhanced by cAMP- and cGMP-dependent protein kinases, and by calmodulin. The cAMP-dependent protein kinase enhanced the phosphorylation of 28 and 22 kDA proteins, while the cGMP-dependent protein kinase phosphorylated the 35 kDa protein. The phosphorylation of 100, 75, 65, 41 and 22 kDa proteins was enhanced by Ca²⁺ and calmodulin. These results indicate that cAMP- and cGMP-dependent protein kinases as well as calmodulin play important roles in Ca²⁺ transport in the sarcolemma, and that the phosphorylated proteins may be associated with an enhancement of Ca²⁺ transport in the sarcolemma.