Journal of Andrology, Vol 15, Issue 2 100-109, Copyright © 1994 by The American Society of Andrology

JOURNAL ARTICLE

Atrial natriuretic factor inhibits the phosphorylation of protein kinase C in plasma membrane preparations of cultured Leydig tumor cells

K. N. Pandey
Department of Biochemistry and Molecular Biology, Medical College of Georgia, School of Medicine, Augusta 30912.

The peptide hormone atrial natriuretic factor (ANF) exerts its effect in a receptor-mediated fashion and the membrane-bound form of guanylate cyclase represents a biologically active ANF receptor; thus, cGMP has been considered a second messenger of ANF. To understand the mechanisms of ANF action, we have studied its effect on protein phosphorylation in the plasma membrane preparations of murine Leydig tumor (MA-10) cells, which overexpress guanylate cyclase-coupled ANF receptor molecules in high density. After pretreatment of the plasma membranes with ANF (100 nM), a marked decrease in phosphorylation of the 78-kDa protein kinase C (PKC) and the 240-kDa protein was observed. Phosphorylation of the 78-kDa PKC was also inhibited by cGMP (0.1 mM); however, phosphorylation of the 240-kDa protein was not affected by cGMP. The quantitative analyses, as determined by densitometric scanning, revealed that both ANF and cGMP inhibited phosphorylation of the 78-kDa PKC by approximately 75% and 45%, respectively. The inhibitory effect of ANF on phosphorylation of the 240-kDa protein was almost 90%, but cGMP did not show any discernible effect on its phosphorylation in plasma membranes of MA-10 cells. Phosphorylation of the 78-kDa PKC was stimulated by Ca2+ and phospholipids, and it immunologically cross-reacted with antiserum against brain PKC. Furthermore, in these plasma membrane preparations, the 78-kDa PKC was immunoprecipitated and its phosphorylation was inhibited by ANF. These data provide evidence for a new signal transduction mechanism of ANF that negatively regulates phosphorylation of the 78-kDa PKC and the 240-kDa protein in a cGMP-dependent and -independent manner in Leydig cells.

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