Effects of protein kinase C modulators on Na+/K+ adenosine triphosphatase activity and phosphorylation in aortae from rats with cirrhosis.

Protein kinase C (PKC) modulates the activity and phosphorylation of the catalytic alpha-subunit of sodium-potassium-adenosine triphosphatase (Na+/K+ ATPase) in normal arteries. Because PKC is altered in cirrhotic aortae, Na+/K+ ATPase may also be altered in these arteries. The aim of the present study was to investigate alpha-subunit activity and phosphorylation in aortae from normal and cirrhotic rats, under baseline conditions and during exposure to PKC modulators. Alpha-subunit activity was assessed by measuring the amount of 32P released by hydrolysis of [gamma-32P]ATP in freshly isolated cell membranes (in the absence of PKC modulators only) and membrane depolarization caused by ouabain-induced alpha-subunit inhibition in isolated aortae (in the absence and presence of PKC modulators). Alpha-subunit phosphorylation was assessed by incorporation of 32P into alpha-subunits. Staurosporine, a PKC inhibitor, and phorbol 12,13-dibutyrate (PDBU), a PKC activator, were used. In addition, alpha-subunit expression was studied by Western blot analysis. In the absence of PKC modulators, the amount of 32P released by hydrolysis of [gamma-32P]ATP and ouabain-induced membrane depolarization were significantly lower in cirrhotic than in normal aortae. Staurosporine suppressed ouabain-induced membrane depolarization in cirrhotic and normal arteries. Ouabain-induced membrane depolarization was similar in cirrhotic aortae exposed to PDBU and in normal arteries studied under baseline conditions. Alpha-subunit phosphorylation was significantly lower in cirrhotic than in normal aortae, in aortae under baseline conditions, and in arteries exposed to staurosporine. Phosphorylation of the alpha-subunit was similar in cirrhotic aortae exposed to PDBU and in normal arteries under baseline conditions. Western blot analysis showed that the amount of alpha-subunit did not significantly differ between cirrhotic and normal aortae. In conclusion, a decrease in baseline Na+/K+ ATPase alpha-subunit activity occurs in aortae from cirrhotic rats as a result of reduced basal PKC activity. This PKC-dependent decreased alpha-subunit activity may be caused by a reduction in PKC-induced alpha-subunit phosphorylation.