Modification of Ca(2+)-handling in cardiomyocytes by redox sensitive mechanisms in response to ouabain.

We examined the role of redox-sensitive signal transduction mechanisms in modifying the changes in [Ca(2+)](i) produced by ouabain upon incubating adult rat cardiomyocytes with antioxidants or inhibitors of different protein kinases and monitoring alterations in fura-2 fluorescence. Ouabain increased basal [Ca(2+)](i), augmented the KCl-induced increase in [Ca(2+)](i), and promoted oxyradical production in cardiomyocytes. These actions of ouabain were attenuated by an oxyradical scavenging mixture (superoxide dismutase plus catalase), and the antioxidants (N-acetyl-L-cysteine and N-(2-mercaptoproprionyl)glycine). An inhibitor of MAP kinase (PD98059) depressed the ouabain-induced increase in [Ca(2+)], whereas inhibitors of tyrosine kinase (tyrphostin and genistein) and PI3 kinase (Wortmannin and LV294002) enhanced the ouabain-induced increase in [Ca(2+)](i). Inhibitors of protein kinase C (calphostin and bisindolylmalaimide) augmented the ouabain-induced increase in [Ca(2+)](i), whereas stimulation of protein kinase C by a phorbol ester (phorbol 12-myristate 13-acetate) depressed the action of ouabain. These results suggest that ouabain-induced inhibition of Na (+)-K(+) ATPase may alter the redox status of cardiomyocytes through the production of oxyradicals, and increase the activities of various protein kinases. Thus, these redox-sensitive signal transduction mechanisms involving different protein kinases may modify Ca(2+)-handling sites in cardiomyocytes and determine the magnitude of net increase in [Ca(2+)](i) in response to ouabain.