Peroxovanadate induces alpha 1B-adrenoceptor phosphorylation and association with protein kinase C

Peroxovanadate induced a marked increase in the phosphorylation state of alpha(1B)-adrenoceptors. The effect was dose-dependent (EC(50) approximately 2 microM) and rapid, reaching its maximum in 5 min and remaining at this level for 30 min. Hydrogen peroxide also increased alpha(1B)-adrenoceptor phosphorylation but to a lesser extent, in an ephemeral fashion, and only at high (millimolar) concentrations. The effect of peroxovanadate was blocked by inhibitors of protein kinase C such as staurosporine and rottlerin and only partially reduced by genistein and inhibitors of phosphoinositide 3-kinase. Protein kinase C alpha, delta and epsilon are associated with the alpha(1B)-adrenoceptor under basal conditions, as reflected by coimmunoprecipitation. Such association was increased by peroxovanadate for all isoforms. In contrast, hydrogen peroxide increased only the association of the epsilon isoform to the adrenoceptor. Peroxovanadate decreased the ability of noradrenaline to increase intracellular calcium, indicating that the receptor phosphorylation induced has functional consequences.