Hypoxia increases the sensitivity of the L-type Ca2+ current to β-adrenergic receptor stimulation via a C2 region-containing protein kinase C isoform

Abstract

The effects of hypoxia on the L-type Ca2+ current (I(Ca-L)) in the absence and presence of the β-adrenergic receptor agonist isoproterenol (ISO) were examined. Exposing guinea pig ventricular myocytes to hypoxia alone resulted in a reversible inhibition of basal I(Ca-L). When cells were exposed to ISO in the presence of hypoxia, the K05 for activation of I(Ca-L) by ISO was significantly decreased from 5.3±0.7 to 1.6±0.1 nmol/L. The membrane-impermeant thiol-specific oxidizing compound 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) attenuated the inhibition of basal I(Ca-L) by hypoxia 81.3±9.4% but had no effect on the increase in sensitivity of I(Ca-L) to ISO. In addition, DTT mimicked the effects of hypoxia on basal I(Ca-L) and the increase in sensitivity to ISO. Neither the inhibitors of guanylate cyclase LY-83583 or methylene blue nor the NO synthase inhibitor N(G)-monomethyl-L-arginine monoacetate had any effect on the basal inhibition of I(Ca-L) or the decrease in K05 for activation of I(Ca-L) by ISO during hypoxia. However, the protein kinase C (PKC) inhibitors bisindolylmaleimide I and Go 7874 significantly attenuated the increase in sensitivity of I(Ca-L) to ISO. More specifically, the response was attenuated when cells were dialyzed with a peptide inhibitor of the C2 region-containing classical PKC isoforms. The same effect was not observed with the PKCε peptide inhibitor. These results suggest that hypoxia regulates I(Ca-L) through the following 2 distinct mechanisms: direct inhibition of basal I(Ca-L) and an indirect effect on the sensitivity of the channel to β-adrenergic receptor stimulation that is mediated through a classical PKC isoform.