PKCδ mediates testosterone-induced increases in coronary smooth muscle Ca_v1.2

Sex hormones have emerged as important modulators of cardiovascular physiology and pathophysiology. Our previous studies demonstrated that testosterone increases expression and activity of L-type, voltage-gated calcium channels (Ca_v1.2) in coronary arteries of males. The purpose of the present study was to determine whether testosterone (T) alters coronary protein kinase Cδ (PKCδ) expression and whether PKCδ plays a role in coronary Ca_v1.2 expression. For in vitro studies, porcine right coronary arteries (RCA) and post-confluent (passages 3-6) 5-day, serum-restricted coronary smooth muscle cell cultures (CSMC) were incubated in the presence and absence of T or dihydrotestosterone (10 and 100 nM) for 18 h at 37 °C in a humidified chamber. For sex and endogenous testosterone- dependent effects, RCA were obtained from intact males, castrated males, castrated males with T replacement, and intact females. In vitro T and dihydrotestosterone caused an ∼2-3-fold increase in PKCδ protein levels, ∼1.5-2-fold increase in PKCδ kinase activity, and localization of PKCδ toward the plasma membrane and nuclear envelope. PKCδ protein levels were higher in coronary arteries of intact males compared with intact females. Elimination of endogenous testosterone by castration reduced RCA PKCδ protein levels, an effect partially (-45%) reversed by exogenous T (castrated males with T replacement). In CSMC, PKC inhibition with either the general PKC inhibitor, cheylerythrine, or the putative PKCδ inhibitor, rottlerin, completely inhibited the T-mediated increase in coronary Ca _v1.2 protein levels. Conversely, Go6976, a conventional PKC isoform inhibitor, failed to inhibit T-induced increases in coronary Ca_v1.2 protein levels. PKCδ short interference RNA completely blocked T-induced increases in Ca_v1.2 protein levels in CSMC. These results demonstrate for the first time that 1) endogenous T is a primary modulator of coronary PKCδ protein and activity in males and 2) T increases Ca_v1.2 protein expression in a PKCδ-dependent manner.