Activation of angiotensin II type I receptor promotes protein kinase C translocation and cell proliferation in human cultured breast epithelial cells

The effect of angiotensin II (Ang II) on Ca2+ signalling in human primary cultured breast epithelial cells was investigated by using fura-2 as the Ca2+ probe. Ang II (0.1-1000 nM) induced an intraceflular free calcium ([Ca2+](i)) transient peak which was unchanged by external Ca2+ removal. In Ca2+-free medium pretreatment with thapsigargin abolished Ang II-induced Ca2+ release. Suppression of 1,4,5-inositol trisphosphate formation by U73122, a phospholipase C inhibitor, blocked the Ang II-induced Ca2+ response. Losartan (DuP753), an inhibitor of Ang II type I receptor (AT1), decreased the [Ca2+](i) increase evoked by Ang II, while CGP4221A, an inhibitor of Ang II type II receptor (AT2) did not. AT1 desensitisation was demonstrated with respect to the Ca2+ response after subsequent exposure of cells to Ang II and also after pretreatment for 25 min with 1000 nM phorbol 12-myristate 13-acetate. Staurosporine, an inhibitor of protein kinases C (PKC), inhibited the AT1 desensitisation. Epithelial breast cells expressed PKC-alpha, -beta1, -delta and -zeta isozymes, and Ang II provoked translocation from the cytosol to the membranes of PKC-alpha, -beta1, and -delta (but not -zeta). Ang II was also able to stimulate cell proliferation in a dose-dependent manner; this effect was blocked by Go 6976, a specific inhibitor of PKC-alpha and -beta1, the Ca2+-dependent isozymes. The main conclusion of this study is that the the Ang II signalling mechanism in breast epithelial cells is based on the elevation of [Ca2+](i) released from intracellular stores through AT1 activation. In addition, Ang II stimulates cell proliferation by the activation of PKC isozymes.