Endothelin-1 (ET-1) and angiotensin-II (AT-II) participate in the pathophysiology of cardiovascular diseases. Regulation of gap junctional intercellular communication may influence heart function and its response to cardiac injury. In this study, we examined the effects of ET-1 and AT-II on connexin43 (Cx43) and connexin40 (Cx40) in cultured neonatal rat ventricular cardiomyocytes (NRCs) and the role of mitogen-activated protein kinase signaling in the ET-1- and AT-II-induced responses. NRCs were incubated for 24 h with either ET-1 or AT-II (each at concentrations ranging from 10 to 1000 nM), and Cx43 expression and phosphorylation increased with increasing concentrations of both. ET-1 effects were significantly blocked by ETA (BQ123), but not by ETB (BQ788), receptor antagonists. AT-II-induced Cx43 induction could be completely inhibited by the AT1 receptor antagonist losartan. In contrast to Cx43, Cx40 expression did not change in either ET-1- or in AT-II-treated NRCs. Thus, these two connexins were differentially regulated. ET-1 and AT-II increased the gap junctional conductance between the cardiomyocytes in culture as measured using a dual-cell voltage clamp. Mitogen-activated protein kinase inhibition revealed that ERK1/2 was critical for up-regulation of Cx43 in response to ET-1, whereas both ERK and p38 signal pathways were involved in the regulation of Cx43 by AT-II. Thus, stimulation of the ERK and p38 signal pathways via ETA and AT1 receptors may partcipate in the regulation of cardiac gap junctions under (patho)physiological conditions.

Gap junction channels are essential for intercellular communication. Among the most abundant gap junction channel proteins is connexin 43 (Cx43). The goal of our study was to find out, whether Cx43 content may be regulated via adenylyl cyclase (AC)/cAMP/protein kinase A (PKA), protein kinase C (PKC) pathways or by a tyrosine kinase coupled pathway, i.e. TNF alpha-receptor dependent pathway. Therefore, we used HeLa cells transfected with Cx43 and exposed these cells for 24 h to either db-cAMP (10(-4)M), forskolin (10(-5)M), the phorbolester phorbol-12,13-didecanoate PDD (10(-7)M) (or its inactive form 4 alpha-PDD), TNF alpha (10 U/ml) with or without additional treatment with the MAP kinase inhibitors SB203580 (10(-5) M, p38 MAP-kinase inhibitor) or the MEK1-inhibitor PD98059 (10(-5)M). Cx43 content was analysed using Western blot analysis. All results were confirmed by a second series of identical experiments using Cx43 immunohistochemistry. We found significantly enhanced Cx43 content in cells treated with db-cAMP, forskolin, PDD or TNF alpha (p<0.05), while 4 alpha-PDD or the solvent DMSO exerted no effect. These increases in Cx43 content could be completely suppressed by SB203580 (p<0.05) but not by PD98059. In absence of a stimulating drug, these inhibitors (SB203580 or PD98059) did not affect Cx43 content. Additional PCR experiments revealed increases in Cx43-mRNA under the influence of db-cAMP, forskolin, PDD or TNFalpha (p<0.05), which all could be completely suppressed by SB203580. From these results we conclude that 1.Cx43 content can be regulated via AC/cAMP/PKA, PKC and TNF alpha-receptor-dependent pathways 2. Activation of p38 MAP kinase is a common pathway for regulation of Cx43 content in HeLa cells