Connexin43 (Cx43) is the predominant intercellular gap junction protein in the heart providing intercellular communication for the cell-to-cell transfer of electrical activation. In a previous study, we could show that α-adrenoceptor stimulation can affect Cx43 expression and function. We now wanted to elucidate which α1-adrenoceptor subtype might be involved. Cultured neonatal rat cardiomyocytes were exposed to various concentrations of phenylephrine (0.1-1,000 nM) for 24 h (n=6). Thereafter, cells were harvested, and after lysis, Cx43 content was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot. Results were normalised to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Finally, we determined the effect of this treatment on intercellular gap junction conductivity using dual whole-cell voltage clamp. Similarly, we tested the effect of an additional treatment with either 10 nM prazosin or, to assess the subtypes, 10 nM of the α1A-antagonist RS17053 (n=6), 500 nM of the α1B-antagonist AH1111OA (n=6), or 50 nM of the α1D-antagonist BMY7378 (n=6). Moreover, we incubated the cells for 24 h with the α1A-adrenoceptor agonist A61603 (10 nM). Phenylephrine led to enhanced Cx43 expression with a pEC50 8.00±0.06. The other cardiac connexins, Cx40 and Cx45, as well as GAPDH were not affected. This increase in Cx43 expression resulted in enhanced gap-junction conductance (44±4 nS vs 26±4 nS). As expected, the increased Cx43 expression could be antagonized by prazosin. Moreover, it was nearly completely inhibited by BMY7378 but was not significantly affected by RS17053. AH1111OA led to a moderate but incomplete inhibition. In contrast, β-actin expression was also up-regulated by phenylephrine but was inhibited by prazosin or RS17053, while it was not affected by BMY7378 or AH1111OA. About 24 h exposure to the α1A-adrenoceptor agonist A61603 led to a twofold increase in β-actin but did not affect Cx43. The low pEC50 value of about 1 nM for noradrenaline reported in our earlier study fits well to the hypothesis of an effect mediated predominantly via α1D-adrenoceptors, which is further supported by the finding of a nearly complete antagonisation of the phenylephrine effect by BMY7378. Thus, we conclude that cardiac Cx43 expression seems to be regulated via α1-adrenoceptors predominantly by subtype α1D-adrenoceptors, while other proteins like β-actin seem to be regulated via α1A-adrenoceptors.
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