Alpha-1-adrenoceptor subtype selective regulation of connexin 43 expression in rat cardiomyocytes.
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 alpha-adrenoceptor stimulation can affect Cx43 expression and function. We now wanted to elucidate which alpha1-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 alpha(1A)-antagonist RS17053 (n=6), 500 nM of the alpha(1B)-antagonist AH1111OA (n=6), or 50 nM of the alpha(1D)-antagonist BMY7378 (n = 6). Moreover, we incubated the cells for 24 h with the alpha(1A)-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, beta-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 alpha(1A)-adrenoceptor agonist A61603 led to a twofold increase in beta-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 alpha(1D)-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 alpha(1)-adrenoceptors predominantly by subtype alpha(1D)-adrenoceptors, while other proteins like beta-actin seem to be regulated via alpha(1A)-adrenoceptors.
Rojas Gomez
Diana Marcela
DM
Clinic for Cardiac Surgery, University of Leipzig, Struempellstr. 39, 04289 Leipzig, Germany.
Schulte
Jan Sebastian
JS
Mohr
Friedrich-Wilhelm
FW
Dhein
Stefan
S
D000199:Actins / Q000096:biosynthesis; D058646:Adrenergic alpha-1 Receptor Agonists; D058668:Adrenergic alpha-1 Receptor Antagonists; D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D002478:Cells, Cultured; D018031:Connexin 43 / Q000096:biosynthesis*; D004553:Electric Conductivity; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010656:Phenylephrine / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D018340:Receptors, Adrenergic, alpha-1 / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000199:Actins; D058646:Adrenergic alpha-1 Receptor Agonists; D058668:Adrenergic alpha-1 Receptor Antagonists; D018031:Connexin 43; D018340:Receptors, Adrenergic, alpha-1; D010656:Phenylephrine