ALPHA(1)-ADRENOCEPTOR ACTIVATION INCREASES ECTO-5'-NUCLEOTIDASE ACTIVITY AND ADENOSINE RELEASE IN RAT CARDIOMYOCYTES BY ACTIVATING PROTEIN-KINASE-C

Background Adenosine is an important regulator of many cardiac functions and is synthesized primarily by ecto- and cytosolic 5'-nucleotidase. We have previously reported that alpha(1)-adrenoceptor blockade attenuates adenosine release from ischemic myocardium, raising the possibility that alpha(1)-adrenoceptor activation activates 5'-nucleotidase. This study tested whether activation of protein kinase C by alpha(1)-adrenoceptor activation increases 5'-nucleotidase activity and augments adenosine release. Methods and Results Cardiomyocytes were isolated from adult male Wistar rats and suspended in modified HEPES-Tyrode's buffer solution. After stabilization, the cardiomyocytes were incubated with and without an exposure to norepinephrine (10(-9) to 10(-5) mol/L) while being treated with propranolol and yohimbine or with and without an exposure to methoxamine (10(-9) to 10(-5) mol/L). Ecto-5'-nucleotidase activity was increased by norepinephrine and methoxamine during 30 minutes in a dose-dependent manner, whereas cytosolic 5'-nucleotidase was not activated. These increases in ecto-5'nucleotidase activity were inhibited by GF109203X, an inhibitor of protein kinase C, and mimicked by phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C. The increase in ecto-5'-nucleotidase was not prevented by cycloheximide. When ecto-5'-nucleotidase activity increased, adenosine release was augmented in methoxamine- and PMA-treated cardiomyocytes (1299+/-252% and 1372+/-149%, respectively) compared with the untreated group (578+/-26%). The increase in adenosine release was blunted by GF109203X and alpha,beta-methyleneadenosine 5'-diphosphate, an inhibitor of ecto-5'-nucleotidase. Conclusions Thus, we conclude that alpha(1)-adrenoceptor-mediated increases in ecto-5'-nucleotidase activity are attributed to activation of protein kinase C in rat cardiomyocytes.