Stimulation of alpha 1-adrenoceptors and protein kinase C-mediated activation of ecto-5'-nucleotidase in rat hearts in vivo.

Abstract

1. To determine whether protein kinase C (PKC)-mediated activation of ecto-5'-nucleotidase would increase interstitial adenosine concentrations in the rat heart in vivo, we made use of the microdialysis technique and a flexibly mounted probe, which was implanted in the left ventricular myocardium and perfused with Tyrode solution. 2. The baseline level of dialysate adenosine was 0.51 +/- 0.09 microM (n = 16). Perfusion of adenosine 5'-monophosphate (AMP, 100 microM) through the probe increased the dialysate adenosine concentration markedly to 9.25 +/- 0.46 microM (n = 15). alpha, beta-Methyleneadenosine 5'-diphosphate (AOPCP, 100 microM), an inhibitor of ecto-5'-nucleotidase, abolished the AMP-induced increase in dialysate adenosine, but did not affect the baseline level of adenosine. These observations suggest that the dialysate adenosine obtained during the perfusion with AMP, but not the baseline levels of adenosine, originated from the dephosphorylation of AMP by ecto-5'-nucleotidase. Thus, the level of adenosine measured during AMP perfusion gives an index of the activity of ecto-5'-nucleotidase in the tissue. 3. Noradrenaline (10 microM) increased the adenosine concentration measured in the presence of 100 microM AMP (i.e. the activity of ecto-5'-nucleotidase) by 38.7 +/- 9.6% (n = 5, P < 0.05), an increase which was inhibited by an antagonist of the alpha 1-adrenoceptor (prazosin, 50 microM) or of PKC (chelerythrine, 10 microM). Further application of either the alpha 1-adrenoceptor agonist methoxamine (100 microM) or the diacylglycerol analogue 1,2-dioctanoyl-sn-glycerol (DOG, 100 microM) also increased the adenosine concentration by 35.1 +/- 10.0% (n = 6, P < 0.05) or 40.6 +/- 8.3% (n = 5, P < 0.05), respectively. 4. The presence of okadaic acid (50 microM), an inhibitor of protein phosphatase, enhanced the noradrenaline-induced increase in adenosine concentration by 112.4 +/- 35.9% (n = 4, P < 0.05), to a level significantly (P < 0.05) greater than the increase caused by noradrenaline alone (38.7 +/- 9.6%). 5. These data provide the first evidence that alpha 1-adrenoceptor stimulation and the subsequent activation of PKC can increase adenosine concentrations in interstitial spaces of ventricular muscle in vivo, through activation of endogenous ecto-5'-nucleotidase.