Abstract
We examined the role of cyclic ADP-ribose (cADP-ribose) as a second messenger downstream of adrenergic receptors in the heart after excitation of sympathetic neurons. To address this question, ADP-ribosyl cyclase activity was measured as the rate of [(3)H]cADP-ribose formation from [(3)H]NAD(+) in a crude membrane fraction of rat ventricular myocytes. Isoproterenol at 1 microM increased ADP-ribosyl cyclase activity by 1.7-fold in ventricular muscle; this increase was inhibited by propranolol. The stimulatory effect on the cyclase was mimicked by 10 nM GTP and 10 microM guanosine 5'-3-O-(thio)triphosphate, whereas 10 microM GTP inhibited the cyclase. Cholera toxin blocked the activation of the cyclase by isoproterenol and GTP. The above effects of isoproterenol and GTP in ventricular membranes were confirmed by cyclic GDP-ribose formation fluorometrically. These results demonstrate the existence of a signal pathway from beta-adrenergic receptors to membrane-bound ADP-ribosyl cyclase via G protein in the ventricular muscle cells and suggest that increased cADP-ribose synthesis is involved in up-regulation of cardiac function by sympathetic stimulation.
Highlights
Sympathetic nerve excitation stimulates -adrenergic receptors on cardiac myocytes by release of noradrenaline, leading to an increase in the contractility
To verify that the above 3H accumulation in cADP-ribose fractions is mainly due to accumulation of [3H]cADP-ribose produced by ventricular ADP-ribosyl cyclase, the reaction mixtures were either boiled or kept on ice. 3H counts collected in the cADP-ribose fractions from the heat-inactivated product were reduced to 8.7 Ϯ 0.29% (n ϭ 3) of that of non-heat-treated ones
The results show that the adrenergic agonist activates ADPribosyl cyclase activity in crude membrane preparation of rat ventricular myocytes, which were measured by both radioisotopic and fluorometric assay
Summary
Sympathetic nerve excitation stimulates -adrenergic receptors on cardiac myocytes by release of noradrenaline, leading to an increase in the contractility. We measured ADP-ribosyl cyclase activity in crude membrane fractions of rat ventricular myocytes in the presence or absence of an adrenergic agonist and GTP. Autoradiography of TLC with [3H]NADϩ—The same reaction mixture used for the ADP-ribosyl cyclase assay containing 0.06 or 0.36 Ci of -[3H]NADϩ was incubated with membranes of rat ventricular muscle.
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