USE OF THE CYCLIC-AMP ANTAGONIST, RP-CAMPS, TO DISTINGUISH BETWEEN CYCLIC-AMP-DEPENDENT AND CYCLIC AMP-INDEPENDENT CONTRACTILE RESPONSES INRAT VENTRICULAR CARDIOMYOCYTES

Conventional inhibitors of cyclic AMP-dependent protein kinase lack me mbrane-permeability or selectivity, or both. The Rp diastereomer of ad enosine cyclic 3',5'-phosphorothioate, Rp-cAMPS, is a novel membrane-p ermeable antagonist of cyclic AMP. We have assessed the ability of thi s compound to distinguish between cyclic AMP-dependent and cyclic AMP- independent contractile responses elicited in ventricular cardiomyocyt es isolated from the hearts of adult rats. Cardiomyocytes were stimula ted to contract at 0.5 Hz in the presence of calcium ion (2 mM) and ad enosine deaminase (5 units/ml). Contractile shortening was expressed a s maximum shortening relative to prestimulus cell length (delta L%). I n the presence of a maximally-effective concentration of isoprenaline (100 nM), which acts by a cyclic AMP-dependent mechanism, Rp-cAMPS inh ibited the contractile response in a concentration-dependent and time- dependent manner. Following preincubation for 30 min with Rp-cAMPS (10 0 mu M), the contractile response to isoprenaline (100 nM) was 14% of that elicited in the absence of this inhibitor. An incubation time of 30 min was chosen for all subsequent studies. Rp-cAMPS(less than or eq ual to 200 mu M) inhibited the contractile response to isoprenaline (1 00 nM) significantly and in a concentration-dependent manner, but fail ed to inhibit the contractile responses elicited by phenylephrine (2 m u M) and calcium ion (7 mM) which act by cyclic AMP-independent mechan isms. In the presence of Rp-cAMPs (200 mu M), the contractile response to isoprenaline (100 nM) was 24% of that in the absence of inhibitor. Rp-cAMPS was used subsequently to investigate the contractile-couplin g mechanisms associated with some novel inotropic agents. Rp-cAMPS (le ss than or equal to 200 mu M) also inhibited the contractile responses to secretin (20 nM) and VIP (20 nM) significantly. In the presence of Rp-cAMPS (200 mu M), the contractile response elicited by secretin (2 0 nM) was 19% of that in the absence of inhibitor, while that elicited by VTP (20 nM) was abolished completely. Rp-cAMPS (less than or equal to 200 mu M) failed to inhibit the contractile response elicited by C GRP (1 nM). In summary, Rp-cAMPS is a membrane-permeable, selective an tagonist of cyclic AMP in ventricular cardiomyocytes and can be used, in conjunction with the bioassay of the intracellular accumulation of cyclic AMP, to distinguish between cyclic AMP-dependent and cyclic AMP -independent contractile coupling mechanisms in these cells. On this b asis, it is concluded that secretin and VIP elicit contractile respons es in rat ventricular cardiomyocytes which are mainly, if not entirely , attributable to cyclic AMP-dependent mechanisms, while the contracti le response to CGRP is mediated by a cyclic AMP-independent mechanism.