Conventional inhibitors of cyclic AMP-dependent protein kinase lack membrane-permeability or selectively, or both. The Rp diastereomer of adenosine cyclic 3′,5′-phosphorothioate, Rp-cAMPS, is a novel membrane-permeable antagonist of cyclic AMP. We have assessed the ability of this compound to distinguish between cyclic AMP-dependent and cyclic AMP-independent contractile responses elicited in ventricular cardiomyocytes isolated from the hearts of adult rats. Cardiomyocytes were stimulated to contract at 0.5 Hz in the presence of calcium ion (2 mM) and adenosine deaminase (5 units/ml). Contractile shortening was expressed as maximum shortening relative to prestimulus cell length (δL%). In the presence of a maximally-effective concentration of isoprenaline (100 nM), which acts by a cyclic AMP-dependent mechanism, Rp-cAMPS inhibited the contractile response in a concentration-dependent and time-dependent manner. Following preincubation for 30 min with Rp-cAMPS (100 μM), 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 (≤200 μM) inhibited the contractile response to isoprenaline (100 nM) significantly and in a concentration-dependent manner, but failed to inhibit the contractile responses elicited phenylephrine (2 μM) and calcium ion (7 mM) which act by cyclic AMP-independent mechanisms. In the presence of Rp-cAMPs (200 μM), 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-coupling mechanisms associated with some novel inotropic agents. Rp-cAMPS (≤200 μM) also inhibited the contractile responses to secretin (20 nM) and VIP (20 nM) significantly. In the presence of Rp-cAMPS (200 μM), the contractile response elicited by secretin (20 nM) was 19% of that in the absence of inhibitor, while that elicited by VIP (20 nM) was abolished completely. Rp-cAMPS (≤200 μM) failed to inhibit the contractile response elicited by CGRP (1 nM).

In summary, Rp-cAMPS is a membrane-permeable, selective antagonist 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 basis, it is concluded that secretin and VIP elicit contractile responses in rat ventricular cardiomyocytes which are mainly, if not entirely, attributable to cyclic AMP-dependent mechanisms, while the contractile response to CGRP is mediated by a cyclic AMP-independent mechanism.