A Specific Pattern of Phosphodiesterases Controls the cAMP Signals Generated by Different G _s -Coupled Receptors in Adult Rat Ventricular Myocytes

Abstract

Abstract-Compartmentation of cAMP is thought to generate the specificity of G s -coupled receptor action in cardiac myocytes, with phosphodiesterases (PDEs) playing a major role in this process by preventing cAMP diffusion. We tested this hypothesis in adult rat ventricular myocytes by characterizing PDEs involved in the regulation of cAMP signals and L-type Ca 2ϩ current (I Ca,L ) on stimulation with ␤ 1 -adrenergic receptors (␤ 1 -ARs), ␤ 2 -ARs, glucagon receptors (Glu-Rs) and prostaglandin E 1 receptors (PGE 1 -Rs). All receptors but PGE 1 -R increased total cAMP, and inhibition of PDEs with 3-isobutyl-1-methylxanthine strongly potentiated these responses. When monitored in single cells by high-affinity cyclic nucleotide-gated (CNG) channels, stimulation of ␤ 1 -AR and Glu-R increased cAMP, whereas ␤ 2 -AR and PGE 1 -R had no detectable effect. Selective inhibition of PDE3 by cilostamide and PDE4 by Ro 20-1724 potentiated ␤ 1 -AR cAMP signals, whereas Glu-R cAMP was augmented only by PD4 inhibition. PGE 1 -R and ␤ 2 -AR generated substantial cAMP increases only when PDE3 and PDE4 were blocked. For all receptors except PGE 1 -R, the measurements of I Ca,L closely matched the ones obtained with CNG channels. Indeed, PDE3 and PDE4 controlled ␤ 1 -AR and ␤ 2 -AR regulation of I Ca,L , whereas only PDE4 controlled Glu-R regulation of I Ca,L thus demonstrating that receptor-PDE coupling has functional implications downstream of cAMP. PGE 1 had no effect on I Ca,L even after blockade of PDE3 or PDE4, suggesting that other mechanisms prevent cAMP produced by PGE 1 to diffuse to L-type Ca 2ϩ channels. These results identify specific functional coupling of individual PDE families to G s -coupled receptors as a major mechanism enabling cardiac cells to generate heterogeneous cAMP signals in response to different hormones. Key Words: cAMP Ⅲ heart Ⅲ G-protein-coupled receptor Ⅲ phosphodiesterase C ardiac myocytes express a number of G s -coupled receptors (G s PCRs) that raise intracellular cAMP levels and activate cAMP-dependent protein kinase (PKA) but exert different downstream effects. For instance, ␤ 1 -adrenergic receptor (␤ 1 -AR) stimulation produces a major and sustained increase in force of contraction, accelerates relaxation, and stimulates glycogen phosphorylase. 1 ␤ 2 -AR stimulation also increases contractile force but does not activate glycogen phosphorylase 2 and does not accelerate relaxation 1,3 (see also ref. 2); glucagon receptor (Glu-R) stimulation activates phosphorylase and exerts positive inotropic and lusitropic effects, but the contractile effects fade with time. 4 Finally, prostaglandin E 1 (PGE 1 ) has no effect on contractile activity or glycogen metabolism. 5,6 Such observations led to the proposal that activation of different G s PCRs results in the accumulation of cAMP and phosphorylation of hormone target proteins in distinct compartments. 7 The discovery of A-kinase anchoring proteins, responsible for the subcellular distribution of particulate PKA, 8 and the development of new method...

Phosphodiesterase 4B in the cardiac L-type Ca2+ channel complex regulates Ca2+ current and protects against ventricular arrhythmias in mice

“…A similar regulation of I Ca,L by PDE4 was found previously in human atrial and rat ventricular myocytes, thus clearly indicating that PDE4 is a major negative regulator of I Ca,L in the heart (14,20,33). Similar to what was reported earlier in the rat (34), PDE4 activity in the mouse ventricle was due to the expression of PDE4A, PDE4B, and PDE4D variants.…”

Cytoplasmic cAMP concentrations in intact cardiac myocytes

“…Other studies have used cyclic nucleotide-gated (CNG) ion channels as reporters of cAMP activity in the subsarcolemmal space of various cell types, including cardiac myocytes (23)(24)(25). Responses detected by isoforms of these channels that have an EC 50 for activation by cAMP of ϳ10 M suggest that levels can reach high micromolar concentrations (23).…”

Real-time optical recording of β1-adrenergic receptor activation reveals supersensitivity of the Arg389 variant to carvedilol

“…Thus, while we did not find a significantly enhanced capacity of the Arg389-β 1 -AR variant to form cAMP under basal conditions in HEK cells (Supplemental Figure 2), expression of the 2 receptor variants in primary rat cardiomyocytes revealed differences as to their chronotropic effect. This is most likely due to the cardiomyocyte environment, where localized submembrane cAMP has been shown to regulate channel activity (47,48), but cannot yet be resolved by Epac1-camps determination.…”