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...

Intracardiac cAMP levels are modulated by hormones and neuromediators with specific effects on contractility and metabolism. To understand how the same second messenger conveys different information, mutants of the rat olfactory cyclic nucleotide-gated (CNG) channel ␣-subunit CNGA2, encoded into adenoviruses, were used to monitor cAMP in adult rat ventricular myocytes. CNGA2 was not found in native myocytes but was strongly expressed in infected cells. In whole cell patchclamp experiments, the forskolin analogue L-858051 (L-85) elicited a non-selective, Mg 2؉ -sensitive current observed only in infected cells, which was thus identified as the CNG current (I CNG ). The ␤-adrenergic agonist isoprenaline (ISO) also activated I CNG , although the maximal efficiency was Ϸ5 times lower than with L-85. However, ISO and L-85 exerted a similar maximal increase of the L-type Ca 2؉ current. The use of a CNGA2 mutant with a higher sensitivity for cAMP indicated that this difference is caused by the activation of a localized fraction of CNG channels by ISO. cAMP-dependent protein kinase (PKA) blockade with H89 or PKI, or phosphodiesterase (PDE) inhibition with IBMX, dramatically potentiated ISO-and L-85-stimulated I CNG . A similar potentiation of ␤-adrenergic stimulation occurred when PDE4 was blocked, whereas PDE3 inhibition had a smaller effect (by 2-fold). ISO and L-85 increased total PDE3 and PDE4 activities in cardiomyocytes, although this effect was insensitive to H89. However, in the presence of IBMX, H89 had no effect on ISO stimulation of I CNG . This study demonstrates that subsarcolemmal cAMP levels are dynamically regulated by a negative feedback involving PKA stimulation of subsarcolemmal cAMP-PDE.Recent evidence indicates that multimolecular signaling complexes between cell surface receptors and intracellular targets are essential for the speed and specificity of signal transduction events (1, 2, 3). However, how such modules maintain specificity when small diffusible molecules are generated during the signaling cascade is difficult to investigate. This question is particularly relevant for cAMP in the heart, where this cyclic nucleotide second messenger exerts diverse effects in response to a number of different neuromediators and hormones. For instance, the ␤-adrenergic agonist isoprenaline (ISO), 1 prostaglandin E 1 (PGE 1 ), and glucagon-like peptide 1 (GLP-1) elevate intracardiac cAMP levels with different effects on contractility; ISO augments the force of contraction, PGE 1 does not, and GLP-1 exerts a negative inotropic effect (4, 5). In order to explain these results, subcellular compartmentation of cAMP was proposed more than 20 years ago (6).Localized cAMP signals may be generated by the interplay between discrete production sites and restricted diffusion within the cytoplasm. In addition to specialized membrane structures that may circumvent cAMP spreading (6, 7), degradation of cAMP into 5Ј-AMP by cyclic nucleotide phosphodiesterases (PDEs) appears critical for the formation of dynamic microdomain...

(PDEs) regulate local cAMP concentration in cardiomyocytes, with PDE4 being predominant for the control of β-AR-dependent cAMP signals. Three genes encoding PDE4 are expressed in mouse heart: Pde4a, Pde4b, and Pde4d. Here we show that both PDE4B and PDE4D are tethered to the LTCC in the mouse heart but that β-AR stimulation of the L-type Ca 2+ current (I Ca,L ) is increased only in Pde4b -/-mice. A fraction of PDE4B colocalized with the LTCC along T-tubules in the mouse heart. Under β-AR stimulation, Ca 2+ transients, cell contraction, and spontaneous Ca 2+ release events were increased in Pde4b -/-and Pde4d -/-myocytes compared with those in WT myocytes. In vivo, after intraperitoneal injection of isoprenaline, catheter-mediated burst pacing triggered ventricular tachycardia in Pde4b -/-mice but not in WT mice. These results identify PDE4B in the Ca V 1.2 complex as a critical regulator of I Ca,L during β-AR stimulation and suggest that distinct PDE4 subtypes are important for normal regulation of Ca 2+ -induced Ca 2+ release in cardiomyocytes. IntroductionDuring the cardiac action potential, Ca 2+ influx through sarcolemmal L-type Ca 2+ channels (LTCCs) triggers Ca 2+ release from juxtaposed ryanodine receptor 2 (RyR2) located in the sarcoplasmic reticulum (SR). This allows a rapid and synchronous Ca 2+ elevation throughout the cell, which activates contraction. During cardiac relaxation, Ca 2+ is rapidly extruded by the Na + /Ca 2+ exchanger and re-sequestered into the SR by the Ca 2+ -ATPase, SERCA2 (1). This process is highly regulated, in particular, by the sympathetic nervous system. β-Adrenergic receptors (β-ARs) exert strong inotropic and lusitropic effects by increasing intracellular cAMP levels and activating cAMP-dependent PKA. PKA then phosphorylates the key proteins of the excitation-contraction coupling (ECC) process, including LTCC and RyR2 but also phospholamban (PLB), which controls Ca 2+ reuptake by SERCA2, as well as the myofilament proteins troponin I and myosin binding protein C (1).The cardiac LTCC consists of the central pore-forming subunit α 1C (Ca V 1.2) and auxiliary β and α 2 -δ subunits that modulate its function (2). Upon β-AR stimulation, phosphorylation of Ca V 1.2, the auxiliary β 2 subunit, or the closely associated protein AHNAK by PKA increases channel activity, thus enhancing the L-type Ca 2+ current (I Ca,L ) (3-5). This regulation involves physical