Regulation of cardiac contraction by neurotransmitters and hormones is often correlated with regulation of the L-type Ca2(+)-channel current (ICa) through the opposite actions of two second messengers, cyclic AMP and cyclic GMP. While cyclic AMP stimulation of ICa is mediated by the activation of cyclic AMP-dependent protein kinase, inhibition of ICa by cyclic GMP in frog heart is largely mediated by activation of cyclic AMP phosphodiesterase. The present patch-clamp study reveals that, in rat ventricular cells, cyclic GMP can also regulate ICa via activation of endogenous cyclic GMP-dependent protein kinase (cGMP-PK). Indeed, the effect of cyclic GMP on ICa was mimicked by intracellular perfusion with the proteolytic active fragment of purified cGMP-PK. Moreover, cGMP-PK immunoreactivity was detected in pure rat ventricular myocytes by using a specific polyclonal antibody. These results demonstrate a dual mechanism for the inhibitory action of cyclic GMP in heart, as well as a physiological role for cGMP-PK in the control of mammalian heart function.

A current challenge in cellular signaling is to decipher the complex intracellular spatiotemporal organization that any given cell type has developed to discriminate among different external stimuli acting via a common signaling pathway. This obviously applies to cAMP and cGMP signaling in the heart, where these cyclic nucleotides determine the regulation of cardiac function by many hormones and neuromediators. Recent studies have identified cyclic nucleotide phosphodiesterases as key actors in limiting the spread of cAMP and cGMP, and in shaping and organizing intracellular signaling microdomains. With this new role, phosphodiesterases have been promoted from the rank of a housekeeping attendant to that of an executive officer.

The role of cAMP subcellular compartmentation in the progress of beta-adrenergic stimulation of cardiac L-type calcium current (ICa) was investigated by using a method based on the use of whole-cell patch-clamp recording and a double capillary for extracellular microperfusion. Frog ventricular cells were sealed at both ends to two patch-clamp pipettes and positioned approximately halfway between the mouths of two capillaries that were separated by a 5-micron thin wall. ICa could be inhibited in one half or the other by omitting Ca2+ from one solution or the other. Exposing half of the cell to a saturating concentration of isoprenaline (ISO, 1 microM) produced a nonmaximal increase in ICa (347 +/- 70%; n = 4) since a subsequent application of ISO to the other part induced an additional effect of nearly similar amplitude to reach a 673 +/- 130% increase. However, half-cell exposure to forskolin (FSK, 30 microM) induced a maximal stimulation of ICa (561 +/- 55%; n = 4). This effect was not the result of adenylyl cyclase activation due to FSK diffusion in the nonexposed part of the cell. To determine the distant effects of ISO and FSK on ICa, the drugs were applied in a zero-Ca solution. Adding Ca2+ to the drug-containing solutions allowed us to record the local effect of the drugs. Dose-response curves for the local and distant effects of ISO and FSK on ICa were used as an index of cAMP concentration changes near the sarcolemma. We found that ISO induced a 40-fold, but FSK induced only a 4-fold, higher cAMP concentration close to the Ca2+ channels, in the part of the cell exposed to the drugs, than it did in the rest of the cell. cAMP compartmentation was greatly reduced after inhibition of phosphodiesterase activity with 3-isobutyl-methylxanthine, suggesting the colocalization of enzymes involved in the cAMP cascade. We conclude that beta-adrenergic receptors are functionally coupled to nearby Ca2+ channels via local elevations of cAMP.

Background— Cyclic guanosine monophosphate (cGMP) is the common second messenger for the cardiovascular effects of nitric oxide (NO) and natriuretic peptides, such as atrial or brain natriuretic peptide, which activate the soluble and particulate forms of guanylyl cyclase, respectively. However, natriuretic peptides and NO donors exert different effects on cardiac and vascular smooth muscle function. We therefore tested whether these differences are due to an intracellular compartmentation of cGMP and evaluated the role of phosphodiesterase (PDE) subtypes in this process. Methods and Results— Subsarcolemmal cGMP signals were monitored in adult rat cardiomyocytes by expression of the rat olfactory cyclic nucleotide–gated (CNG) channel α-subunit and recording of the associated cGMP-gated current ( I _CNG ). Atrial natriuretic peptide (10 nmol/L) or brain natriuretic peptide (10 nmol/L) induced a clear activation of I _CNG , whereas NO donors ( S -nitroso- N -acetyl-penicillamine, diethylamine NONOate, 3-morpholinosydnonimine, and spermine NO, all at 100 μmol/L) had little effect. The I _CNG current was strongly potentiated by nonselective PDE inhibition with isobutyl methylxanthine (100 μmol/L) and by the PDE2 inhibitors erythro-9-(2-hydroxy-3-nonyl)adenine (10 μmol/L) and Bay 60-7550 (50 nmol/L). Surprisingly, sildenafil, a PDE5 inhibitor, produced a dose-dependent increase of I _CNG activated by NO donors but had no effect (at 100 nmol/L) on the current elicited by atrial natriuretic peptide. Conclusions— These results indicate that in rat cardiomyocytes (1) the particulate cGMP pool is readily accessible at the plasma membrane, whereas the soluble pool is not; and (2) PDE5 controls the soluble but not the particulate pool, whereas the latter is under the exclusive control of PDE2. Differential spatiotemporal distributions of cGMP may therefore contribute to the specific effects of natriuretic peptides and NO donors on cardiac function.

CAMP is a powerful second messenger whose known general effector is protein kinase A (PKA). The identification of a cAMP binding protein, Epac, raises the question of its role in Ca²⁺ signalling in cardiac myocytes. In this study, we analysed the effects of Epac activation on Ca²⁺ handling by using confocal microscopy in isolated adult rat cardiomyocytes. [Ca²⁺]_i transients were evoked by electrical stimulation and Ca²⁺ sparks were measured in quiescent myocytes. Epac was selectively activated by the cAMP analogue 8‐(4‐chlorophenylthio)‐2′‐O‐methyladenosine‐3′,5′‐cyclic monophosphate (8‐CPT). Patch‐clamp was used to record the L‐type calcium current (I_Ca), and Western blot to evaluate phosphorylated ryanodine receptor (RyR). [Ca²⁺]_i transients were slightly reduced by 10 μm 8‐CPT (F/F₀: decreased from 4.7 ± 0.5 to 3.8 ± 0.4, P < 0.05), an effect that was boosted when cells were previously infected with an adenovirus encoding human Epac. I_Ca was unaltered by Epac activation, so this cannot explain the decreased [Ca²⁺]_i transients. Instead, a decrease in the sarcoplasmic reticulum (SR) Ca²⁺ load underlies the decrease in the [Ca²⁺]_i transients. This decrease in the SR Ca²⁺ load was provoked by the increase in the SR Ca²⁺ leak induced by Epac activation. 8‐CPT significantly increased Ca²⁺ spark frequency (Ca²⁺ sparks s⁻¹ (100 μm)⁻¹: from 2.4 ± 0.6 to 6.9 ± 1.5, P < 0.01) while reducing their amplitude (F/F₀: 1.8 ± 0.02 versus 1.6 ± 0.01, P < 0.001) in a Ca²⁺/calmodulin kinase II (CaMKII)‐dependent and PKA‐independent manner. Accordingly, we found that Epac increased RyR phosphorylation at the CaMKII site. Altogether, our data reveal a new signalling pathway by which cAMP governs Ca²⁺ release and signalling in cardiac myocytes.