The role of cAMP subcellular compartmentation in the progress of f3-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-,im 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 ,IM) 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 ,LM) 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 13-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 ␣-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 mol/L) had little effect. The I CNG current was strongly potentiated by nonselective PDE inhibition with isobutyl methylxanthine (100 mol/L) and by the PDE2 inhibitors erythro-9-(2-hydroxy-3-nonyl)adenine (10 mol/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. Key Words: cyclic GMP Ⅲ natriuretic peptides Ⅲ nitric oxide Ⅲ phosphodiesterases Ⅲ sildenafil C yclic guanosine monophosphate (cGMP) is a ubiquitous intracellular second messenger in the cardiovascular system. In the heart, acute elevation of cGMP concentration usually exerts negative metabolic as well as inotropic effects, 1,2 whereas chronic elevation prevents and reverses cardiac hypertrophy. 3-5 cGMP synthesis is controlled by 2 types of guanylyl cyclases (GC) that differ in their cellular location and activation by specific ligands: a particulate GC (pGC) present at the plasma membrane, which is activated by natriuretic peptides such as atrial (ANP), brain (BNP), and C-type natriuretic peptide 6 -8 ; and a soluble guanylyl cyclase (sGC) present in the cytosol and activated by nitric oxide (NO). 8,9 Clinical Perspective p 2228Although NO and natriuretic peptides use cGMP as a common second messenger, there are many instances in which activation of pGC and sGC leads to different functional effects. 10 -14 One explanation for these divergent effects is that cGMP rises in specific subcellular...