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.

Abstract-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. Key Words: cAMP Ⅲ cGMP Ⅲ heart Ⅲ G protein-coupled receptor Ⅲ phosphodiesterase T he cyclic nucleotides cyclic adenosine 3Ј,5Ј-monophosphate (cAMP) and cyclic guanosine 3Ј,5Ј-monophosphate (cGMP) were identified more than 4 decades ago. 1,2 Since then, many studies have appeared on how these 2 second messengers are synthesized or degraded, what makes their level go up or down, what they do to target effectors by either covalent (phosphorylation) or noncovalent (direct binding to proteins, such as ion channels or guanine-nucleotide-exchange factors) mechanisms, and how they affect a countless number of cellular functions. [3][4][5][6] In certain tissues and organs, the cyclic nucleotide pathways have been so fully explored over the years that one can wonder what else is there to be found. This is the case for cAMP in the heart, where it plays a key role in the sympathetic nerve/␤-adrenergic receptor (␤-AR)/adenylyl cyclase (AC)/protein kinase A (PKA) axis that serves to stimulate cardiac rhythm (chronotropy) as well as contractile force (inotropy) and relaxation (lusitropy). 7 Yet, there are a number of questions that have always made us wonder but have only lately begun to receive the attention they deserve: how so many different receptors coupled to cAMP or cGMP signaling pathway manage to achieve specific cellular responses? What is the purpose of the different adenylyl and guanylyl cyclases present in the same cell? Why do Original

β 3 -adrenergic receptor (β 3 -AR) activation produces a negative inotropic effect in human ventricles. Here we explored the role of β 3 -AR in the human atrium. Unexpectedly, β 3 -AR activation increased human atrial tissue contractility and stimulated the L-type Ca 2+ channel current (I Ca,L ) in isolated human atrial myocytes (HAMs). Right atrial tissue specimens were obtained from 57 patients undergoing heart surgery for congenital defects, coronary artery diseases, valve replacement, or heart transplantation. The I Ca,L and isometric contraction were recorded using a whole-cell patch-clamp technique and a mechanoelectrical force transducer. Two selective β 3 -AR agonists, SR58611 and BRL37344, and a β 3 -AR partial agonist, CGP12177, stimulated I Ca,L in HAMs with nanomolar potency and a 60%-90% efficacy compared with isoprenaline. The β 3 -AR agonists also increased contractility but with a much lower efficacy (~10%) than isoprenaline. The β 3 -AR antagonist L-748,337, β 1 -/β 2 -AR antagonist nadolol, and β 1 -/β 2 -/β 3 -AR antagonist bupranolol were used to confirm the involvement of β 3 -ARs (and not β 1 -/β 2 -ARs) in these effects. The β 3 -AR effects involved the cAMP/PKA pathway, since the PKA inhibitor H89 blocked I Ca,L stimulation and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) strongly increased the positive inotropic effect. Therefore, unlike in ventricular tissue, β 3 -ARs are positively coupled to L-type Ca 2+ channels and contractility in human atrial tissues through a cAMP-dependent pathway.