cAMP compartmentation is responsible for a local activation of cardiac Ca2+ channels by beta-adrenergic agonists.

Abstract

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.

The human near‐term myometrial β3‐adrenoceptor but not the β2‐adrenoceptor is resistant to desensitisation after sustained agonist stimulation

“…This increase might be too weak, with regard to the 10-fold cAMP increase observed in forskolin-treated myometrial cells, to induce PDE4 de novo synthesis (Mehats et al, 1999). Another reason for the lack of PDE4 upregulation observed in our study might be linked to specific cAMP intracellular compartmentalisation, as already described (Jurevicius & Fischmeister, 1996;Zaccolo & Pozzan, 2002).…”

Effects of forskolin on inotropic performance and phospholamban phosphorylation in exercise-trained hypertensive myocardium

“…Although both isoproterenol and forskolin are known to increase LV inotropic and lusitropic function via increasing cAMP (8,18,22), it is hypothesized that cellular compartmentation allows for a more profuse increase in [Ca 2ϩ ] i and contractility with isoproterenol (18,23,48). Patchclamp experiments in LV cardiomyocytes show L-type Ca 2ϩ current is more localized to the ␤-AR with isoproterenol, whereas L-type Ca 2ϩ current is more widely dispersed throughout the entire sarcolemma with forskolin (25). Thus, although speculative, another potential mechanism is that training may alter cellular compartmentation of the ␤-AR system, such that the trained myocardium elicits a more localized and prolific response to ␤-AR agonism compared with SHR-SED hearts.…”

“…Forskolin activates adenylate cyclase by binding to the cytosolic domain of the membrane-bound enzyme and leads to cAMP generation in a reversible manner, irrespective of cell surface receptors (44). This is of particular interest because, although both direct ␤-AR agonism with isoproterenol and downstream signaling with forskolin result in a dosedependent increase in intracellular cAMP, differences in cellular compartmentation of these systems allow for a more prolific rise in intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) and inotropy with isoproterenol (18,23,25,45,48). Moreover, since PKA can phosphorylate and uncouple ␤-AR signaling, we also examined whether concurrent forskolin and isoproterenol infusion would induce a differential inotropic response in the hypertensive sedentary and trained myocardium.…”

Negative Feedback Exerted by cAMP-dependent Protein Kinase and cAMP Phosphodiesterase on Subsarcolemmal cAMP Signals in Intact Cardiac Myocytes

“…PDE Subtypes That Regulate cAMP Signals-Among the various targets of PKA that could control cAMP levels, PDEs appear as obvious candidates (8,12,13,14). Thus, in a first series of experiments, the effect of the non-selective PDE inhibitor IBMX on cAMP homeostasis reported by I CNG was investigated.…”