CAMP COMPARTMENTATION IS RESPONSIBLE FOR A LOCAL ACTIVATION OF CARDIAC CA2-ADRENERGIC AGONISTS( CHANNELS BY BETA)

The role of cAMP subcellular compartmentation in the progress of beta- adrenergic stimulation of cardiac L-type calcium current (I-Ca) was in vestigated by using a method based on the use of whole-cell patch-clam p recording and a double capillary for extracellular microperfusion, F rog ventricular cells were sealed at both ends to two patch-clamp pipe ttes and positioned approximately halfway between the mouths of two ca pillaries that were separated by a 5-mu m thin wall. I-Ca could be inh ibited 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 mu M) produced a nonmaximal increase in I-Ca (347 +/- 70%; n = 4) since a subsequent application of ISO to the other pa rt induced an additional effect of nearly similar amplitude to reach a 673 +/- 130% increase. However, half-cell exposure to forskolin (FSK, 30 mu M) induced a maximal stimulation of I-Ca (561 +/- 55%; n = 4), This effect was not the result of adenylyl cyclase activation due to F SK diffusion in the nonexposed part of the cell. To determine the dist ant effects of ISO and FSK on I-Ca, the drugs were applied in a zero-C a solution. Adding Ca2+ to the drug-containing solutions allowed us to record the local effect of the drugs, Dose-response curves for the lo cal and distant effects of ISO and FSK on I-Ca were used as an index o f cAMP concentration changes near the sarcolemma. We found that ISO in duced a 40-fold, but FSK induced only a 4-fold, higher cAMP concentrat ion 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 -isobulyl-methylxanthine, suggesting the colocalization of enzymes inv olved in the cAMP cascade. We conclude that beta-adrenergic receptors are functionally coupled to nearby Ca2+ channels via local elevations of cAMP.