MODULATION OF BASAL L-TYPE CA2+ CURRENT BY ADENOSINE IN FERRET ISOLATED RIGHT-VENTRICULAR MYOCYTES

1. The whole-cell configuration of the gigaohm seal voltage clamp and an internal perfusion technique were used to study the effects of adenosine on the basal L-type Ca2+ current (I(Ca)) in enzymatically isolated right ventricular myocytes of ferrets. Basal L-type I(Ca) was isolated by using a Na+- and K+-free saline (replacement by N-methyl-D-glucamine+, Cs+ and TEA+, respectively). All experiments were conducted at room temperature (22-24-degrees-C). 2. Basal I(Ca) was markedly reduced during exposure to adenosine in a concentration-dependent manner with a half-inhibitory concentration (IC50) of 0.3 muM and maximum inhibition of 35 %. This effect was completely abolished by 50 nM 8-cyclopentyl-1,3-dipropylxanthine (CPDPX), a specific A1 adenosine receptor antagonist with an inhibition constant, K(i) = 0.48 nM. Inhibition was also observed in the presence of 1 muM atropine. 3. Adenosine decreased basal I(Ca) by decreasing the peak amplitude of I(Ca) without significantly altering (i) the voltage dependence of the current-voltage relationship, (ii) the apparent reversal potential, (iii) the voltage dependence of steady-state activation and inactivation, (iv) the kinetics of inactivation at 0 mV, and (v) the kinetics of recovery from inactivation at -70 mV. 4. Pretreatment of cells with 0.4 mum/ml pertussis toxin (PTX) for 4 h at 37-degrees-C produced greater than 90 % ADP ribosylation of PTX-sensitive G proteins. PTX pretreatment significantly attenuated the adenosine-mediated decrease in I(Ca) (35 % in control; 4.6 % after PTX pretreatment). 5. The peptide inhibitor (PKI) of cyclic AMP-dependent protein kinase A at a concentration of 2 muM neither inhibited basal I(Ca) nor attenuated the effects of adenosine on basal I(Ca). However, PKI decreased the stimulatory effects of 100 muM cAMP on I(Ca). 6. Increasing intracellular cAMP to a supra-saturable level by using 10 mM cAMP and 100 muM papaverine did not prevent adenosine from inhibiting I(Ca). 7. Consistent with the reduction of basal I(Ca), adenosine produced an inhibitory effect on the action potential under basal conditions, i.e. hyperpolarization of the plateau phase and marked shortening of action potential duration. These effects were concentration dependent. 8. These results demonstrate a reduction of the basal L-type I(Ca) by adenosine in ferret ventricular myocardium. This reduction is not mediated by modification of voltage-dependent properties of macroscopic I(Ca). The shortening of action potentials may be explained in part by the reduction in I(Ca). This change in I(Ca) may be responsible in part for the negative inotropic effect of adenosine observed in ferret ventricular myocardium. 9. In ferret ventricular myocardium regulation Of I(Ca) by adenosine involves binding to an A1-specific receptor and activation of a PTX-sensitive G protein. Our data strongly suggest that the adenosine-mediated inhibition of basal I(Ca) in isolated ferret right ventricular myocytes does not occur through inhibition of basal protein kinase A activity.