Figure 3. The effect of DNP on the inactivation curve of the volt- age-gated L-type Ca2+ currents, ICa,L in adult rabbit ventricular myocytes. The voltage protocol was elicited by 300 ms pulses with 10 mV incre- ments from a holding potential of -80 mV in the range of -40 to +50 mV following a depolarizing pulse of 0 mV in the absence or presence of DNP (0.1 μM). The interval between voltage steps was 5 seconds. To build up an inactivation curve, we measured the peak current at the sec- ond depolarizing pulse of 0 mV. The current values were normalized to the maximum level observed under control conditions for each experiment. To determine whether changes in the kinetics of the Ca2+ channels could account for the decrease of ICa,L, we analyzed the steady-state inactivation characteristics in the absence or presence of DNP (Figure 3). For these experiments, the following voltage protocol was applied. From a holding potential of -80 mV, 500 ms pulses with 10 mV increments in the range of -80 to +30 mV were applied, followed by a depolarizing pulse of 0 mV in the absence or presence of DNP (0.1 μM). DNP, although decreasing the peak ICa,L density, had no effect on the steady state inactivation characteristics of ICa,L. Thus, the DNP-induced ICa,L inhibition is most likely due to a decrease in open probability of the Ca2+ channels, rather than to a modification of their voltage dependence. DNP is known to augment the formation of cGMP in aortic endothelial and smooth muscle cells and to be associated with marked increase of cGMP in its natriuretic and diuretic effects (Schweitz et al., 1992; Collins et al., 2000). Considering these results, we hypothesized that the DNP-induced ICa,L inhibition could be mediated via PKG pathway. To confirm the involvement of PKG in the DNP-induced inhibition of ICa,L, we examined the effect of DNP on the ICa,L in the presence of a PKG inhibitor, KT5823 (Figure 4). DNP at a concentration of 0.1 μM failed to suppress further the Ca2+ channel current in the rabbit ventricular myocytes pretreated with KT5823 (1 μM) for 30 min. This finding indicates that the DNP-induced inhibition of the Ca2+ channel current is likely to be mediated by PKG activation. Cardiac L-type Ca2+ channel protein, α1c is phosphorylated by DNP Cardiac L-type Ca2+ channels are composed of α1c, Inhibition of calcium channel by DNP 365 Figure 4. The influence of KT5823 (1 μM) on the inhibitory effect of DNP on the voltage-gated L-type Ca2+ currents, ICa,L in adult rabbit ventricular myocytes. The ventricular myocytes were pretreated for 30 min with a protein kinase inhibitor KT5823 (1 μM) at room temperature. The cur- rents were elicited by 300 ms pulses with 10 mV increments from a hold- ing potential of -80 mV in the range of -40 to +50 mV in the absence (A) or presence (B) of DNP (0.1 μM). The interval between voltage steps was 5 seconds. The current-voltage relationships in the absence or pres- ence of DNP (0.1 μM). The current values were normalized to the max- imum level observed under control conditions for each experiment. β2 and α2δ subunits, and both the α1c and β2 have been demonstrated to be the direct targets of phosphorylation (Gao et al., 1997; Puri et al., 1997). In order to test whether or not the DNP-induced inhibition of the Ca2+ channel current was due to phosphorylation of the cardiac L-type Ca2+ channel proteins, we metabolically labeled rabbit ventricular myocytes with [32P]orthophosphate and then incubated the cells with DNP and/or KT5823. α1c subunits immunoprecipitates from the labeled cells treated with DNP (1 μM) for 10 min, contained [32P] radioactivity (Figure 5), indicating that DNP can phosphorylate α1c subunit of L-type Ca2+ channels. This DNP-induced phosphorylation of α1c subunits proteins was completely blocked by KT5823 (1 μM). These α1c subunit proteins had a similar molecular mass independent of drug treatments when analyzed on immunoblot using anti-α1c antibody. This result indicates that DNP phosphorylates the cardiac L-type Ca2+ channel proteins via PKG activation. Effect of DNP on the ventricular action potential It is well known that the inhibition of cardiac L-type Ca2+ channels induces the shortening of cardiac action potential duration. To test whether DNP decreases the cardiac action potential, we examined the effect of DNP on the ventricular action potential in rabbit ventricular myocytes using conventional microelectrode technique (Figure 6). The resting membrane potentials of ventricular muscles was -80.5 ± 2.1 mV (n = 6) and action potential durations (APD) at 100% repolarization was 193.9 ± 5.2 ms (n = 6) in ventricular muscles stimulated at 2 Hz. The maximum effect of DNP on action potential