364 Exp. Mol. Med. Vol. 44(6), 363-368, 2012 Figure 1. The inhibitory effect of DNP and d-DNP on the voltage-gated L-type Ca2+ currents, ICa,L in adult rabbit ventricular myocytes. (A, B) Superimposed current traces were elicited by 300 ms pulses with 10 mV increments from a holding 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 be- tween voltage steps was 5 seconds. (C) The current-voltage relation- ships of peak current in the absence or presence of DNP or C-termi- nal-deleted DNP (d-DNP; Des-Arg30, Des-Pro31-DNP) (0.1 μM). The cur- rent values were normalized to the maximum level observed under con- trol conditions for each experiment. controlled by extrinsic factors such as hormones and by intrinsic factors such as cellular pH or ATP levels. L-type Ca2+ channel activity is increased by activation of cyclic AMP-dependent protein kinase (PKA), whereas is decreased by activation of cGMP-dependent protein kinase (PKG) (Sumii and Sperelakis, 1995). Additionally, DNP augments the formation of 3’, 5’ cyclic guanosine monophosphate (cGMP), a second messenger for the other natriuretic peptides, in aortic endothelial and smooth muscle cells (Schweitz et al., 1992). Considering these, DNP could regulate the cardiac function by inhibiting the L-type Ca2+ channels through PKG activation. However, no investigations have been made into a casual relation between DNP, cyclic GMP or PKG, and the cardiac Ca2+ channels with cardiac action potential duration. In the present study, we examined the effect of DNP on the cardiac L-type Ca2+ channels in rabbit ventricular myocytes. The present study clearly showed that DNP inhibits the cardiac L-type Ca2+ channel activity through PKG activation. Figure 2. Concentration-dependence of DNP-induced ICa,L inhibition in adult rabbit ventricular myocytes. (A) The current-voltage relationships of peak current in the absence or presence of DNP (10-1000 nM). The cur- rent values were normalized to the maximum level observed under con- trol conditions for each experiment. (B) Concentration-dependent in- hibition of the ICa,L current evoked by a depolarizing pulse of 0 mV. The amplitudes of peak currents were normalized to the maximum current ob- tained in control, and plotted against DNP concentrations. Data were fit- ted with a Hill equation. Each point with vertical bars represents mean ± SEM and each data was obtained from 5 myocytes. Results Voltage-gated L-type Ca2+ currents, ICa,L are suppressed by DNP The actions of DNP on ICa,L were examined in adult rabbit ventricular myocytes. ICa,L were elicited by depolarizing steps of 300 ms duration from a holding potential of -80 mV. DNP at a concentration of 0.1 μM decreased ICa,L density to 40.2 ± 7.0% of the control at 0 mV (P ＜ 0.001, n = 6; Figure 1). Figure 1C shows relations between the peak current and the potential of depolarizing pulse. The potential at which ICa,L was greatest was somewhat variable (either 0 or +10 mV) from myocyte to myocyte. However, DNP did not shift the potentials for the threshold (-40 mV) and the maximum peak current (0 mV). In other words, DNP did not affect the voltage dependence of activation of ICa,L. Interestingly, C-terminal-deleted DNP (d-DNP; Des-Arg30, Des-Pro31-DNP, 0.1 μM) also inhibited ICa,L to 53.6 ± 7.6% of the control at 0 mV (P ＜ 0.001, n = 6; Figure 1C). These results indicate that C-terminal of DNP (Arg30 and Pro31 residues of DNP) is not essential in inhibiting L-type Ca2+ channels. Figure 2 shows the concentration dependence of DNP-induced ICa,L inhibition in rabbit ventricular myocytes. DNP inhibited ICa,L at a concentration starting as low as 10 nM. Increasing the concentration of DNP led to progressively greater inhibition of ICa,L (Figure 2), where IC50 of DNP was 25.5 ± 4.6 nM (n = 5).