366 Exp. Mol. Med. Vol. 44(6), 363-368, 2012 Figure 5. Phosphorylation of cardiac voltage-sensitive Ca2+ channel pro- tein, α1c, by protein kinase G in rabbit ventricular myocytes. Ventricular myocytes were incubated with [32P]orthophosphate for 3h prior to treat- ment with DNP or KT5823. The labeled cells were treated with DNP for 10 min and/or with KT5823 for 1h. α1c subunit of L-type Ca2+ channels was immunoprecipitated by anti-α1c antibody. The immunoprecipitates were separated on SDS-polyacrylamide gels and the dried gels were ex- posed to XAR film (upper panel). After exposure to XAR film, the dried gel was swelled with distilled water and transferred into NC paper. The paper was blotted with anti-α1c antibody (lower panel). duration was observed about 30 min after exposure to the drug and remained relatively unchanged with further perfusion. Therefore, we exposed the tissue to the drugs for ~30 min or until the plateau effect of the drug was present. DNP (0.1 μM) decreased the action potential duration at 90% repolarization (APD90) by 22.4 ± 4.2% of APD90 before DNP (n = 6), which was completely blocked by KT5823 (1 μM). These results suggest that DNP can cause the shortening of action potential duration through inhibition of L-type Ca2+ channel via PKG activation. Discussion In the present study, we have examined the effects of DNP on the cardiac L-type Ca2+ channels, and clarified its action mechanism at molecular level. Our results clearly showed that DNP inhibited the L-type Ca2+ channel activity by phosphorylating the L-Ca2+ channel α1c protein via PKG activation. This is the first report showing the DNP-induced inhibition of L-type Ca2+ channels and its molecular mechanism. In the present study, DNP inhibited the cardiac L-type Ca2+ channel activity through PKG activation in isolated rabbit ventricular myocytes. DNP is a 38-amino acid peptide that contains a 17-amino acid disulfide ring structure similar to ANP, BNP, and CNP (Schweitz et al., 1992), all of which mediate biologic actions through particulate guanylyl cyclase receptors and generation of cGMP (Schweitz et al., 1992). DNP substantially augments the formation of cGMP in aortic endothelial and smooth muscle cells (Schweitz et al., 1992; Collins et al., 2000). And, it has been proposed that cGMP play a role antagonistic to cAMP in regulating cardiac Ca2+ channels (Goldberg et al., 1975). This is supported by several reports that the cardiac L-type Ca2+ channel activities were inhibited by 8-bromo-cyclic GMP, a potent activator of PKG, as well as PKG Figure 6. Effects of DNP (0.1 μM) on action potentials in isolated rabbit ventricular muscles. (A) A typical representation of superimposed action potentials in the presence of DNP (a, n = 6) and in the combined pres- ence of KT5823 (1 μM, b). (B) Averaged effects of DNP (0.1 μM) on APD90 of isolated rabbit ventricular muscles. Each column with vertical bar denotes the mean with S.E.M. from six observations. *P ＜ 0.05. (Sumii and Sperelakis, 1995). Furthermore, Tohse et al. (1995) found that ANP, structurally similar to DNP, inhibited the cardiac L-type Ca2+ channel activity through the intracellular production of cGMP and then activation of PKG. In the present study, we found that DNP inhibited the cardiac L-type Ca2+ channel activity through PKG activation and phosphorylated the pore-forming subunit of cardiac L-type Ca2+ channel protein, α1c, which was completely blocked by PKG inhibitor. These results clearly indicate that DNP inhibits the cardiac L-type Ca2+ channel activity by phosphorylating the voltage-sensitive Ca2+ channel protein through PKG activation. However, further experiments are required to clarify which amino acids of cardiac Ca2+ channel α1c should be phosphorylated by DNP to inhibit its activity. During inhibiting the cardiac L-type Ca2+ channels, DNP did not affect the voltage dependence of activation and the inactivation characteristics of ICa,L, indicating that 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. Additionally, there was no significant difference between the inhibitory effects of DNP and C-terminal-deleted DNP (Des-Arg30, Des-Pro31-DNP) on ICa,L, indicating that C-terminal of DNP (Arg30 and Pro31 residues of DNP) is not essential in inhibiting L-type Ca2+ channels. Schirger et al. (1999) have reported that DNP-like immunoreactivity is present in human plasma and