20-HETE-Induced Changes In Inotropic Effects Depend On The Activation Or Inactivation Of L-type Calcium Channels By PKA And\or PKC In Rat Ventricular Myocytes

The arachidonic acid metabolite generated by cytochrome P450 (CYP)ω-hydroxylases, 20-hydroxyeicosatetraenoic acid (20-HETE), has important roles in regulating vascular tone, but the functions and specific pathways involved in modulating myocardial inotropic effects have not been reported. In the present study, Langendorff method was used to research the effects of 20-HETE on the isolated heart. In low doses, 20-HETE (0.1-3 nmol/L) had a positive function of increasing the pressure of the reperfused heart; however, in the high-dose group, 20-HETE (10 nmol/L) obviously had a negative effect. Furthermore, using fura-2-acetoxymethyl ester (AM) fluorescence imaging and patch-clamp techniques, we studied the effects of 20-HETE on intracellular calcium levels ([Ca2+]i) and the L-type Ca2+ channel in cardiomyocytes isolated from rats. We found that, in low doses, 20-HETE (0.01, 0.03, 0.1μmol/L) elevated intercellular Ca2+ levels in a dose-dependent manner; however, in high doses, 20-HETE (0.3, 1μmol/L) induced decreases in [Ca2+]i in the cardiomyocytes. Low-dose 20-HETE (0.1μmol/L) increased the amplitude of the whole-cell L-type Ca2+ channel current (ICaL); however, the high dose of 20-HETE (1μmol/L) had the opposite effect. When Ca2+ transients were measured using a laser confocal microscope, the amplitudes were increased with low levels of 20-HETE (0.01μmol/L, 0.1μmol/L), but decreased with high 20-HETE (1.0μmol/L). The descending rates of Ca2+ transients were decreased significantly by 20-HETE of a different concentration. The 20-HETE-induced increases in [Ca2+]i, whole-cell L-type Ca2+ channel currents and the amplitudes of Ca2+ transients were blocked by extracellular application of chelerythrine (an inhibitor of protein kinase C, PKC) and H-89 (an inhibitor of cAMP-dependent protein kinase, PKA). PKC and PKA activity assays showed that 0.03μmol/L of 20-HETE increased PKC and PKA activity in isolated cardiomyocytes, while chelerythrine (5μmol/L) and H-89 (5μmol/L) inhibited these effects. Our results indicate that 20-HETE regulates myocardial inotropic effects by activating or inhibiting the L-type calcium channel to modulate [Ca2+]i and that PKC and PKA contribute to these effects.