Effects of the endogenous cannabinoid anandamide on voltage-dependent sodium and calcium channels in rat ventricular myocytes.

BACKGROUND AND PURPOSE: The endocannabinoid anandamide (N-arachidonoyl ethanolamide; AEA) exerts negative inotropic and antiarrhythmic effects in ventricular myocytes. EXPERIMENTAL APPROACH: Whole-cell patch-clamp technique and radioligand-binding methods were used to analyse the effects of anandamide in rat ventricular myocytes. KEY RESULTS: In the presence of 1-10 µM AEA, suppression of both Na(+) and L-type Ca(2+) channels was observed. Inhibition of Na(+) channels was voltage and Pertussis toxin (PTX) - independent. Radioligand-binding studies indicated that specific binding of [(3) H] batrachotoxin (BTX) to ventricular muscle membranes was also inhibited significantly by 10 µM metAEA, a non-metabolized AEA analogue, with a marked decrease in Bmax values but no change in Kd . Further studies on L-type Ca(2+) channels indicated that AEA potently inhibited these channels (IC50 0.1 µM) in a voltage- and PTX-independent manner. AEA inhibited maximal amplitudes without affecting the kinetics of Ba(2+) currents. MetAEA also inhibited Na(+) and L-type Ca(2+) currents. Radioligand studies indicated that specific binding of [(3) H]isradipine, was inhibited significantly by metAEA. (10 µM), changing Bmax but not Kd . CONCLUSION AND IMPLICATIONS: Results indicate that AEA inhibited the function of voltage-dependent Na(+) and L-type Ca(2+) channels in rat ventricular myocytes, independent of CB1 and CB2 receptor activation.

Effects of endogenous cannabinoid anandamide on cardiac Na⁺/Ca²âº exchanger.

Endocannabinoid anandamide (N-arachidonoyl ethanolamide; AEA) has been shown to cause negative inotropic and antiarrhythmic effects in ventricular myocytes. In this study, using whole-cell patch clamp technique, we have investigated the effects of AEA on cardiac Na(+)/Ca(2+) exchanger (NCX1)-mediated currents. AEA suppressed NCX1 with an IC50 value of 4.7 µM. Both inward and outward components of exchanger currents were suppressed by AEA equally. AEA inhibition was mimicked by the metabolically stable analogue, methanandamide (metAEA, 10 µM) while it was not influenced by inhibition of fatty acid amide hydrolase with 1 µM URB597 incubation. The effect of AEA, was not altered in the presence of cannabinoid receptor 1 and 2 antagonists AM251 (1 µM) and AM630 (1 µM), respectively. In addition, inhibition by AEA remained unchanged after pertussis toxin (PTX, 2 µg/ml) treatment or following the inclusion of GDP-ß-S (1 mM) in pipette solution. Currents mediated by NCX1 expressed in HEK-293 cells were also inhibited by 10 µM AEA a partially reversible manner. Confocal microscopy images indicated that the intensity of YFP-NCX1 expression on cell surface was not altered by AEA. Collectively, the results indicate that AEA directly inhibits the function of NCX1 in rat ventricular myocytes and in HEK-293 cells expressing NCX1.