Effects of the Endocannabinoid Anandamide On Excitability and Calcium Signaling In Rat Ventricular Myocytes
Abstract
Endogenous cannabinoids (endocannabinoids) exert a wide range of Biological effects. In addition to having their well-known neurobehavioral effects, a role for the major endocannabinoid anandamide (N-arachidonoyl ethanol amide; AEA), in the cardiovascular system in various pathological conditions has been Reported. The aim of this thesis is to explore the effects of AEA on contractility, Ca2+ signaling, and action potential (AP) characteristics in rat ventricular myocytes. A video edge detection system was used to measure myocyte shortening. Intracellular Ca2+was measured in cells loaded with the fluorescent indicator fura-2 AM. AEA (I µM) caused a significant decrease in the amplitude of electrically-evoked myocyte shortening. The effect of AEA was not altered in the presence of pertussis toxin (PTX), AM251 and SR141716 (CB1 m1tagonists) or AM630 m1d SR 144528 (CB2 antagonists). AEA also caused a significant decrease in the amplitudes of electrically-evoked Ca2+ transients. However, the amplitudes of caffeine-evoked Ca2+ transients and the rate of recovery of electrically-evoked Ca2+transients following caffeine application were not altered. In the whole-cell mode of patch-clamp technique, AEA (I µM) significantly decreased the duration of APs. The inhibition was not altered in the presence of PTX, AM251 and AM630. Furthermore, AEA inhibited voltage-activated inward Na+ (INa) and Ca2+ (I, L.ca) currents; major ionic currents shaping the APs in ventricular myocytes, in a voltage and PTX-independent manner. Cardiac Na+/Ca2+ exchanger (NCXl) mediated currents were also suppressed by AEA. The effect of AEA was not influenced by the inhibition of fatty acid amide hydrolase (F AAH) or in the presence of PTX, AM251 and AM630 or following the inclusion of GDP-β-S in pipette solution. The results of this study indicate for the first time that impaired Ca2+ signaling underlies the negative inotropic actions of AEA in rat ventricular myocytes, and that the direct interaction of AEA with ion channel(s) shaping APs, mediates, at least in part, the effects of AEA on myocyte contractility. In addition, the results indicate for the first time that, under normal conditions, AEA can directly inhibit the activity of NCX1 in ventricular myocytes. In view of the massive release of various N- acylethanolamines (NAEs), including AEA, during cardiac ischemia and hypoxic conditions, further understanding of their mechanism(s) of action and target proteins is essential in the development of better treatment modalities under pathological conditions.