Uncoupling of sarcoplasmic reticulum Ca2+-ATPase by N-arachidonoyl dopamine. Members of the endocannabinoid family as thermogenic drugs

BACKGROUND AND PURPOSE The sarcoplasmic reticulum Ca2+-ATPase (SERCA) plays a role in thermogenesis. The exogenous compound capsaicin increased SERCA-mediated ATP hydrolysis not coupled to Ca2+ transport. Here, we have sought to identify endogenous compounds that may function as SERCA uncoupling agents. EXPERIMENTAL APPROACH Using isolated SR vesicles from rabbits, we have screened for endogenous compounds that uncouple SERCA. We have also studied their ability to deplete cytoplasmic ATP from human skeletal muscle cells in culture. KEY RESULTS Studies on SR vesicles showed that the endogenous lipid metabolite N-arachidonoyl dopamine (NADA) was a potent stimulator of SERCA uncoupling. NADA stabilized an E1-like pump conformation that had a lower dephosphorylation rate, low affinity for Ca2+ at the luminal sites and a specific proteinase K cleavage pattern involving protection of the C-terminal p83C fragment from further cleavage. Moreover, we found a significantly decreased cytoplasmic ATP levels following treatment of skeletal muscle cells with 100 nM NADA. This effect was dependent on the presence of glucose and abolished by pretreatment with the specific SERCA inhibitor thapsigargin, regardless of the presence of glucose. CONCLUSIONS AND IMPLICATIONS NADA is an endogenous molecule that may function as SERCA uncoupling agent in vivo. Members of the endocannabinoid family exert concerted actions on several Ca2+-handling proteins. Uncoupling of SERCA by exogenous compounds could be a novel post-mitochondrial strategy for reduction of cellular ATP levels. In addition, signalling networks leading to SERCA uncoupling can be explored to study the importance of this ion pump in pathophysiological conditions related to metabolism.