title: Uncoupling of sarcoplasmic reticulum Ca²⁺-ATPase by N-arachidonoyl dopamine. Members of the endocannabinoid family as thermogenic drugs.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  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
  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.
authors.affiliation: Department of Biomedicine, University of Aarhus, Aarhus C, Denmark. yam@biophys.au.dk
authors.forename: Y A
authors.initials: YA
authors.lastname: Mahmmoud
authors.forename: M
authors.initials: M
authors.lastname: Gaster
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000328:Adult; D000818:Animals; D001095:Arachidonic Acids / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D004298:Dopamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D008875:Middle Aged; D018485:Muscle Fibers, Skeletal / Q000187:drug effects* / Q000378:metabolism; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism*
chemical_list: D001095:Arachidonic Acids; C474941:arachidonyl dopamine; D000255:Adenosine Triphosphate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium; D004298:Dopamine