title: A2B adenosine receptors inhibit superoxide production from mitochondrial complex I in rabbit cardiomyocytes via a mechanism sensitive to Pertussis toxin.
abstract: OBJECTIVE
  A(2B) adenosine receptors protect against ischaemia/reperfusion injury by activating survival kinases including extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K). However, the underlying mechanism(s) and signalling pathway(s) remain undefined.
  
  
  METHODS
  HEK 293 cells stably transfected with human A(2B) adenosine receptors (HEK-A(2B) ) and isolated adult rabbit cardiomyocytes were used to assay phosphorylation of ERK by Western blot and cation flux through cAMP-gated channels by patch clamp methods. Generation of reactive oxygen species (ROS) by mitochondria was measured with a fluorescent dye.
  
  
  RESULTS
  In HEK-A(2B) cells, the selective A(2B) receptor agonist Bay 60-6583 (Bay 60) increased ERK phosphorylation and cAMP levels, detected by current through cAMP-gated ion channels. However, increased cAMP or its downstream target protein kinase A was not involved in ERK phosphorylation. Pertussis toxin (PTX) blocked ERK phosphorylation, suggesting receptor coupling to G(i) or G(o) proteins. Phosphorylation was also blocked by inhibition of PI3K (with wortmannin) or of ERK kinase (MEK1/2, with PD 98059) but not by inhibition of NO synthase (NOS). In cardiomyocytes, Bay 60 did not affect cAMP levels but did block the increased superoxide generation induced by rotenone, a mitochondrial complex I inhibitor. This effect of Bay 60 was inhibited by PD 98059, wortmannin or PTX. Inhibition of NOS blocked superoxide production because NOS is downstream of ERK.
  
  
  CONCLUSIONS
  Activation of A(2B) adenosine receptors reduced superoxide generation from mitochondrial complex I through G(i/o) , ERK, PI3K, and NOS, all of which have been implicated in ischaemic preconditioning.
authors.affiliation: Department of Physiology Pharmacology Medicine, University of South Alabama, College of Medicine, Mobile, AL 36688, USA.
authors.forename: Xiulan
authors.initials: X
authors.lastname: Yang
authors.forename: Wenkuan
authors.initials: W
authors.lastname: Xin
authors.forename: Xi-Ming
authors.initials: XM
authors.lastname: Yang
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kuno
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Rich
authors.forename: Michael V
authors.initials: MV
authors.lastname: Cohen
authors.forename: James M
authors.initials: JM
authors.lastname: Downey
mesh_terms: D000818:Animals; D015153:Blotting, Western; D000242:Cyclic AMP / Q000378:metabolism; D042967:Electron Transport Complex I / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D008929:Mitochondria, Heart / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D018408:Patch-Clamp Techniques; D037342:Pertussis Toxin / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011817:Rabbits; D043704:Receptor, Adenosine A2B / Q000235:genetics / Q000502:physiology*; D013481:Superoxides / Q000378:metabolism*; D014162:Transfection
chemical_list: D000242:Cyclic AMP; D042967:Electron Transport Complex I; D048049:Extracellular Signal-Regulated MAP Kinases; D009569:Nitric Oxide; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases; D043704:Receptor, Adenosine A2B; D013481:Superoxides