Rabbit erythrocytes possess adenylyl cyclase type II that is activated by the heterotrimeric G proteins Gs and Gi.
Adenosine triphosphate (ATP) release from rabbit erythrocytes occurs in response to deformation or reduced oxygen tension. A signal transduction pathway that relates these stimuli to ATP release has been proposed. This pathway includes the heterotrimeric G proteins, Gs and Gi, adenylyl cyclase, protein kinase A, and the cystic fibrosis transmembrane conductance regulator. Importantly, adenylyl cyclase types II, IV and VII have been reported to be activated by both Gs and Gi. Here, we demonstrate that rabbit erythrocytes possess an adenylyl cyclase subtype that is activated both by the alpha subunit and the betagamma subunit of Gs and Gi, respectively. Washed rabbit erythrocytes released ATP when exposed to the beta adrenergic receptor-mediated activator of Gs, isoproterenol (ISO, 10 microM, n = 8, p < 0.05) as well as in response to incubation with a direct activator of Gi, mastoparan 7 (MAS7, 10 microM, n = 12, p < 0.05). In contrast, an inactive mastoparan derivative, mastoparan 17 (MAS 17, 10 microM, n = 6) did not stimulate ATP release. Importantly, incubation of washed rabbit erythrocytes with either isoprotenerol (ISO) (10 microM, n = 7) or MAS7 (10 microM, n = 11) resulted in increases in cyclic adenosine monophosphate (cAMP) (p < 0.01).Western analysis was used to determine if an adenylyl cyclase capable of being activated by both Gs and Gi was a component of rabbit erythrocyte membranes. We identified adenylyl cyclase type II with two antibodies generated against different epitopes of the protein. These results provide support for the hypothesis that, in rabbit erythrocytes, activation of either Gs or Gi results in the stimulation of adenylyl cyclase resulting in increases in cAMP leading, ultimately, to the release of ATP.
Sprague
Randy
R
Department of Pharmacological and Physiological Science, Saint Louis University, School of Medicine, 1402 South Grand Blvd, Saint Louis, Missouri 63104, USA. spraguer@slu.edu
Bowles
Elizabeth
E
Stumpf
Madelyn
M
Ricketts
Gregory
G
Freidman
Alberto
A
Hou
Wei-Hsien
WH
Stephenson
Alan
A
Lonigro
Andrew
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D000242:Cyclic AMP / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D010455:Peptides / Q000494:pharmacology; D011817:Rabbits
D036341:Intercellular Signaling Peptides and Proteins; C084070:Mas7 protein, synthetic; D010455:Peptides; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases; C100622:adenylyl cyclase 2; D007545:Isoproterenol