title: Homeostasis of extracellular ATP in human erythrocytes.
abstract: We explored the intra- and extracellular processes governing the kinetics of extracellular ATP (ATPe) in human erythrocytes stimulated with agents that increase cAMP. Using the luciferin-luciferase reaction in off-line luminometry we found both direct adenylyl cyclase activation by forskolin and indirect activation through β-adrenergic stimulation with isoproterenol-enhanced [ATP]e in a concentration-dependent manner. A mixture (3V) containing a combination of these agents and the phosphodiesterase inhibitor papaverine activated ATP release, leading to a 3-fold increase in [ATP]e, and caused increases in cAMP concentration (3-fold for forskolin + papaverine, and 10-fold for 3V). The pannexin 1 inhibitor carbenoxolone and a pannexin 1 blocking peptide ((10)Panx1) decreased [ATP]e by 75-84%. The residual efflux of ATP resulted from unavoidable mechanical perturbations stimulating a novel, carbenoxolone-insensitive pathway. In real-time luminometry experiments using soluble luciferase, addition of 3V led to an acute increase in [ATP]e to a constant value of ∼1 pmol × (10(6) cells)(-1). A similar treatment using a surface attached luciferase (proA-luc) triggered a rapid accumulation of surface ATP levels to a peak concentration of 2.4 pmol × (10(6) cells)(-1), followed by a slower exponential decay (t(&frac12;) = 3.7 min) to a constant value of 1.3 pmol × (10(6) cells)(-1). Both for soluble luciferase and proA-luc, ATP efflux was fully blocked by carbenoxolone, pointing to a 3V-induced mechanism of ATP release mediated by pannexin 1. Ecto-ATPase activity was extremely low (∼28 fmol × (10(6) cells min)(-1)), but nevertheless physiologically relevant considering the high density of erythrocytes in human blood.
authors.affiliation: IQUIFIB, Department of Biological Chemistry, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, CIII3AAD, Argentina.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Montalbetti
authors.affiliation: IQUIFIB, Department of Biological Chemistry, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, CIII3AAD, Argentina.
authors.forename: Maria F
authors.initials: MF
authors.lastname: Leal Denis
authors.affiliation: Laboratory of Molecular Endocrinology and Signal Transduction, Institute of Biology and Experimental Medicine-CONICET, Vuelta de Obligado 2490, CP 1428 Buenos Aires, Argentina; Department of Biological Chemistry, School of Sciences, University of Buenos Aires, Buenos Aires, CIII3AAD, Argentina.
authors.forename: Omar P
authors.initials: OP
authors.lastname: Pignataro
authors.affiliation: Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
authors.forename: Eiry
authors.initials: E
authors.lastname: Kobatake
authors.affiliation: Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina 27599-7248.
authors.forename: Eduardo R
authors.initials: ER
authors.lastname: Lazarowski
authors.affiliation: IQUIFIB, Department of Biological Chemistry, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, CIII3AAD, Argentina. Electronic address: pjs@qb.ffyb.uba.ar.
authors.forename: Pablo J
authors.initials: PJ
authors.lastname: Schwarzbaum
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry*; D000262:Adenylyl Cyclases / Q000737:chemistry; D000818:Animals; D002229:Carbenoxolone / Q000737:chemistry; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004912:Erythrocytes / Q000378:metabolism*; D006706:Homeostasis; D006801:Humans; D006868:Hydrolysis; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D010208:Papaverine / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D014981:Xenopus
chemical_list: D010455:Peptides; D005576:Colforsin; D000255:Adenosine Triphosphate; D010208:Papaverine; D000242:Cyclic AMP; D008156:Luciferases; D000262:Adenylyl Cyclases; D007545:Isoproterenol; D002229:Carbenoxolone