title: Extracellular osmolarity modulates G protein-coupled receptor-dependent ATP release from 1321N1 astrocytoma cells.
abstract: We previously reported that ATP release from 1321N1 human astrocytoma cells could be stimulated either by activation of G protein-coupled receptors (GPCR) or by hypotonic stress. Cheema et al. (Cheema TA, Ward CE, Fisher SK. J Pharmacol Exp Ther 315: 755-763, 2005) have demonstrated that thrombin activation of protease-activated receptor 1 (PAR1) in 1321N1 cells and primary astrocytes acts synergistically with hypotonic stress to gate the opening of volume-sensitive organic osmolyte and anion channels (VSOAC) and that hypertonic stress strongly inhibits PAR1 gating of VSOAC. We tested the hypothesis that a VSOAC-type permeability might comprise a GPCR-regulated pathway for ATP export by determining whether PAR1-sensitive ATP release from 1321N1 cells is similarly potentiated by hypotonicity but suppressed by hypertonic conditions. Strong hypotonic stress by itself elicited ATP release and positively modulated the response to thrombin. Thrombin-dependent ATP release was also potentiated by mild hypotonic stress that by itself did not stimulate ATP export. Notably, PAR1-sensitive ATP export was greatly inhibited in hypertonic medium. Neither the potency nor efficacy of thrombin as an activator of proximal PAR1 signaling was affected by hypotonicity or hypertonicity. 1,9-Dideoxyforskolin and carbenoxolone similarly attenuated PAR1-dependent ATP release and suppressed the PAR1-independent ATP elicited by strong hypotonic stress. Probenecid attenuated PAR1-stimulated ATP release under isotonic but not mild hypotonic conditions and had no effect on PAR1-independent release stimulated by strong hypotonicity. PAR1-dependent ATP export under all osmotic conditions required concurrent signaling by Ca(2+) mobilization and Rho-GTPase activation. In contrast, PAR1-independent ATP release triggered by strong hypotonicity required neither of these intracellular signals. Thus, we provide the new finding that GPCR-regulated ATP release from 1321N1 astrocytoma cells is remarkably sensitive to both positive and negative modulation by extracellular osmolarity. This supports a model wherein GPCR stimulation and osmotic stress converge on an ATP release pathway in astrocytes that exhibits several features of VSOAC-type channels.
authors.affiliation: Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 44120, USA.
authors.forename: Andrew E
authors.initials: AE
authors.lastname: Blum
authors.forename: B Corbett
authors.initials: BC
authors.lastname: Walsh
authors.forename: George R
authors.initials: GR
authors.lastname: Dubyak
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000838:Anions; D001254:Astrocytoma / Q000378:metabolism*; D020013:Calcium Signaling; D002229:Carbenoxolone / Q000494:pharmacology; D045744:Cell Line, Tumor; D002614:Chelating Agents / Q000494:pharmacology; D005576:Colforsin / Q000031:analogs & derivatives / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation; D045604:Extracellular Fluid / Q000378:metabolism*; D006801:Humans; D006982:Hypertonic Solutions; D007038:Hypotonic Solutions; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D009994:Osmolar Concentration; D011339:Probenecid / Q000494:pharmacology; D044463:Receptor, PAR-1 / Q000187:drug effects / Q000378:metabolism*; D013744:Tetanus Toxin / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000378:metabolism
chemical_list: D000838:Anions; D002614:Chelating Agents; D006982:Hypertonic Solutions; D007038:Hypotonic Solutions; D007473:Ion Channels; D044463:Receptor, PAR-1; D013744:Tetanus Toxin; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D005576:Colforsin; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D013917:Thrombin; D020741:rho GTP-Binding Proteins; D002229:Carbenoxolone; C047983:1,9-dideoxyforskolin; D011339:Probenecid