title: On the role of ATP release, ectoATPase activity, and extracellular ADP in the regulatory volume decrease of Huh-7 human hepatoma cells.
abstract: Hypotonicity triggered in human hepatoma cells (Huh-7) the release of ATP and cell swelling, followed by volume regulatory decrease (RVD). We analyzed how the interaction between those processes modulates cell volume. Cells exposed to hypotonic medium swelled 1.5 times their basal volume. Swelling was followed by 41% RVD(40) (extent of RVD after 40 min of maximum), whereas the concentration of extracellular ATP (ATP(e)) increased 10 times to a maximum value at 15 min. Exogenous apyrase (which removes di- and trinucleotides) did not alter RVD, whereas exogenous Na(+)-K(+)-ATPase (which converts ATP to ADP in the extracellular medium) enhanced RVD(40) by 2.6 times, suggesting that hypotonic treatment alone produced a basal RVD, whereas extracellular ADP activated RVD to achieve complete volume regulation (i.e., RVD(40) ≈100%). Under hypotonicity, addition of 2-(methylthio)adenosine 5'-diphosphate (2MetSADP; ADP analog) increased RVD to the same extent as exposure to Na(+)-K(+)-ATPase and the same analog did not stimulate RVD when coincubated with MRS2211, a blocker of ADP receptor P2Y(13). RT-PCR and Western blot analysis confirmed the presence of P2Y(13). Cells exhibited significant ectoATPase activity, which according to RT-PCR analysis can be assigned to ENTPDase2. Both carbenoxolone, a blocker of conductive ATP release, and brefeldin A, an inhibitor of exocytosis, were able to partially decrease ATP(e) accumulation, pointing to the presence of at least two mechanisms for ATP release. Thus, in Huh-7 cells, hypotonic treatment triggered the release of ATP. Conversion of ATP(e) to ADP(e) by ENTPDase 2 activity facilitates the accumulated ADP(e) to activate P2Y(13) receptors, which mediate complete RVD.
authors.affiliation: Instituto de Química y Fisicoquímica Biológicas (Facultad de Farmacia y Bioquímica), Universidad de Buenos Aires, Buenos Aires, Argentina. victoria@qb.ffyb.uba.ar
authors.forename: María V
authors.initials: MV
authors.lastname: Espelt
authors.forename: Felicitas
authors.initials: F
authors.lastname: de Tezanos Pinto
authors.forename: Cora L
authors.initials: CL
authors.lastname: Alvarez
authors.forename: Germán Sanchez
authors.initials: GS
authors.lastname: Alberti
authors.forename: Jeremías
authors.initials: J
authors.lastname: Incicco
authors.forename: María F
authors.initials: MF
authors.lastname: Leal Denis
authors.forename: Carlos
authors.initials: C
authors.lastname: Davio
authors.forename: Pablo J
authors.initials: PJ
authors.lastname: Schwarzbaum
mesh_terms: D000244:Adenosine Diphosphate / Q000031:analogs & derivatives; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001391:Azo Compounds / Q000494:pharmacology; D020126:Brefeldin A / Q000494:pharmacology; D002229:Carbenoxolone / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D045744:Cell Line, Tumor; D048429:Cell Size; D005089:Exocytosis / Q000187:drug effects; D006801:Humans; D007038:Hypotonic Solutions; D047908:Intracellular Signaling Peptides and Proteins; D008113:Liver Neoplasms / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D058919:Purinergic P2 Receptor Antagonists / Q000494:pharmacology; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D018048:Receptors, Purinergic P2
chemical_list: D001391:Azo Compounds; D007038:Hypotonic Solutions; D047908:Intracellular Signaling Peptides and Proteins; C556691:MRS 2211; D011500:Protein Synthesis Inhibitors; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D020126:Brefeldin A; D011732:Pyridoxal Phosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D002229:Carbenoxolone
keywords: P2Y13 receptor; cell volume regulation; human hepatoma; nucleotides; purinergic signaling