INVESTIGADORES
ESPELT Maria Victoria
artículos
Título:
11. On the role of ATP release, ectoATPase activity and extracellular ADP in the regulatory volume decrease of Huh-7 human hepatoma cells.
Autor/es:
ESPELT M.V; DE TEZANOS PINTO F; ALVAREZ C.L.; SANCHEZ ALBERTI G; INCICCO, J; M. F. LEAL DENIS; DAVIO, C; P.J. SCHWARZBAUM
Revista:
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY
Editorial:
AMER PHYSIOLOGICAL SOC
Referencias:
Lugar: Bethesda; Año: 2013 p. 1013 - 1026
ISSN:
0363-6143
Resumen:
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% RVD40 (extent of RVD after 40 min of maximum), whereas the concentration of extracellular ATP (ATPe) 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 RVD40 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., RVD40 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 P2Y13. RT-PCR and Western blot analysis confirmed the presence of P2Y13. 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 ATPe 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 ATPe to ADPe by ENTPDase 2 activity facilitates the accumulated ADPe to activate P2Y13 receptors, which mediate complete RVD.
