Publication Date:
2013-05-16
Description:
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.
Print ISSN:
0363-6143
Electronic ISSN:
1522-1563
Topics:
Medicine
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