ATP release and RVD in goldfish hepatocytes under hypotonic shock

PAFUNDO D. E., FAILLACE, M. P., KRUMSCHNABEL, G., AND SCHWARZBAUM, P. J

Universidad Favaloro, Buenos Aires

Congreso; XXII Latinoamerican and First Ibero-American congress of physiological sciences; 2006

Sociedad Argentina de Fisiologia

ATP release and RVD in Goldfish hepatocytes under hypotonic shock. D. E. Pafundo, M. P. Faillace, G. (1) Krumschnabel,,y P. J. Schwarzbaum, IQUIFIB. Facultad de Farmacia y Bioquimica (UBA), Buenos Aires, Argentina 1 Department of Zoology and Limnology, University of Innsbruck, Insbruck, Austria   In most cells, hypotonic swelling is followed by a compensatory shrinkage called regulatory volume decrease (RVD). This effect is thought to prevent cell disruption and thus extend cell viability. However, when goldfish (Carassius auratus) hepatocytes are challenged with a 40% hypotonic medium, cells increase their volume 1.7 times and remain swollen with no significant loss of cell viability. According to previous work in fish hepatocytes (Pafundo et al. 2004), hypotonic exposure cause cells to increase their volume, followed by ATP release. Once in the extracellular medium, ATP may act on P2 receptors to initiate a cascade of events which end up with efflux of osmolytes (mainly K+ and Cl-) and water and recovery of cell volume. In the present work, we show that goldfish hepatocytes are capable of 41.95+4.27 % cell volume regulation in presence of 1 mM ATPgS. The effect of ATPgS was concentration dependent (1–50 mM, K0.5=570nM) and is abolished by antagonists of P2 receptors (64% inhibition). We also show that they release up to 725 + 166 nM ATP even though these cells do not perform RVD in hypotonic conditions. To solve the previous discrepancy, we found that goldfish hepatocytes are capable of RVD when the experiments are performed in a small medium volume, allowing the release ATP to accumulate in the extracellular space. That is: under 40 ml hypotonic medium (which resulted in a column of media of 100 um above the cells), cells had 55.63+4.55 % RVD This RVD was abolished by P2 antagonists with a 72.8 % inhibition. In conclusion, we determine that goldfish hepatocytes, presumed to have no RVD, do release ATP under hypotonic stress, and that this ATP can induce RVD when accumulated in the extracellular space.