Submitted on July 19, 2007
Accepted on October 8, 2007
Kinetics of ATP release and cell volume regulation of hyposmotically challenged goldfish hepatocytes
Diego Esteban Pafundo1, Osvaldo Chara2, Maria Paula Faillace1, Gerhard Krumschnabel3, and PABLO J SCHWARZBAUM1*
1 Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, IQUIFIB, Buenos Aires, Argentina
2 Universidad Nacional de La Plata, IFLYSIB, La Plata, Buenos Aires, Argentina
3 Zoology and Limnology, University of Innsbruck, Technikerstr. 25, Innsbruck, A-6020, Austria; Division of Developmental Immunology, Innsbruck Medical University, Innsbruck, Austria
* To whom correspondence should be addressed. E-mail: pablos{at}qb.ffyb.uba.ar.
In most animal cells, hypotonic swelling is followed by a regulatory volume decrease (RVD) thought to prevent cell death. In contrast, goldfish hepatocytes challenged with hypotonic medium (180 mosM, HYPO) increase their volume 1.7 times but remain swollen and viable for at least 5 hours. Incubation with ATP
S (an ATP analog) in HYPO triggers a 42 % volume decrease. This effect is concentration dependent (K1/2=760nM), and partially abolished by P2 receptor antagonists (64% inhibition). A similar induction of RVD is observed with ATP, UTP and UDP, whereas adenosine inhibits RVD. Goldfish hepatocytes release more than 500 nM ATP during the first minutes of HYPO with no induction of RVD. The fact that similar concentrations of ATPS did trigger RVD could be explained by showing that ATPS induced ATP release. Finally, we observed that in a very small extracellular volume hepatocytes do show a 56 % RVD. This response was diminished by P2 receptor antagonists (73 %) and increased (73 %) when the extracellular ATP hydrolysis was inhibited 72 %.
Using a mathematical model, we predict that during the first 2 min of HYPO exposure the extracellular [ATP] is mainly governed by ATP diffusion and by both non-lytic and lytic ATP release, with almost no contribution from ecto-ATPase activity. We show that goldfish hepatocytes under standard HYPO (large volume) do not display RVD unless this is triggered by addition of micromolar concentrations of nucleotides. However, under very low assay volumes, sufficient endogenous extracellular [ATP] can build up to induce RVD.
