Volume regulatory decrease mediated by extracellular nucleotides

PAFUNDO, DE, CHARA, O., SCHWARZBAUM PJ

Hotel Radisson, Montevideo, Uruguay

Simposio; Symposium on water transport in epithelium; 2007

International Union of Pure and Applied Biophysics (IUPAB)

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 with no loss of viability. Incubation of goldfish hepatocytes with ATPgS (an ATP analog) in HYPO triggers a 42 % volume decrease. This effect is dependent on its presence during the first 5 min of HYPO exposure, is concentration dependent (K1/2=570nM), and is partially abolished by P2 receptor antagonists (64% inhibition). A similar induction of RVD is observed with micromolar concentrations of the P2 receptor agonists ATP, UTP and UDP, whereas ADP fails to induce RVD and adenosine inhibits RVD. A luciferin-luciferase-based luminiscence method used to estimate the release of ATP to the extracellular medium showed that goldfish hepatocytes release more than 500 nM ATP with no concomitant RVD. The fact that a similar concentration of ATPgS did trigger RVD could be explained by showing that it induced a release of ATP by hepatocytes. Finally, in a very small extracellular volume hepatocytes display a 56 % RVD. This was diminished by P2 antagonists (73 %) and increased (73 %) when the extracellular hydrolysis of ATP was inhibited by 72 %. Using a mathematical model we predict that during the first 2 min of HYPO exposure the concentration of ATPe is mainly governed by ATP diffusion and by both non-lytic and lytic ATP release, with almost no contribution from ecto-ATPase activity. Later on, ecto-ATPase activity becomes important in defining the time dependent decay of extracellular ATP levels. Our study shows that goldfish hepatocytes under standard HYPO (large volume) do not display RVD unless this is triggered by addition of micromolar concentrations of various nucleotides. However, under very low assay volumes, sufficient endogenous [ATPe] can build up to induce RVD.