Volume regulatory decrease mediated by extracellular nucleotides

PAFUNDO DIEGO; CHARA OSVALDO; SCHWARZBAUM PABLO JULIO

Montevideo

Congreso; 5th Southern Cone Biophysics Congress. 6th International Conference of Biological Physics.; 2007

IUPAP - SAB

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 viablefor 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 withmicromolar 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 goldfishhepatocytes release more than 500 nM ATP with no concomitant RVD. The fact that a similar concentration of ATPgS did trigger RVDcould be explained by showing that it induced a release of ATP by hepatocytes. Finally, in a very small extracellular volume hepatocytesdisplay a 56 % RVD. This was diminished by P2 antagonists (73 %) and increased (73 %) when the extracellular hydrolysis of ATP wasinhibited by 72 %. Using a mathematical model we predict that during the first 2 min of HYPO exposure the concentration of ATPe ismainly 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 thatgoldfish hepatocytes under standard HYPO (large volume) do not display RVD unless this is triggered by addition of micromolarconcentrations of various nucleotides. However, under very low assay volumes, sufficient endogenous [ATPe] can build up to induce RVD.Resumen 267 http://icbp2007.congresoselis.info/resumenAmpliado.php?idTL=7611 de