Kinetics of ATP release and cell volume regulation of hyposmotically challenged goldfish hepatocytes.
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 h. Incubation with ATPgammaS (an ATP analog) in HYPO triggers a 42% volume decrease. This effect is concentration dependent (K(1/2) = 760 nM) 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 ATPgammaS did trigger RVD could be explained by showing that ATPgammaS 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 nonlytic 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 the addition of micromolar concentrations of nucleotides. However, under very low assay volumes, sufficient endogenous extracellular [ATP] can build up to induce RVD.
Pafundo
Diego E
DE
Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina.
Chara
Osvaldo
O
Faillace
María P
MP
Krumschnabel
Gerhard
G
Schwarzbaum
Pablo J
PJ
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D006054:Goldfish / Q000502:physiology*; D022781:Hepatocytes / Q000378:metabolism* / Q000473:pathology*; D006863:Hydrogen-Ion Concentration; D008954:Models, Biological; D009997:Osmotic Pressure; D058906:Purinergic P1 Receptor Agonists; D058915:Purinergic P1 Receptor Antagonists; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D013997:Time Factors
D058906:Purinergic P1 Receptor Agonists; D058915:Purinergic P1 Receptor Antagonists; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D000255:Adenosine Triphosphate