Kinetics of ATP release and cell volume regulation of hyposmotically challenged goldfish hepatocytes

Abstract

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 ATP␥S (an ATP analog) in HYPO triggers a 42% volume decrease. This effect is concentration dependent (K1/2 ϭ 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 ATP␥S did trigger RVD could be explained by showing that ATP␥S 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. extracellular ATP; water transport; ectonucleotidases MOST VERTEBRATE CELLS, when suddenly exposed to hypotonic conditions, rapidly swell due to the influx of water but subsequently, despite continuous osmotic perturbation, this swelling is opposed by the compensatory efflux of osmolytes and water. The resulting cell volume decrease is termed regulatory volume decrease, or RVD (20). RVD is mediated to a large extent by KCl loss through both K ϩ and Cl Ϫ channels, parallel activity of K ϩ -H ϩ and Cl Ϫ -HCO 3 Ϫ exchangers or KCl cotransporters (5, 21). However, little is known on the extracellular factors inducing RVD.In the past few years, a growing body of evidence has shown that extracellular nucleotides, mainly ATP, play a significant role in the control of cell volume regulation by binding to specific cell surface molecules termed "P" receptors (purinic and pyrimidinic receptors) (29). P1 receptors bind adenosine and other nucleosides, whereas P2 receptors bind mainly dinucleotides and trinucleotides. The P2 receptor family (8, 32, 36, 38) consists of two main subtypes, P2X and P2Y, representing ligand-gated cation channels and G protein-coupled receptors (linked to phospholipase C), respectively. At the cell membrane, the availability of agonists of P receptors is tightly regulated by specific membrane-bound enzymes located at the surface of the cell. These include E-NTPDases, a family of enzymes that hydrolyze nucleoside diphosphates and triphosphates, and also...

Homeostasis of Extracellular ATP in Human Erythrocytes

“…Real-time luminometry measurements were performed with cells on coverslips that were mounted in the assay chamber of a custom-built luminometer as described earlier (19). Because luciferase activity at 37°C is only 10% of that observed at 20°C (20), to maintain full luciferase activity real-time ATP measurements were performed in a cool chamber acclimated at 20°C.…”

“…To this end we monitored cell volume of erythrocytes exposed to 3V, both in the presence and absence of carbenoxolone. Relative cell volumes before and after addition of pharmacological agents were estimated at room temperature using the BCECF fluorescence quenching method, as described (34,37,38). Under quenching conditions, fluorescence intensity of human erythrocytes decreased with the increase in fluorophore concentration.…”

On the role of ATP release, ectoATPase activity, and extracellular ADP in the regulatory volume decrease of Huh-7 human hepatoma cells

“…Swollen cells of liver and hepatoma cells lose Cl Ϫ and K ϩ through electroneutral ion transport pathways or by the separate activation of K ϩ and anion channels (27). Although many intracellular signaling processes of cell volume regulation are now well characterized (29), comparatively little is known regarding the extracellular factors controlling RVD (14,48,49). In this respect, mechanical stimuli such as shear stress (7), mechanical strain (55), and hypotonic stress (45) have been shown to induce the release of ATP in the absence of lysis from a variety of cells (36,44,51,52,61), whereas addition of exogenous ATP to hypotonic cells suspensions was able to stimulate RVD (32,49).…”

A Volume Regulatory Response Can Be Triggered by Nucleosides in Human Erythrocytes, a Perfect Osmometer No Longer

“…Similarly, RVD was activated by UTP in salivary gland duct cells (17) and by ATP, UTP, UDP, or ATP␥S in trout and goldfish hepatocytes (18,19).…”

“…Moreover, extracellular adenosine, derived from hydrolysis of extracellular ATP, was shown to inhibit RVD via P1 activation in fish hepatocytes (18,19). Thus, it seems clear that understanding volume regulation requires an understanding of events involved in nucleoside signaling on the cell surface.…”