Effects of hypo-osmotic stress on ATP release in isolated turbot (Scophthalmus maximus) hepatocytes

Background information. ATP is released from many cell types exposed to hypo-osmotic shock and is involved in RVD (regulatory volume decrease). Purinergic signalling events have been extensively investigated in mammals, but not in marine teleosteans. Results. The effect of hypo-osmotic shock on ATP release was examined in isolated hepatocytes from turbot (Scophthalmus maximus), a marine flatfish. Hypo-osmotic stress (240 mOsm·kg−1) induced a significant increase in ATP efflux, and was inhibited by a potential CFTR (cystic fibrosis transmembrane conductance regulator) inhibitor, glibenclamide, but not by the MDR1 (multidrug resistance 1) P-glycoprotein inhibitor, verapamil. ATP efflux could be a cAMP-dependent process, as IBMX (isobutylmethylxanthine) and forskolin triggered the process under iso-osmotic conditions. Protein kinases, including protein kinase C, could also be involved, as staurosporine and chelerythrine inhibited the mechanism. Calcium could contribute to ATP efflux as ionomycin, a calcium ionophore, elicited a rapid release under iso-osmotic conditions, and chelation using EGTA abolished ATP release under hypo-osmotic conditions. RVD was partially abolished by apyrase, an ATP scavenger, and suramin, a purinoceptor antagonist. Moreover, hypo-osmotic shock induced a rise in intracellular calcium which could be involved in RVD. Since extracellular ATP triggered an increase in cellular free-calcium content under iso-osmotic conditions, our results could indicate that hypo-osmotic-induced ATP efflux contributes to RVD in turbot hepatocytes by stimulating purinergic receptors, which may lead to activation of a calcium signalling pathway. Conclusions. These data provide the first evidence of volume-sensitive ATP signalling for volume maintenance in a marine teleost fish cell type.