Mechanisms of the ATP potentiation of hyposmotic taurine release in Swiss 3T3 fibroblasts

Reducing osmolarity by 35% increased (3)H-taurine efflux from Swiss 3T3 fibroblasts from 0.5% to a peak of 5.7%. The presence of ATP (10-100 microM; EC(50) 1.5 microM) increased taurine efflux up to 10%, and decreased the set point for hyposmotically stimulated taurine release (HTR). ATP potentiation was mimicked by UTP, reduced by addition of suramin and pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and unaffected by ADP, beta,gamma-methylene-ATP (beta,gamma-ATP) or 2-methylthio-ATP (Me-ATP), suggesting its mediation by purinergic P2Y(2) and P2Y(4) metabotropic receptors. Under isosmotic conditions ATP increased the cytosolic [Ca(2+)] ([Ca(2+)](i)) markedly, but did not increase taurine release. HTR was independent of external Ca(2+) but was reduced (by 56-59%) by BAPTA-AM, thapsigargin-induced depletion of intracellular Ca(2+) stores, or phospholipase C (PLC) inhibition. Blockade of calmodulin (CaM) or calmodulin kinase II (CaMKII) reduced HTR by 54% and 76%, respectively. The ATP-mediated potentiation was prevented fully by all these treatments. HTR was reduced by 30-50% by blockers of protein tyrosine kinases (AG18), phosphoinositide 3-kinase (PI3K) (wortmannin), p21rho (toxin B), p21rho-kinase (Y27632) and the stress-activated kinase p38 (PD169316). ATP-mediated potentiation was reduced similarly by these blockers. Simultaneous inhibition of PI3K and CaMKII abolished HTR. Altogether, these results suggest a modulatory effect of ATP, probably exerted by a potentiation of the Ca(2+)-dependent fraction of HTR. This fraction has as signalling elements a PLC-dependent [Ca(2+)](i) increase, resulting from Ca(2+) released from thapsigargin-sensitive internal stores, followed by activation of CaM/CaMKII reactions. The Ca(2+)/ATP effect operates only when the Ca(2+)-independent, tyrosine kinase-mediated pathway is already activated. Suggested elements of cross-talk between the two pathways are PLC, PI3K and CaMKII.