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.
Department of Biophysics, Institute of Cell Physiology, National University of Mexico, Apartado Postal 70-253, 04510 Mexico City, Mexico.
Rodrigo
R
Franco
Rafael
R
Rodríguez
Herminia
H
Pasantes-Morales
D016475:3T3 Cells; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000378:metabolism; D004357:Drug Synergism; D007038:Hypotonic Solutions / Q000494:pharmacology*; D051379:Mice; D009997:Osmotic Pressure; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D058473:Receptors, Purinergic P2Y2; D013654:Taurine / Q000378:metabolism*; D014882:Water-Electrolyte Balance / Q000502:physiology*
D018118:Chloride Channels; D007038:Hypotonic Solutions; C546386:P2ry2 protein, mouse; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; C104749:purinoceptor P2Y4; D013654:Taurine; D000255:Adenosine Triphosphate; D019869:Phosphatidylinositol 3-Kinases; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium