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

  • Cell and Molecular Physiology
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Abstract

Reducing osmolarity by 35% increased 3H-taurine efflux from Swiss 3T3 fibroblasts from 0.5% to a peak of 5.7%. The presence of ATP (10–100 µM; EC50 1.5 µM) 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, β,γ-methylene-ATP (β,γ-ATP) or 2-methylthio-ATP (Me-ATP), suggesting its mediation by purinergic P2Y2 and P2Y4 metabotropic receptors. Under isosmotic conditions ATP increased the cytosolic [Ca2+] ([Ca2+]i) markedly, but did not increase taurine release. HTR was independent of external Ca2+ but was reduced (by 56–59%) by BAPTA-AM, thapsigargin-induced depletion of intracellular Ca2+ 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 Ca2+-dependent fraction of HTR. This fraction has as signalling elements a PLC-dependent [Ca2+]i increase, resulting from Ca2+ released from thapsigargin-sensitive internal stores, followed by activation of CaM/CaMKII reactions. The Ca2+/ATP effect operates only when the Ca2+-independent, tyrosine kinase-mediated pathway is already activated. Suggested elements of cross-talk between the two pathways are PLC, PI3K and CaMKII.

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Acknowledgements

We wish to thank Claudia Peña-Segura and Alicia Sampieri for their excellent technical assistance. This study was supported in part by grants No. IN206403-3 from DGAPA, UNAM; and 3586-N and 34886-M from CONACyT.

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  1. Department of Biophysics, Institute of Cell Physiology, National University of Mexico (UNAM), Apartado Postal 70-253, 04510, Mexico City, Mexico

    Rodrigo Franco, Rafael Rodríguez & Herminia Pasantes-Morales

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Correspondence to Herminia Pasantes-Morales.

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Franco, R., Rodríguez, R. & Pasantes-Morales, H. Mechanisms of the ATP potentiation of hyposmotic taurine release in Swiss 3T3 fibroblasts. Pflugers Arch - Eur J Physiol 449, 159–169 (2004). https://doi.org/10.1007/s00424-004-1322-1

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