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

Abstract

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.

Osmoregulation of Taurine Efflux from Cultured Human Breast Cancer Cells: Comparison with Volume Activated Cl^- Efflux and Regulation by Extracellular ATP

“…Several groups have studied the association between extracellular ATP and volumesensitive taurine efflux and the relationship appears to be complex and dependent upon cell type: some studies have reported a stimulation of taurine release by ATP [19,30,31], some have found that ATP inhibits taurine efflux [24] whereas others have found no effect [26]. The present study shows that MDA-MB-231 and MCF-7 cells differed in their response to extracellular ATP.…”

Activation of P2Y receptors causes strong and persistent shrinkage of C11-MDCK renal epithelial cells

“…It has become increasingly clear that agonists of these receptors, and ATP in particular, may accelerate cell-volume recovery under anisosmotic conditions and potently modulate volume-sensitive channels as well as transporters in cells, even in the absence of cell-volume changes (15,28,30,54). It has also been established that signaling linked to G protein-coupled receptors may impact cell volume, as, for example, in mouse Means Ϯ SE values (in nmol/mg of protein) from experiments performed in triplicate are shown.…”

Two conventional protein kinase C isoforms, α and βI, are involved in the ATP‐induced activation of volume‐regulated anion channel and glutamate release in cultured astrocytes

“…However, a number of laboratories have demonstrated that stimulation of GPCRs, including several metabotropic P2Y receptors for ATP and ADP, produces limited activation of a VRAC-like pathway in non-swollen cells and greatly increases its activity in cells exposed to hypoosmotic medium (Jeremic et al 2001;Mongin and Kimelberg 2002;Mongin and Kimelberg 2003;Loveday et al 2003;Darby et al 2003;Franco et al 2004). Depending on the experimental system and the GPCR involved, PKCs have been found to play major or minor roles in the regulation of VRAC activity by GPCR agonists (Loveday et al 2003;Falktoft and Lambert 2004;Mongin and Kimelberg 2005;Heacock et al 2006;Cheema et al 2007;Ramos-Mandujano et al 2007).…”

“…An early hypothesis that cell swelling activates VRACs indirectly, via an autocrine release of ATP and subsequent activation of P2Y receptors (Wang et al 1996), was later rejected based on contradicting experimental observations in numerous cell lines (see Hazama et al 1999;Mongin and Kimelberg 2002;Mongin and Kimelberg 2003, and references therein). Nonetheless, GPCR-related signaling increases VRAC sensitivity to cell swelling and accelerates the process of cell volume regulation (Dezaki et al 2000;Mongin and Kimelberg 2002;Loveday et al 2003;Franco et al 2004), thus potentially aiding in cell volume restoration under normal and pathological conditions. VRACs are also functionally important for cell proliferation and apoptosis.…”

“…Several groups discovered that ATP and agonists of several other G-protein coupled receptors (GPCR) strongly increase VRAC activity in swollen cells. GPCR agonists may also produce limited VRAC activation in non-swollen cells of both neural and non-neural origin (Tilly et al 1994;Mongin and Kimelberg 2002;Loveday et al 2003;Darby et al 2003;Franco et al 2004;Falktoft and Lambert 2004;Heacock et al 2006;Cheema et al 2007). Regulation of VRAC activity by several purinergic (ATP) receptors may be especially important in the brain.…”