Extracellular osmolarity modulates G protein-coupled receptor-dependent ATP release from 1321N1 astrocytoma cells

Abstract

We previously reported that ATP release from 1321N1 human astrocytoma cells could be stimulated either by activation of G protein-coupled receptors (GPCR) or by hypotonic stress. Cheema et al. (Cheema TA, Ward CE, Fisher SK. J Pharmacol Exp Ther 315: 755-763, 2005) have demonstrated that thrombin activation of protease-activated receptor 1 (PAR1) in 1321N1 cells and primary astrocytes acts synergistically with hypotonic stress to gate the opening of volume-sensitive organic osmolyte and anion channels (VSOAC) and that hypertonic stress strongly inhibits PAR1 gating of VSOAC. We tested the hypothesis that a VSOAC-type permeability might comprise a GPCR-regulated pathway for ATP export by determining whether PAR1-sensitive ATP release from 1321N1 cells is similarly potentiated by hypotonicity but suppressed by hypertonic conditions. Strong hypotonic stress by itself elicited ATP release and positively modulated the response to thrombin. Thrombin-dependent ATP release was also potentiated by mild hypotonic stress that by itself did not stimulate ATP export. Notably, PAR1-sensitive ATP export was greatly inhibited in hypertonic medium. Neither the potency nor efficacy of thrombin as an activator of proximal PAR1 signaling was affected by hypotonicity or hypertonicity. 1,9-Dideoxyforskolin and carbenoxolone similarly attenuated PAR1-dependent ATP release and suppressed the PAR1-independent ATP elicited by strong hypotonic stress. Probenecid attenuated PAR1-stimulated ATP release under isotonic but not mild hypotonic conditions and had no effect on PAR1-independent release stimulated by strong hypotonicity. PAR1-dependent ATP export under all osmotic conditions required concurrent signaling by Ca(2+) mobilization and Rho-GTPase activation. In contrast, PAR1-independent ATP release triggered by strong hypotonicity required neither of these intracellular signals. Thus, we provide the new finding that GPCR-regulated ATP release from 1321N1 astrocytoma cells is remarkably sensitive to both positive and negative modulation by extracellular osmolarity. This supports a model wherein GPCR stimulation and osmotic stress converge on an ATP release pathway in astrocytes that exhibits several features of VSOAC-type channels.

Vesicular and conductive mechanisms of nucleotide release

“…VRAC Cl − channel activity is activated in response to osmotic cell swelling and inhibited by a broad spectrum of reagents, including glibenclamide, verapamil, tamoxifen, fluoxetine, nordihydroguaiaretic acid, dideoxyforskolin, niflumic acid, quinine, NPPB, DIDS, and SITS [101,105]. VRAC inhibitors suppressed ATP release in aortic endothelial cells [102] and 1321N1 astrocytoma cells [105], but not in Intestine 407 cells [106] or alveolar or airway epithelial cells [73]. Recently, bradykinin-stimulated astrocytes exhibited VRAC-like activity that was associated with the release of glutamate but not ATP [107].…”

Evidence for sustained ATP release from liver cells that is not mediated by vesicular exocytosis

“…Similarly, changes in the extracellular osmolarity induced by hypotonic solution may also stimulate interaction of pannexin1 hemichannels with other membrane proteins in liver cells, and this may result in the decreased sensitivity to CBX and probenecid. A recent study in 1321 N1 astrocytes provides evidence for this explanation [38]. The authors found that extracellular osmolarity modulates the sensitivity of thrombin-dependent ATP release to probenecid.…”

Transforming Growth Factor β1 Alters Calcium Mobilizing Properties and Endogenous ATP Release in A549 Cells: Possible Implications for Cell Migration

“…Regulated ATP release occurs in most mammalian cells under stressed or stimulated conditions such as mechanical stress (8,9), hypoxia (10,11), lysophospholipids (12), and agonist stimulation (9). Static unstimulated cells also release ATP (13), and it was shown that the extracellular ATP concentration ([ATP] o ) was three times higher in thyroid cancer cell lines than in normal thyroid cells (14).…”