Rho-family GTPases modulate Ca(2+) -dependent ATP release from astrocytes

• Published in: American Journal of Physiology: Cell Physiology 2008-07, Vol.295 (1), p.C231
• Main Authors: Blum, Andrew E, Joseph, Sheldon M, Przybylski, Ronald J, Dubyak, George R
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - secretion; ADP Ribose Transferases - pharmacology; Antigens, CD - metabolism; Apyrase - metabolism; Astrocytes - drug effects; Astrocytes - physiology; Bacterial Proteins - pharmacology; Bacterial Toxins - pharmacology; Botulinum Toxins - pharmacology; Brefeldin A - pharmacology; Calcium - physiology; Carbachol - pharmacology; Carbenoxolone - pharmacology; Cell Line; Cell Membrane Permeability; Humans; Receptor, Muscarinic M3 - metabolism; Receptor, PAR-1 - metabolism; Receptors, Lysophosphatidic Acid - metabolism; rho GTP-Binding Proteins - antagonists & inhibitors; rho GTP-Binding Proteins - physiology; Secretory Vesicles - physiology; Thrombin - metabolism
• ISSN: 0363-6143
• DOI: 10.1152/ajpcell.00175.2008

Previously, we reported that activation of G protein-coupled receptors (GPCR) in 1321N1 human astrocytoma cells elicits a rapid release of ATP that is partially dependent on a G(q)/phophospholipase C (PLC)/Ca(2+) mobilization signaling cascade. In this study we assessed the role of Rho-family GTPase signaling as an additional pathway for the regulation of ATP release in response to activation of protease-activated receptor-1 (PAR1), lysophosphatidic acid receptor (LPAR), and M3-muscarinic (M3R) GPCRs. Thrombin (or other PAR1 peptide agonists), LPA, and carbachol triggered quantitatively similar Ca(2+) mobilization responses, but only thrombin and LPA caused rapid accumulation of active GTP-bound Rho. The ability to elicit Rho activation correlated with the markedly higher efficacy of thrombin and LPA, relative to carbachol, as ATP secretagogues. Clostridium difficile toxin B and Clostridium botulinum C3 exoenzyme, which inhibit Rho-GTPases, attenuated the thrombin- and LPA-stimulated ATP release but did not decrease carbachol-stimulated release. Thus the ability of certain G(q)-coupled receptors to additionally stimulate Rho-GTPases acts to strongly potentiate a Ca(2+)-activated ATP release pathway. However, pharmacological inhibition of Rho kinase I/II or myosin light chain kinase did not attenuate ATP release. PAR1-induced ATP release was also reduced twofold by brefeldin treatment suggesting the possible mobilization of Golgi-derived, ATP-containing secretory vesicles. ATP release was also markedly repressed by the gap junction channel inhibitor carbenoxolone in the absence of any obvious thrombin-induced change in membrane permeability indicative of hemichannel gating.