Rho-family GTPases modulate Ca(2+) -dependent ATP release from astrocytes.
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.
Blum
Andrew E
AE
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
Joseph
Sheldon M
SM
Przybylski
Ronald J
RJ
Dubyak
George R
GR
D036002:ADP Ribose Transferases / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D015703:Antigens, CD / Q000378:metabolism; D001081:Apyrase / Q000378:metabolism; D001253:Astrocytes / Q000187:drug effects / Q000502:physiology*; D001426:Bacterial Proteins / Q000494:pharmacology; D001427:Bacterial Toxins / Q000494:pharmacology; D001905:Botulinum Toxins / Q000494:pharmacology; D020126:Brefeldin A / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D002217:Carbachol / Q000494:pharmacology; D002229:Carbenoxolone / Q000494:pharmacology; D002460:Cell Line; D002463:Cell Membrane Permeability; D006801:Humans; D043587:Receptor, Muscarinic M3 / Q000378:metabolism; D044463:Receptor, PAR-1 / Q000378:metabolism; D049368:Receptors, Lysophosphatidic Acid / Q000378:metabolism; D022142:Secretory Vesicles / Q000502:physiology; D013917:Thrombin / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*
D015703:Antigens, CD; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D043587:Receptor, Muscarinic M3; D044463:Receptor, PAR-1; D049368:Receptors, Lysophosphatidic Acid; C031815:toxB protein, Clostridium difficile; D020126:Brefeldin A; D000255:Adenosine Triphosphate; D002217:Carbachol; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D013917:Thrombin; D001905:Botulinum Toxins; D001081:Apyrase; C067869:CD39 antigen; D020741:rho GTP-Binding Proteins; D002229:Carbenoxolone; D002118:Calcium