Thrombin promotes release of ATP from lung epithelial cells through coordinated activation of rho- and Ca2+-dependent signaling pathways.
Extracellular ATP controls key aspects of lung function via activation of epithelial cell purinergic receptors, but how ATP is released from cells remains poorly understood. To identify mechanistic components upstream of ATP release, we examined the effect of selected G protein coupled-receptor activation on ATP release from lung epithelial cells. The protease-activated receptor (PAR) agonist thrombin elicited a rapid Ca(2+)-dependent release of ATP from A549 cells. In contrast, the P2Y(2) receptor agonist UTP caused negligible ATP release, despite promoting a robust Ca(2+) response. Agonist-elicited ATP release was associated with Rho activation and was reduced in cells transfected with dominant negative mutants of p115-Rho GEF or RhoA, and by inhibitors of Rho kinase (ROCK). However, RhoA activation alone did not promote ATP release if temporally separated from Ca(2+) mobilization. PAR3 was the only PAR subtype detected in A549 cells by reverse transcription-PCR. Transfection of cells with human PAR3 cDNA increased thrombin-promoted ATP release, inositol phosphate formation, and RhoA activation. Conversely, small interference RNA against PAR3 diminished thrombin-evoked responses. Thrombin-elicited ATP release was accompanied by an enhanced cellular uptake of propidium iodide in a Ca(2+)- and ROCK-dependent manner and was inhibited by connexin/pannexin hemichannel blockers. Our data suggest that thrombin promotes ATP release from A549 cells via Rho- and Ca(2+)-dependent activation of connexin/pannexin hemichannels. The relevance of these findings is highlighted by the observation that exposure of primary cultures of well differentiated human bronchial epithelial cells to thrombin resulted in robust ATP release, which was inhibited by ROCK inhibitors and by connexin/pannexin hemichannel blockers.
Department of Cell and Molecular Physiology, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Lucia
L
Seminario-Vidal
Silvia
S
Kreda
Lisa
L
Jones
Wanda
W
O'Neal
Joann
J
Trejo
Richard C
RC
Boucher
Eduardo R
ER
Lazarowski
D000255:Adenosine Triphosphate / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D017630:Connexins / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism*; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D008168:Lung / Q000166:cytology*; D044463:Receptor, PAR-1 / Q000378:metabolism; D018179:Receptors, Thrombin / Q000378:metabolism; D020545:Respiratory Mucosa / Q000166:cytology / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013917:Thrombin / Q000494:pharmacology*; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D017630:Connexins; D007295:Inositol Phosphates; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D000255:Adenosine Triphosphate; D054460:rho-Associated Kinases; D013917:Thrombin; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; C112908:protease-activated receptor 4; D002118:Calcium