ATP stimulates Ca2+-waves and gene expression in cultured human pulmonary fibroblasts
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Given that extracellular ATP is markedly elevated in inflammation and is known to modulate fibroblast function, we examined the effects of exogenously added ATP on Ca(2+)-handling and gene expression in human pulmonary fibroblasts. Cells were loaded with the Ca(2+)-indicator dye fluo-4 and studied using confocal fluorimetry. Standard RT-PCR was used to probe gene expression. ATP (10(-5)M) evoked recurring Ca(2+)-waves which were completely occluded by cyclopiazonic acid (depletes the internal Ca(2+)-store) or the phospholipase inhibitor U73122. Pretreatment with ryanodine (10(-5)M), however, had no effect on the ATP-evoked responses. Regarding the receptor through which ATP acted, we found the ATP-response to be mimicked by UTP or ADP but not by adenosine or alpha,beta-methylene-ATP, and to be blocked by the purinergic receptor blocker PPADS. The ATP-evoked response was greater and longer lasting within the nucleus than in the non-nuclear portion of the cytosol. RT-PCR showed that ATP also rapidly and dramatically increased gene expression of P2Y(4) receptors, the cytokine TGF-beta (an important modulator of wound repair) and two matrix proteins (collagen A1 and fibronectin) approximately 4-5 times above baseline: this increase was not significantly affected by ryanodine but was abolished by PPADS. We conclude that, in human pulmonary fibroblasts, ATP acts upon P2Y receptors to liberate internal Ca(2+) through ryanodine-insensitive channels, leading to a Ca(2+)-wave which courses throughout the cell and modulates gene expression.
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