ATP stimulates Ca(2+)-waves and gene expression in cultured human pulmonary fibroblasts

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.