Capacitative Ca2+ Entry in Pulmonary Myocyte Growth

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DESCRIPTION (provided by applicant): Pulmonary vasoconstriction and vascular smooth muscle proliferation greatly contribute to the elevated pulmonary vascular resistance and arterial pressure in patients with primary pulmonary hypertension (PPH), a fatal disease with unknown causes. A common theory is that vasoconstriction and cell proliferation use overlapping signaling processes that result in parallel intracellular events. A rise in cytosolic Ca2+ ([Ca2+]cyt) stimulates cell contraction and proliferation. Thus, intracellular Ca2+ may serve as a shared signal transduction element that leads to pulmonary vasoconstriction and vascular remodeling in PPH. [Ca2+]cyt about pulmonary artery smooth muscle cells (PASMC) is increased by Ca2+ release from the sarcoplasmic reticulum (SR) and Ca2+ influx through sarcolemmal Ca2+-permeable channels. The mitogen-induced changes in [Ca2+]cyt consist of an initial release of Ca2+ from the SR followed by a sustained Ca2+ influx. Depletion of the SR Ca2+ induces capacitative Ca2+ entry (CCE), which maintains the sustained Ca2+ influx and refills Ca2+ into the SR. The TRP-encoded proteins may form the Ca2+-permeable channels that are responsible for CCE. In human PASMC, the mRNA and protein levels of TRP1 were significantly higher in proliferating cells than in growth-arrested cells. The enhanced TRP1 mRNA and protein expression was associated with increases in [Ca2+]cyt due to Ca2+ release from the SR and CCE. These results imply that the up-regulation o TRP1 may contribute to the increased CCE, and elevated [Ca2+]cyt and intracellularly-stored [Ca2+] in the SR ([Ca2+]SR). Based on these data, we hypothesize that up-regulation of TRP genes leads to an increase in the activity of a TRP-encoded Ca2+ channel. This channel would then serve as a critical Ca2+ entry pathway to raise [Ca2+]cyt and refill Ca2+ into the SR, both of which are necessary for pulmonary vasoconstriction and PASMC proliferation. The up-regulated TRP genes, augmented Ca2+ release-activated (store depletion-mediated) Ca2+ currents (ICRAC), and enhanced CCE may thus play a critical role in the elevated pulmonary vascular resistance in PPH patients. Three Specific Aims are addressed to test the hypotheses: 1) to characterize the TRP gene expression, ICRAC, CCE, and [Ca2+]SR in normal human PASMC, and to compare these parameters between growth-arrested and proliferating cells; 2) to investigate whether functional expression of TRPs facilitates cell proliferation by increasing resting [Ca2+]cyt, CCE, and [Ca2+]SR in normal human PASMC; and 3) to investigate and compare ICRAC, molecular expression of TRP channels, spatial and temporal changes of [Ca2+]cyt through CCE, and [Ca2+]SR in PASMC from normal subjects and patients with non-pulmonary hypertension diseases, secondary pulmonary hypertension, and PPH.