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Elementary TRPV4 sparklets in the endothelium dilate mouse pulmonary arteries
Author(s) -
Marziano Corina,
Hong Kwangseok,
Cope Eric L,
Sonkusare Swapnil K
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.lb609
Subject(s) - trpv4 , transient receptor potential channel , endothelium , pulmonary edema , vasodilation , pulmonary artery , chemistry , mesenteric arteries , extracellular , lung , medicine , cardiology , anesthesia , artery , receptor , biochemistry
Endothelial dysfunction is a hallmark of pulmonary disorders including pulmonary hypertension, pulmonary edema, acute lung injury and ischemia. Ca 2+ influx through TRPV4 (transient receptor potential vanilloid 4) channels plays an important role in regulating the function of pulmonary endothelium, and abnormal TRPV4 channel activity has been associated with lung injury, ischemia, and pulmonary edema. However, the function of TRPV4 channels and their regulation by physiological stimuli in the pulmonary endothelium remain unknown. The objectives of this study were to elucidate the regulation of TRPV4 channels in pulmonary endothelium under physiological conditions; and to identify the Ca 2+ ‐sensitive targets that mediate TRPV4‐induced vasodilation in these arteries. Third or higher order pulmonary arteries (PAs) were isolated, slit‐open and pinned down, and the Ca 2+ signals were recorded using a spinning disk confocal imaging system. In the presence of cyclopiazonic acid to deplete the internal stores of Ca 2+ , TRPV4 channel function was recorded as Ca 2+ influx events‐TRPV4 sparklets (Sonkusare et al., Science , 2012). The activity of TRPV4 sparklets, estimated as fluorescence integral over a field of view, was increased by 3‐ and 17‐fold in the presence of 3 and 10 nM GSK1016790A (TRPV4 agonist), respectively, when compared to the baseline. The TRPV4 sparklets were inhibited by TRPV4 channel inhibitor HC067047 (1 μM); and were absent in the presence of 0 mM extracellular Ca 2+ in both the endothelium‐denuded arteries and the arteries from TRPV4 knockout mice. A multi‐Gaussian curve fit to the all‐points histogram of the fractional fluorescence traces revealed a single channel amplitude of 0.18 ΔF/F 0 in the mice that express Ca 2+ biosensor GCaMP2 selectively in the endothelium, and 0.28 ΔF/F 0 in the arteries loaded with fluo‐4AM. The TRPV4 sparklet amplitudes were not affected by a change in the agonist concentration, or in the presence of TRPV4 inhibitor. The effect of TRPV4 channel activation on arterial diameter was studied in the PAs cannulated and pressurized to 10–20 mm Hg and pre‐constricted with U46619 (50–100 nM). GSK101 (30 nM) induced a 67% vasodilation which was mostly inhibited by N G ‐nitro‐L‐Arginine (nitric oxide synthase (NOS) inhibitor, 100 μM). Our results support the concept that TRPV4 sparklets in PA endothelium represent single TRPV4 channel openings that cause vasodilation via NOS activation. These results will enable direct studies of TRPV4 channel regulation in PAs under healthy and diseased conditions. Support or Funding Information Supported by NIH award HL121484 to SKS