We have previously shown a pathophysiological role for NGF in pulmonary hypertension, particularly in pulmonary arterial (PA) hyperreactivity. Our experiments have suggested different mechanisms activated by NGF depending on a short–or a long-term PA treatment. The aim of the present study was to determine the mechanisms activated after a NGF short–term PA treatment (1h) leading to PA hyperreactivity.
Basal and agonist-induced calcium responses were evaluated in vitro in primary rat PA smooth muscle cells (rPASMC) using calcium imaging (Fura-2, Fluo-4), in the absence or presence of NGF (100ng/ml, 1h). Calcium sources involved in NGF-increased calcium responses were determined using endoplasmic reticulum calcium stores depleters (thapsigargin, 1μM or cyclopiazonic acid, 10μM), or a calcium–free extracellular medium. NGF–induced phosphorylation of myosin phosphatase target subunit 1 (MYPT1) was evaluated in vitro by Western blotting in primary human PA smooth muscle cells (hPASMC). Contractions of rat PA were induced ex vivo by endothelin-1 (ET-1, 10-12–10-6M) or phenylephrine (PHE, 10-10–10-6M) in the absence or presence of NGF (100ng/ml, 1h), with or without K252a (TrkA kinase inhibitor, 300nM) or Y–27632 (Rho-associated protein kinase -ROCK-inhibitor, 10μM).
In vitro, NGF short-term treatment increases both basal and agonist-induced intracellular calcium responses in rPASMC, with endoplasmic reticulum being the main calcium source contributing to these effects. In addition, short–term treatment with NGF induced MYPT1 phosphorylation in hPASMC, involving a TrkA/ROCK-dependent pathway. Ex vivo, short-term treatment with NGF increased rat PA reactivity to PHE and ET–1. This effect was totally abolished after treatment with K252a or Y-27632.
In conclusion, our results show that NGF can induce PA hyperreactivity through activation of its TrkA receptor, leading to both modulation of calcium intracellular responses and activation of ROCK-dependent calcium sensitization mechanisms. Such mechanisms may therefore contribute to NGF-dependent PA hyperreactivity in pulmonary hypertension.Le texte complet de cet article est disponible en PDF.
Keyword : Circulation