Involvement of TRPV1 and TRPV4 channels in migration of rat pulmonary arterial smooth muscle cells

Abstract

Pulmonary hypertension, the main disease of the pulmonary circulation, is characterized by an increase in pulmonary vascular resistance, involving proliferation and migration of pulmonary arterial smooth muscle cells (PASMC). However, cellular and molecular mechanisms underlying these phenomena remain to be identified. In the present study, we thus investigated in rat intrapulmonary arteries (1) the expression and the functional activity of TRPV1 and TRPV4, (2) the PASMC migration triggered by these TRPV channels, and (3) the associated reorganization of the cytoskeleton. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated expression of TRPV1 and TRPV4 mRNA in rat intrapulmonary arteries. These results were confirmed at the protein level by western blot. Using microspectrofluorimetry (indo-1), we show that capsaicin and 4α-phorbol-12,13-didecanoate (4α-PDD), selective agonists of TRPV1 and TRPV4, respectively, increased the intracellular calcium concentration of PASMC. Furthermore, stimulation of TRPV1 and TRPV4 induced PASMC migratory responses, as assessed by two different methods (a modified Boyden chamber assay and a wound-healing migration assay). This response cannot seem to be attributed to a proliferative effect as assessed by BrdU and Wst-1 colorimetric methods. Capsaicin- and 4α-PDD-induced calcium and migratory responses were inhibited by the selective TRPV1 and TRPV4 blockers, capsazepine and HC067047, respectively. Finally, as assessed by immunostaining, these TRPV-induced migratory responses were associated with reorganization of the F-actin cytoskeleton and the tubulin and intermediate filament networks. In conclusion, these data point out, for the first time, the implication of TRPV1 and TRPV4 in rat PASMC migration, suggesting the implication of these TRPV channels in the physiopathology of pulmonary hypertension.

TRPV4 channel contributes to serotonin-induced pulmonary vasoconstriction and the enhanced vascular reactivity in chronic hypoxic pulmonary hypertension

“…1A). Preincubation of PA rings with 5 M HC-067047, a concentration used previously on PASMCs (6,47), for 15 min caused significant reduction in the maximal response (E max ϭ 63.0 Ϯ 7.6, n ϭ 10, P Ͻ 0.01) and the pD 2 (7.2 Ϯ 0.08, P Ͻ 0.01) of PE ( Fig. 1, A-C).…”

“…Hence, the participation of TRPV4 in CHPH is solely restricted to pulmonary vasculature and is independent of cardiac function. Recent evidence from others also shows that CH enhances TRPV4-induced Ca 2ϩ release from ryanodine-sensitive Ca 2ϩ stores in rat PASMCs (6) and activation of TRPV4 promotes PASMC migration (47). Taking all these pieces of information together, we postulate that TRPV4 in PASMCs contributes to the development of CHPH through multifaceted influences on Ca 2ϩ influx and release, promoting basal and myogenic tone, cell migration, and vascular reactivity to collectively muster an elevation of pulmonary vascular resistance and vascular remodeling.…”

“…HC-067047 and RN-1747 are the two currently commercially available TRPV4-specific antagonists with IC 50 of 17 nM and 5.9 M, respectively (17,63). Both antagonists have been used to inhibit TRPV4 in different cell types, and HC-067047 has been shown to block TRPV4 currents and Ca 2ϩ signals in PASMCs (6,47). Even though these antagonists are selective for TRPV4 against other TRP channels, they have dramatic off-target effects at the commonly used concentrations on KCl and PE-induced vasoconstriction of PAs and perhaps on other smooth muscles.…”

Analysis of responses to the TRPV4 agonist GSK1016790A in the pulmonary vascular bed of the intact-chest rat

“…It has been shown that TRPV4 channels are expressed on vascular endothelial and smooth muscle cells in a variety of blood vessels and that activation of these channels induces vascular smooth muscle hyperpolarization, the release of NO from the endothelium, and vasodilation (2,5,9,11,12,25). It has been hypothesized that TRPV4 channel activation represents the hyperpolarizing mechanism by which ACh induces endothelium-dependent vasodilation (7,16,17,27).…”

Transient Receptor Potential Channels as Drug Targets: From the Science of Basic Research to the Art of Medicine

“…Pulmonary artery smooth muscle cells also express TRPV1 and TRPV4. TRPV1 and TRPV4 agonists (capsaicin and 4a-PDD, respectively) induced migratory responses in these cells in a manner that was inhibited by both capsazepine (a first-generation TRPV1 antagonist) (Wang et al, 2008a) and the TRPV4 blocker HC-067047 (Martin et al, 2012).…”