Transient Receptor Potential Vanilloid Type 4–Deficient Mice Exhibit Impaired Endothelium-Dependent Relaxation Induced by Acetylcholine In Vitro and In Vivo

Abstract

Abstract-Agonist-induced Ca 2ϩ entry is important for the synthesis and release of vasoactive factors in endothelial cells. The transient receptor potential vanilloid type 4 (TRPV4) channel, a Ca 2ϩ -permeant cation channel, is expressed in endothelial cells and involved in the regulation of vascular tone. Here we investigated the role of TRPV4 channels in acetylcholine-induced vasodilation in vitro and in vivo using the TRPV4 knockout mouse model. The expression of TRPV4 mRNA and protein was detected in both conduit and resistance arteries from wild-type mice. In small mesenteric arteries from wild-type mice, the TRPV4 activator 4␣-phorbol-12,13-didecanoate increased endothelial [Ca 2ϩ ] i in situ, which was reversed by the TRPV4 blocker ruthenium red. In wild-type animals, acetylcholine dilated small mesenteric arteries that involved both NO and endothelium-derived hyperpolarizing factors. In TRPV4-deficient mice, the NO component of the relaxation was attenuated and the endothelium-derived hyperpolarizing factor component was largely eliminated. Compared with their wild-type littermates, TRPV4-deficient mice demonstrated a blunted endothelial Ca 2ϩ response to acetylcholine in mesenteric arteries and reduced NO release in carotid arteries. Acetylcholine (5 mg/kg, IV) decreased blood pressure by 37.0Ϯ6.2 mm Hg in wild-type animals but only 16.6Ϯ2.7 mm Hg in knockout mice. We conclude that acetylcholine-induced endothelium-dependent vasodilation is reduced both in vitro and in vivo in TRPV4 knockout mice. These findings may provide novel insight into mechanisms of variety of agonists such as acetylcholine, bradykinin, and even mechanical stimuli induce a rapid increase in endothelial Ca 2ϩ , leading to the synthesis and release of relaxing factors, including NO, prostacyclin, and endothelium-derived hyperpolarizing factors (EDHFs). 1 In endothelial and other mammalian cells, the Ca 2ϩ increase is usually a consequence of Ca 2ϩ release from intracellular stores of the endoplasmic reticulum and Ca 2ϩ influx through Ca 2ϩ -permeable cation channels in the plasma membrane via store-operated or receptor-operated mechanisms. 2 The influx of Ca 2ϩ from the extracellular space contributes to the sustained increase of the cytosolic Ca 2ϩ concentration. Despite the importance of calcium entry in the synthesis of endothelial relaxing factors, the proximate cause of this critical signaling event remains elusive.The discovery of transient receptor potential (TRP) channels provides new insights into potential mechanisms of Ca 2ϩ entry in endothelial cells. TRP channel-mediated Ca 2ϩ entry has been implicated in diverse responses, including changes in vascular permeability, angiogenesis, vascular remodeling, and vasorelaxation. 3,4 Of many subtypes of TRP channels expressed in endothelial cells, TRP vanilloid type 4 (TRPV4) channels have received increasing attention. These channels are widely expressed in vascular endothelial cells of several species and activated by both chemical and physical stimuli, including hypotonic...

Contraction by Ca2+ Influx via the L-Type Ca2+ Channel Voltage Window in Mouse Aortic Segments is Modulated by Nitric Oxide

“…This is also consistent with old observations that the resting level of cGMP falls after removal of external Ca 2+ (White & Martin, 1989). In other studies, it has been shown that the TRPV4-mediated Ca 2+ signal was required for eNOS activation by ACh (Adapala et al, 2011;Zhang et al, 2009), also illustrating the necessity of Ca 2+ influx for endothelial NO release. Isshiki et al (Isshiki et al, 2004) suggested that agonist-stimulated eNOS activity in the endothelial cells was sensitive to external Ca 2+ -dependent acute changes in intracellular subcortical Ca 2+ signals and that basal eNOS activity was maintained and regulated by subplasmalemmal Ca 2+ equilibrated with extracellular Ca 2+ .…”

Update on vascular endothelial Ca2+signalling: A tale of ion channels, pumps and transporters

“…Although the cellular mechanisms linking membrane receptors to TRPV4 opening are still unclear, UTP utilizes PLA2 to gate the channel [297] , while PKCα mediates Ach-dependent activation [307] . Despite the evidence for TRPV4-evoked vasodilation, the baseline blood pressure is not affected in TRPV4 -/-mice [305,306] . Nevertheless, systemic activation of the channel may induce vasorelaxation in vivo [302,303,308,309] and, eventually, lead to circulatory collapse [303] .…”

“…TRPC3-and TRPC6-induced Ca 2+ inflow underpin the stimulatory effect of VEGF on endothelial proliferation, migration and permeability [18,231,236] , while ATP exploits TRPC3 to activate NF-κB and increase vascular cell adhesion molecule-1 expression [237] . TRPC3-driven NO synthesis and vasore- TRPV1 TRPV2 TRPV3 TRPV4 TRPV5 TRPV6 PCa/PNa and conductance (pS) [202] 10, 35-80 1-3, NM 12, 172 6, 90 > 100, 75 > 100, 40-70 Human coronary artery ECs [300] (+RT-PCR, WB, IHC) Human pulmonary artery ECs [415] +(RT-PCR) +(RT-PCR) +(RT-PCR) Human pulmonary microvascular ECs +(RT-PCR) Human cerebral microvascular ECs [272] +(RT-PCR, IC) Human cerebral arterioles ECs [305] +(RT-PCR, IHC) Human dermal microvascular ECs [319] +(RT-PCR, WB) Breast cancer derived microvascular ECs [319] +(RT-PCR, WB) Human umbilical vein ECs [227,277,296,301] +(RT-PCR) +(WB, IHC) Mouse aortic ECs [280,285,299,306] +(WB) +(RT-PCR, WB, NM, IHC) Mouse pulmonary artery ECs [313] +(WB, IHC) Mouse mesenteric artery ECs [27,280,302] +(RT-PCR, WB, IC) +(RT-PCR, IC) Mouse cerebral microvascular ECs +(RT-PCR) +(RT-PCR, WB) +(RT-PCR) Mouse dermal microvascular ECs [307] +(RT-PCR, WB) Mouse carotid artery ECs [290,296] +(IHC) Rat mesenteric artery ECs [226,275,302] +(WB) +(WB, IC, IHC) Rat femoral artery ECs [312] +(RT-PCR, IC) Rat pulmonary artery ECs [303] +(WB, IHC) Rat renal artery ECs [303] +(IHC) Rat cardiac microvascular ECs [303,…”

“…An alternative mechanism has been unravelled in human coronary arterioles from patients with coronary artery disease, where flow-induced TRPV4 activation causes ECs to release mitochondrial ROS, thereby relaxing the adjoining VSMCs [300] . Under physiological conditions, TRPV4-mediated vasodilation, mainly via NO and EDHF production, is induced by both shear stress [27,290,291,296] and extracellular autacoids, such as Ach and UTP [297,301,305,306] . Although the cellular mechanisms linking membrane receptors to TRPV4 opening are still unclear, UTP utilizes PLA2 to gate the channel [297] , while PKCα mediates Ach-dependent activation [307] .…”

Coordination of Vasomotor Responses by the Endothelium

“…Knockout of the key structural protein caveolin-1 appears to ablate EDHF-mediated relaxation by disrupting Ca 2+ -signaling involving TRPV4 and thus reducing connexin expression, 49 whereas TRPV4-deficient mice also lose EDHF relaxation, have reduced NO-relaxation and decreased blood pressure reduction to systemic ACh. 50 …”