The transient receptor potential vallinoid type 4 (TRPV4) channel has been implicated in the endothelial shear response and flow-mediated dilation, although the precise functions of this channel remain poorly understood. In the present study, we investigated the role of TRPV4 in shear stress-induced endothelial Ca(2+) entry and the potential link between this signaling response and relaxation of small resistance arteries. Using immunohistochemical analysis and RT-PCR, we detected strong expression of TRPV4 protein and mRNA in the endothelium in situ and endothelial cells freshly isolated from mouse small mesenteric arteries. The selective TRPV4 agonist GSK1016790A increased endothelial Ca(2+) and induced potent relaxation of small mesenteric arteries from wild-type (WT) but not TRPV4(-/-) mice. Luminal flow elicited endothelium-dependent relaxations that involved both nitric oxide and EDHFs. Both nitric oxide and EDHF components of flow-mediated relaxation were markedly reduced in TRPV4(-/-) mice compared with WT controls. Using a fura-2/Mn(2+) quenching assay, shear was observed to produce rapid Ca(2+) influx in endothelial cells, which was markedly inhibited by the TRPV4 channel blocker ruthenium red and TRPV4-specific short interfering RNA. Flow elicited a similar TRPV4-mediated Ca(2+) entry in HEK-293 cells transfected with TRPV4 channels but not in nontransfected cells. Collectively, these data indicate that TRPV4 may be a potential candidate of mechanosensitive channels in endothelial cells through which the shear stimulus is transduced into Ca(2+) signaling, leading to the release of endothelial relaxing factors and flow-mediated dilation of small resistance arteries.

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...

Rationale Endothelial derived hydrogen peroxide (H2O2) is a necessary component of the pathway regulating flow-mediated dilation (FMD) in human coronary arterioles (HCA). However H2O2 has never been shown to be the endothelium-dependent transferrable hyperpolarization factor (EDHF) in response to shear stress. Objective We examined the hypothesis that H2O2 serves as the EDHF in HCA to shear stress. Methods and Results Two HCAs were cannulated in series (a donor intact vessel upstream and endothelium-denuded detector vessel downstream). Diameter changes to flow were examined in the absence and presence of PEG-Catalase (PEG-CAT).The open state probability of BKCa channels in smooth muscle cells (SMC) downstream from the perfusate from an endothelium-intact arteriole was examined by patch clamping. In some experiments a cyanogen bromide activated resin column bound with CAT was used to remove H2O2 from the donor vessel. When flow proceeds from donor to detector, both vessels dilate (donor: 68±7%; detector: 45±11%). With flow in the opposite direction, only the donor vessel dilates. PEG-CAT contacting only the detector vessel blocked FMD in that vessel (6 ±4%) but not in donor vessel (61 ±13%). Paxilline inhibited dilation of endothelium-denuded HCA to H2O2. Effluent from donor vessels elicited K+ channel opening in an iberiotoxin - or PEG-CAT sensitive fashion in cell-attached patches, but had little effect on channel opening on inside-out patches. Vasodilation of detector vessels was diminished when exposed to effluent from CAT-column. Conclusions Flow induced endothelial production of H2O2 which acts as the transferrable EDHF activating BKCa channels on the SMC.

In human coronary arterioles (HCAs) from patients with coronary artery disease, flow-induced dilation is mediated by a unique mechanism involving the release of H(2)O(2) from the mitochondria of endothelial cells (ECs). How flow activates ECs to elicit the mitochondrial release of H(2)O(2) remains unclear. Here, we examined the role of the transient receptor potential vanilloid type 4 (TRPV4) channel, a mechanosensitive Ca(2+)-permeable cation channel, in mediating ROS formation and flow-induced dilation in HCAs. Using RT-PCR, Western blot analysis, and immunohistochemical analysis, we detected the mRNA and protein expression of TRPV4 channels in ECs of HCAs and cultured human coronary artery ECs (HCAECs). In HCAECs, 4α-phorbol-12,13-didecanoate (4α-PDD), a selective TRPV4 agonist, markedly increased (via Ca(2+) influx) intracellular Ca(2+) concentration. In isolated HCAs, activation of TRPV4 channels by 4α-PDD resulted in a potent concentration-dependent dilation, and the dilation was inhibited by removal of the endothelium and by catalase, a H(2)O(2)-metabolizing enzyme. Fluorescence ROS assays showed that 4α-PDD increased the production of mitochondrial superoxide in HCAECs. 4α-PDD also enhanced the production of H(2)O(2) and superoxide in HCAs. Finally, we found that flow-induced dilation of HCAs was markedly inhibited by different TRPV4 antagonists and TRPV4-specific small interfering RNA. In conclusion, the endothelial TRPV4 channel is critically involved in flow-mediated dilation of HCAs. TRPV4-mediated Ca(2+) entry may be an important signaling event leading to the flow-induced release of mitochondrial ROS in HCAs. Elucidation of this novel TRPV4-ROS pathway may improve our understanding of the pathogenesis of coronary artery disease and/or other cardiovascular disorders.

Rationale Hydrogen peroxide (H2O2) serves as a key endothelium-derived hyperpolarizing factor mediating flow-induced dilation in human coronary arterioles (HCAs). The precise mechanisms by which H2O2 elicits smooth muscle hyperpolarization are not well understood. An important mode of action of H2O2 involves the oxidation of cysteine residues in its target proteins, including protein kinase G (PKG)-Iα, thereby modulating their activities. Objective Here we hypothesize that H2O2 dilates HCAs through direct oxidation and activation of PKG-Iα leading to the opening of the large-conductance Ca2+-activated K+ (BKCa) channel and subsequent smooth muscle hyperpolarization. Methods and Results Flow and H2O2 induced pressure gradient/concentration-dependent vasodilation in isolated endothelium-intact and -denuded HCAs, respectively. The dilation was largely abolished by iberiotoxin, a BKCa channel blocker. The PKG inhibitor Rp-8-Br-PET-cGMP also markedly inhibited flow- and H2O2-induced dilation, whereas the soluble guanylate cyclase inhibitor ODQ had no effect. Treatment of coronary smooth muscle cells (SMCs) with H2O2 elicited dose-dependent, reversible dimerization of PKG-Iα, and induced its translocation to the plasma membrane. Patch-clamp analysis identified a paxilline-sensitive single-channel K+ current with a unitary conductance of 246-pS in freshly isolated coronary SMCs. Addition of H2O2 into the bath solution significantly increased the probability of BKCa single-channel openings recorded from cell-attached patches, an effect that was blocked by the PKG-Iα inhibitor DT-2. H2O2 exhibited an attenuated stimulatory effect on BKCa channel open probability in inside-out membrane patches. Conclusions H2O2 dilates HCAs through a novel mechanism involving protein dimerization and activation of PKG-Iα and subsequent opening of smooth muscle BKCa channels.

Objective-Hydrogen peroxide (H 2 O 2 ) is an endothelium-derived hyperpolarizing factor in human coronary arterioles (HCAs). H 2 O 2 mediates bradykinin (BK)-induced vasodilation and reduces bioavailability of epoxyeicosatrienoic acids (EETs); however, the cellular and enzymatic source of H 2 O 2 is unknown. Methods and Results-NADPH oxidase expression was determined by immunohistochemistry. Superoxide and H 2 O 2 production was assayed in HCAs and human coronary artery endothelial cells (HCAECs) using dihydroethidium and dichlorodihydrofluorescein histofluorescence, respectively. Superoxide was quantified by HPLC separation of dihydroethidium products. Diameter changes of HCAs were measured by videomicroscopy. NADPH oxidase subunits Nox1, Nox2, Nox4, p22, p47, and p67 were each expressed in HCA endothelium. In HCAs or HCAECs incubated with dihydroethidium and dichlorodihydrofluorescein, BK induced superoxide and H 2 O 2 formation, which was inhibited by gp91ds-tat or apocynin but not by gp91scram-tat or rotenone. HPLC analysis confirmed that BK specifically induced superoxide production. Gp91ds-tat reduced vasodilation to BK but not to papaverine. 14,15-EEZE (an EET antagonist) further reduced the residual dilation to BK in the presence of gp91ds-tat, but had no effect in the presence of gp91scram-tat, suggesting that NADPH oxidase-derived ROS modulate EET bioavailability. Key Words: oxidant stress Ⅲ endothelium Ⅲ coronary circulation Ⅲ neurohumoral control of circulation Ⅲ hypertension-basic studies H 2 O 2 is a reactive oxygen species (ROS) that functions as an endothelium-derived hyperpolarizing factor (EDHF) in some circulatory beds, including HCAs. [1][2][3][4] EDHF is particularly important in the microcirculation, where vascular resistance is regulated. As an EDHF, H 2 O 2 may compensate to maintain adequate perfusion in states of heightened oxidative stress such as cardiovascular disease, where nitric oxide (NO)-mediated vasodilation is impaired. In addition to its direct vasodilator properties, H 2 O 2 can modulate bioavailability of EETs, cytochrome P450 (CYP)-derived metabolites of arachidonic acid that also function as EDHFs. 5 H 2 O 2 arises by enzymatic or spontaneous dismutation of the superoxide anion. Superoxide is generated from numerous intracellular sources, including mitochondria, 4 CYPs, 6 and NADPH oxidases. 7 The canonical Nox2-containing NADPH oxidase was originally identified in phagocytes, where it mediates the microbicidal respiratory burst; however, Nox2 as well as alternative NADPH oxidases containing Nox1 or Nox4 are now recognized as an important source of ROS in vascular tissue as well. 8 Although the pathological role of NADPH oxidase is well defined by its heightened participation in a variety of vascular diseases, 7,9 its possible contribution to physiological vascular stimuli is less clear, especially in the human heart. This study was conducted to examine the putative role of NADPH oxidase in mediating dilation to BK in HCAs. Published data indicate that both flow-induc...

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BackgroundAcetylcholine (ACH) dose‐dependently constricts human coronary atrial arterioles (HCA) but dilates ventricular arterioles in patients with coronary disease. Whether NO mechanisms participate in Ach‐induced dilation in atrial arterioles remains unclear. Since CAD and its risk factors generate ROS that can quench NO, we hypothesized that acetylcholine‐induced dilation can be restored in HCA by restoring redox balance and supporting exogenous NO synthesis.MethodsArterioles (~150 μm) from patients with coronary artery disease were prepared for videomicroscopy. HCA's were pre‐incubated with indomethacin (10‐5), sepiapterin (10‐4), L‐Arginine (10‐3), and Peg‐SOD (300units/ml) for 30 minutes. After preconstruction with endothelin‐1, vasomotor responses to exogenous ACH (10‐9 to 10‐4 M) were evaluated in control or in the presence and absence of N‐nitro‐L‐arginine methyl ester (L‐name; 10‐4)).ResultsACH dilated pretreated compared to control intact arterioles [%max dilation (MD): 80±8% vs. ‐36.4±7%]. Treatment with L‐name abolished dilation (MD:‐0.5±9%).ConclusionsReducing endogenous oxidative stress and providing NOS substrate converts an , ACH‐induced constriction to an NO ‐dependent vasodilation in HCA. There may be a greater sensitivity to ROS in endothelium of atrial vs. ventricular arterioles.