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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 Ca2+ 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 Ca2+ and induced potent relaxation of small mesenteric arteries from wild-type (WT) but not [TRPV4.sup.-/-] 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.sup.2+] quenching assay, shear was observed to produce rapid [Ca.sup.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 [Ca2.sup.+] 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.sup.2+] signaling, leading to the release of endothelial relaxing factors and flow-mediated dilation of small resistance arteries. transient receptor potential; endothelium; endothelium-derived factors; calcium signaling doi: 10.1152/ajpheart.00854.2009

Small coronary arteries (SCA) from diabetic rats exhibit enhanced peroxynitrite ([ONOO.sup.-]) formation and concurrent impairment of voltage-dependent potassium ([K.sub.v]) channel function. However, it is unclear whether [ONOO.sup.-] plays a causative role in this impairment. We hypothesized that functional loss of [K.sub.v] channels in coronary smooth muscle cells (SMC) in diabetes is due to ONOO with subsequent tyrosine nitration of [K.sub.v] channel proteins. Diabetic rats and nondiabetic controls were treated with or without ebselen (Eb) for 4 wk. SCA were prepared for immunohistochemistry (IHC), immunoprecipitation (IP) followed by Western blot (WB), videomicroscopy, and patch-clamp analysis. IHC revealed excess [ONOO.sup.-] in SCA from diabetic rats. IP and WB revealed elevated nitration of the [K.sub.v]1.2 [alpha]-subunit and reduced [K.sub.v]l.2 protein expression in diabetic rats. Each of these changes was improved in Eb-treated rats. Protein nitration and [K.sub.v]1.5 expression were unchanged in SCA from diabetic rats. Forskolin, a direct cAMP activator that induces [K.sub.v]1 channel activity, dilated SCA from nondiabetic rats in a correolide (Cor; a selective [K.sub.v]1 channel blocker)-sensitive fashion. Cor did not alter the reduced dilation to forskolin in diabetic rats; however, Eb partially restored the Cor-sensitive component of dilation. Basal [K.sub.v] current density and response to forskolin were improved in smooth muscle cells from Eb-treated DM rats. We conclude that enhanced nitrosative stress in diabetes mellitus contributes to [K.sub.v]1 channel dysfunction in the coronary microcirculation. Eb may be beneficial for the therapeutic treatment of vascular complications in diabetes mellitus. diabetes; peroxynitrite; oxidative stress; coronary circulation

Platelet endothelial cell adhesion molecule 1 (PECAM-1) is capable of transducing signals in endothelial cells exposed to shear; however, the biological consequences of this signal transduction are unknown. Because shear stress elicits flow-mediated dilation (FMD), we examined whether steady-state FMD in mouse coronary arteries (MCAs) is affected in the PECAM-1 knockout (KO) mouse. MCAs were isolated from wild-type (WT) or KO mice and prepared for videomicroscopy, histofluorescence, Western blotting, and immunohistochemistry. FMD was examined in the absence and presence of [N.sup.[omega]]-nitro-L-arginine methyl ester (L-NAME) and L-NAME + indomethacin (INDO). FMD was reduced in KO relative to WT MCAs, but the L-NAME-inhibitable portion of FMD was similar between the two. The INDO-sensitive component of FMD was diminished in KO MCAs. In contrast, the residual component of dilation, presumably because of endothelium-derived hyperpolarizing factor (EDHF), was abolished in KO MCAs. Histofluorescence showed relatively more superoxide ([O.sup.-.sub.2]*; oxy-ethidium fluorescence) and peroxide production (dihydrochlorofluorescene fluoresecence) in KO MCAs at rest. Flow augmented [O.sup.-.sub.2]* and peroxide production in WT MCAs but had little effect on KO MCAs. Enhanced nitric oxide generation was observed in arteries from KO mice, accompanied with increased eNOS S1177 phosphorylation. In vessels from KO mice, treatment with ebselen decreased peroxynitrite (ONO[O.sup.-]) formation and improved the reduced FMD, largely due to restoration of the presumed EDHF component. These results suggest that PECAM-1 is necessary for normal FMD in the mouse coronary circulation. In the absence of this adhesion and signaling molecule, ONO[O.sup.-] production is increased concomitant with a reduction in both the EDHF and INDO-sensitive components of FMD. flow-induced dilation; oxidative stress

We have shown that short-term exposure of rat small coronary arteries (RSCAs) to high glucose enhances superoxide ([O.sup.-.sub.2] *) formation and impairs cAMP-mediated dilation by reducing voltage-gated [K.sup.+] ([K.sub.v]) channel function. However, it is not clear whether the impairment also occurs in diabetes mellitus (DM), where alternate mechanisms could mask or aggravate vasodilator dysfunction. RSCAs were isolated from control and streptozotocin-induced diabetic rats. Reduced constriction to 4-aminopyridine (4-AP) was observed in RSCAs from DM rats, indicating [K.sub.v] channel impairment. Forskolin increased 4-AP-inhibitable [K.sup.+] channel open-state probability and whole cell [K.sup.+] current density in coronary myocytes from non-DM rats but had little effect on [K.sup.+] current density in cells from DM rats. Diminished dilation to 8-bromo-cAMP, forskolin, or isoproterenol was observed in DM RSCAs. The attenuated dilation to forskolin or isoproterenol in DM RSCAs was partially restored by application of the superoxide dismutase mimetic manganese[III] tetrakis (4-benzoic acid) porphyrin. Histofluorescence studies using hydroethidine revealed a blockage of [O.sup.-.sub.2] * generation by the NADPH oxidase inhibitor apocynin in DM RSCAs. Sepiapterin, a precursor of tetrahydrobiopterin, had little effect on hyperglycemia-induced [O.sup.-.sub.2] * formation. Consistent with the findings from the concurrent fluorescence study, apocynin also partially restored the reduced dilator response to forskolin in DM RSCAs. Forskolin-induced cAMP production was unaltered in DM. We conclude that in diabetes, enhanced [O.sup.-.sub.2] * formation by activation of NADPH oxidase impairs cAMP-medicated dilation in RSCAs by inhibiting [K.sub.v] channel activity. hyperglycemia; superoxide; coronary circulation

In human coronary arterioles (HCAs) from patients with coronary artery disease, flow-induced dilation is mediated by a unique mechanism involving the release of H2O2 from the mitochondria of endothelial cells (ECs). How flow activates ECs to elicit the mitochondrial release of H2O2 remains unclear. Here, we examined the role of the transient receptor potential vanilloid type 4 (TRPV4) channel, a mechanosensitive Ca2+-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 Ca2+ influx) intracellular Ca2+ 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 H2O2-metabolizing enzyme. Fluorescence ROS assays showed that 4α-PDD increased the production of mitochondrial superoxide in HCAECs. 4α-PDD also enhanced the production of H2O2 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 Ca2+ 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. [ABSTRACT FROM AUTHOR]

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 Ca2+ 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 Ca2+ and induced potent relaxation of small mesenteric arteries from wild-type (WT) but not TRPV4-/- mice. Luminal flow elicited endotheliumdependent 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/Mn2+ quenching assay, shear was observed to produce rapid Ca2+ 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 Ca2+ 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 Ca2+ signaling, leading to the release of endothelial relaxing factors and flow-mediated dilation of small resistance arteries. [ABSTRACT FROM AUTHOR]

Mitochondrial H2O2 contributes to flow-mediated dilation (FMD) in human coronary arterioles (HCA). We examined the hypothesis that the endothelial cytoskeleton plays a critical role in transducing endothelial wall shear stress into a stimulus for releasing mitochondrial ROS. Phallacidin together with α-, β-tubulin antibodies and Mito-Tracker Red showed the proximity of F-actin, microtubules and mitochondria in endothelial cells. Cytochalasin D (CytoD) and nocodazole (Noc) disrupted endothelial F-actin and microtubules in HCA, respectively, concurrent with a reduction in the generation of cytosolic O2 and H•− and H2O2 (hydroethidine and dichlorodihydrofluorescein fluorescence) and mitochondrial superoxide (mitoSox) during flow (control: 3.5 ± 1.6, Cyto D: 0.51 ± 0.2, Noc: 0.81 ± 0.6). FMD, but not the dilation to bradykinin or papaverine, was reduced by Cyto D (26 ± 10% vs. 56 ± 3%) or Noc (26 ± 11% vs. 58 ± 7%). These results suggest that cytoskeletal elements are a critical component of the signaling mechanism linking endothelial shear stress and mitochondrial release of ROS in the human coronary microcirculation. [ABSTRACT FROM AUTHOR]

We have shown that short-term exposure of rat small coronary arteries (RSCAs) to high glucose enhances superoxide (O2-·) formation and impairs cAMP-mediated dilation by reducing voltage-gated K+ (Kv) channel function. However, it is not clear whether the impairment also occurs in diabetes mellitus (DM), where alternate mechanisms could mask or aggravate vasodilator dysfunction. RSCAs were isolated from control and streptozotocin-induced diabetic rats. Reduced constriction to 4-aminopyridine (4-AP) was observed in RSCAs from DM rats, indicating Kv channel impairment. Forskolin increased 4-AP-inhibitable K+ channel open-state probability and whole cell K+ current density in coronary myocytes from non-DM rats but had little effect on K+ current density in cells from DM rats. Diminished dilation to 8-bromo-cAMP, forskolin, or isoproterenol was observed in DM RSCAs. The attenuated dilation to forskolin or isoproterenol in DM RSCAs was partially restored by application of the superoxide dismutase mimetic manganese[III] tetrakis (4-benzoic acid) porphyrin. Histofluorescence studies using hydroethidine revealed a blockage of O2-· generation by the NADPH oxidase inhibitor apocynin in DM RSCAs. Sepiapterin, a precursor of tetrahydrobiopterin, had little effect on hyperglycemia-induced O2-·. formation. Consistent with the findings from the concurrent fluorescence study, apocynin also partially restored the reduced dilator response to forskolin in DM RSCAs. Forskolin-induced cAMP production was unaltered in DM. We conclude that in diabetes, enhanced O2-· formation by activation of NADPH oxidase impairs cAMP-medicated dilation in RSCAs by inhibiting Kv channel activity. [ABSTRACT FROM AUTHOR]

Emerging evidence indicates that the vanilloid subfamily of transient receptor potential channels, member 4 (TRPV4) is activated by shear stress leading to Ca2+-influx in vascular endothelial cells. Activation of TRPV4 may represent a novel mechanism by which shear stress activates endothelial cells to induce arterial dilation. We investigated the role of TRPV4 channel in vasomotor responses of small bovine coronary arteries (BCA). Using immunoblots, TRPV4 protein was detected in coronary endothelial cells. Using a Ca2+ assay with fura-2, 4a-phorbol-12,13-didecanoate (4αPDD, 5 mM), a selective TRPV4 opener, increased intracellular Ca2+ ([Ca2+]i) in endothelial cells (mean increase above basal or Δ[Ca2+]i of 153±6.0 nM; n=3), a response that was inhibited by removing extracellular Ca2+ (Δ[Ca2+]i of 20±2.4 nM) or by the TRPV4 blocker ruthenium red (RuR) (Δ[Ca2+]i of 55±5.4 nM). In isolated and cannulated BCA, 4aPDD (50 nM-5 mM) elicited concentration-dependent relaxations (maximal relaxation of 55±8%; n=4). Shear stress (10-150 ml/min) dilated BCA (maximal relaxation of 59±11%; n=4), which were inhibited by RuR (6±10%). It is concluded that TRPV4 channels are expressed in BCA and activation of these channels induces endothelial Ca2+ influx and arterial relaxation. TRPV4 channels may play a role in endothelial shear sensing and flow-induced dilation in these arteries. [ABSTRACT FROM AUTHOR]

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: Endothelial derived hydrogen peroxide (H(2)O(2)) is a necessary component of the pathway regulating flow-mediated dilation (FMD) in human coronary arterioles (HCAs). However, H(2)O(2) has never been shown to be the endothelium-dependent transferrable hyperpolarization factor (EDHF) in response to shear stress., Objective: We examined the hypothesis that H(2)O(2) serves as the EDHF in HCAs 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 polyethylene glycol catalase (PEG-CAT). The open state probability of large conductance Ca(2+)-activated K(+) (BK(Ca)) channels in smooth muscle cells 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 H(2)O(2) 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 HCAs to H(2)O(2). 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 H(2)O(2), which acts as the transferrable EDHF activating BK(Ca) channels on the smooth muscle cells.