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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