NADPH oxidase 4 contributes to TRPV4-mediated endothelium-dependent vasodilation in human arterioles by regulating protein phosphorylation of TRPV4 channels.

Impaired endothelium-dependent vasodilation has been suggested to be a key component of coronary microvascular dysfunction (CMD). A better understanding of endothelial pathways involved in vasodilation in human arterioles may provide new insight into the mechanisms of CMD. The goal of this study is to investigate the role of TRPV4, NOX4, and their interaction in human arterioles and examine the underlying mechanisms. Arterioles were freshly isolated from adipose and heart tissues obtained from 71 patients without coronary artery disease, and vascular reactivity was studied by videomicroscopy. In human adipose arterioles (HAA), ACh-induced dilation was significantly reduced by TRPV4 inhibitor HC067047 and by NOX 1/4 inhibitor GKT137831, but GKT137831 did not further affect the dilation in the presence of TRPV4 inhibitors. GKT137831 also inhibited TRPV4 agonist GSK1016790A-induced dilation in HAA and human coronary arterioles (HCA). NOX4 transcripts and proteins were detected in endothelial cells of HAA and HCA. Using fura-2 imaging, GKT137831 significantly reduced GSK1016790A-induced Ca2+ influx in the primary culture of endothelial cells and TRPV4-WT-overexpressing human coronary artery endothelial cells (HCAEC). However, GKT137831 did not affect TRPV4-mediated Ca2+ influx in non-phosphorylatable TRPV4-S823A/S824A-overexpressing HCAEC. In addition, treatment of HCAEC with GKT137831 decreased the phosphorylation level of Ser824 in TRPV4. Finally, proximity ligation assay (PLA) revealed co-localization of NOX4 and TRPV4 proteins. In conclusion, both TRPV4 and NOX4 contribute to ACh-induced dilation in human arterioles from patients without coronary artery disease. NOX4 increases TRPV4 phosphorylation in endothelial cells, which in turn enhances TRPV4-mediated Ca2+ entry and subsequent endothelium-dependent dilation in human arterioles.

Endothelin-1 potentiates TRPV1-mediated vasoconstriction of human adipose arterioles in a protein kinase C-dependent manner.

BACKGROUND AND PURPOSE: The TRPV cation channels have emerged as important regulators of vascular tone. TRPV1 channels and endothelin-1 are independently associated with the pathophysiology of coronary vasospasm, but the relationship between their vasomotor functions remains unclear. We characterized the vasomotor function of TRPV1 channels in human arterioles and investigated regulation of their vasomotor function by endothelin-1. EXPERIMENTAL APPROACH: Human arterioles (mainly from adipose tissue) were threaded on two metal wires, equilibrated in a physiological buffer at 37°C and exposed to increasing concentrations of capsaicin, with or without SB366791 (TRPV1-selective inhibitor) or GF109203X (PKC-selective inhibitor). Some arterioles were pre-constricted with endothelin-1 or phenylephrine or high potassium buffer. TRPV1 mRNA and protein expression in human arteries were also assessed. KEY RESULTS: TRPV1 transcripts and proteins were detected in human resistance arteries. Capsaicin (1 µM) induced concentration-dependent constriction of endothelium-intact and endothelium-denuded human adipose arterioles (HAA), which was significantly inhibited by SB366791. Pre-constriction of HAA with endothelin-1, but not high potassium buffer or phenylephrine, significantly potentiated capsaicin (0.1 µM)-induced constriction. GF109203X significantly inhibited potentiation of capsaicin-induced constriction by endothelin-1. CONCLUSION AND IMPLICATIONS: TRPV1 channels are expressed in the human vasculature and affect vascular tone of human arterioles on activation. Their vasomotor function is modulated by endothelin-1, mediated in part by PKC. These findings reveal a novel interplay between endothelin-1 signalling and TRPV1 channels in human VSMC, adding to our understanding of the ion channel mechanisms that regulate human arteriolar tone and may also contribute to the pathophysiology of coronary vasospasm.

Transient receptor potential vanilloid 4 (TRPV4) activation by arachidonic acid requires protein kinase A-mediated phosphorylation.

Transient receptor potential vanilloid 4 (TRPV4) is a Ca2+-permeable channel of the transient receptor potential (TRP) superfamily activated by diverse stimuli, including warm temperature, mechanical forces, and lipid mediators such as arachidonic acid (AA) and its metabolites. This activation is tightly regulated by protein phosphorylation carried out by various serine/threonine or tyrosine kinases. It remains poorly understood how phosphorylation differentially regulates TRPV4 activation in response to different stimuli. We investigated how TRPV4 activation by AA, an important signaling process in the dilation of coronary arterioles, is affected by protein kinase A (PKA)-mediated phosphorylation at Ser-824. Wildtype and mutant TRPV4 channels were expressed in human coronary artery endothelial cells (HCAECs). AA-induced TRPV4 activation was blunted in the S824A mutant but was enhanced in the phosphomimetic S824E mutant, whereas the channel activation by the synthetic agonist GSK1016790A was not affected. The low level of basal phosphorylation at Ser-824 was robustly increased by the redox signaling molecule hydrogen peroxide (H2O2). The H2O2-induced phosphorylation was accompanied by an enhanced channel activation by AA, and this enhanced response was largely abolished by PKA inhibition or S824A mutation. We further identified a potential structural context dependence of Ser-824 phosphorylation-mediated TRPV4 regulation involving an interplay between AA binding and the possible phosphorylation-induced rearrangements of the C-terminal helix bearing Ser-824. These results provide insight into how phosphorylation specifically regulates TRPV4 activation. Redox-mediated TRPV4 phosphorylation may contribute to pathologies associated with enhanced TRPV4 activity in endothelial and other systems.

Shaker-related voltage-gated K+ channel expression and vasomotor function in human coronary resistance arteries.

OBJECTIVES: KV channels are important regulators of vascular tone, but the identity of specific KV channels involved and their regulation in disease remain less well understood. We determined the expression of KV 1 channel subunits and their role in cAMP-mediated dilation in coronary resistance arteries from subjects with and without CAD. METHODS: HCAs from patients with and without CAD were assessed for mRNA and protein expression of KV 1 channel subunits with molecular techniques and for vasodilator response with isolated arterial myography. RESULTS: Assays of mRNA transcripts, membrane protein expression, and vascular cell-specific localization revealed abundant expression of KV 1.5 in vascular smooth muscle cells of non-CAD HCAs. Isoproterenol and forskolin, two distinct cAMP-mediated vasodilators, induced potent dilation of non-CAD arterioles, which was inhibited by both the general KV blocker 4-AP and the selective KV 1.5 blocker DPO-1. The cAMP-mediated dilation was reduced in CAD and was accompanied by a loss of or reduced contribution of 4-AP-sensitive KV channels. CONCLUSIONS: KV 1.5, as a major 4-AP-sensitive KV 1 channel expressed in coronary VSMCs, mediates cAMP-mediated dilation in non-CAD arterioles. The cAMP-mediated dilation is reduced in CAD coronary arterioles, which is associated with impaired 4-AP-sensitive KV channel function.

Contribution of KV1.5 Channel to Hydrogen Peroxide-Induced Human Arteriolar Dilation and Its Modulation by Coronary Artery Disease.

RATIONALE: Hydrogen peroxide (H2O2) regulates vascular tone in the human microcirculation under physiological and pathophysiological conditions. It dilates arterioles by activating large-conductance Ca2+-activated K+ channels in subjects with coronary artery disease (CAD), but its mechanisms of action in subjects without CAD (non-CAD) when compared with those with CAD remain unknown. OBJECTIVE: We hypothesize that H2O2-elicited dilation involves different K+ channels in non-CAD versus CAD, resulting in an altered capacity for vasodilation during disease. METHODS AND RESULTS: H2O2 induced endothelium-independent vasodilation in non-CAD adipose arterioles, which was reduced by paxilline, a large-conductance Ca2+-activated K+ channel blocker, and by 4-aminopyridine, a voltage-gated K+ (KV) channel blocker. Assays of mRNA transcripts, protein expression, and subcellular localization revealed that KV1.5 is the major KV1 channel expressed in vascular smooth muscle cells and is abundantly localized on the plasma membrane. The selective KV1.5 blocker diphenylphosphine oxide-1 and the KV1.3/1.5 blocker 5-(4-phenylbutoxy)psoralen reduced H2O2-elicited dilation to a similar extent as 4-aminopyridine, but the selective KV1.3 blocker phenoxyalkoxypsoralen-1 was without effect. In arterioles from CAD subjects, H2O2-induced dilation was significantly reduced, and this dilation was inhibited by paxilline but not by 4-aminopyridine, diphenylphosphine oxide-1, or 5-(4-phenylbutoxy)psoralen. KV1.5 cell membrane localization and diphenylphosphine oxide-1-sensitive K+ currents were markedly reduced in isolated vascular smooth muscle cells from CAD arterioles, although mRNA or total cellular protein expression was largely unchanged. CONCLUSIONS: In human arterioles, H2O2-induced dilation is impaired in CAD, which is associated with a transition from a combined large-conductance Ca2+-activated K+- and KV (KV1.5)-mediated vasodilation toward a large-conductance Ca2+-activated K+-predominant mechanism of dilation. Loss of KV1.5 vasomotor function may play an important role in microvascular dysfunction in CAD or other vascular diseases.