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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 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.

Zhang DX, Gutterman DD. Mitochondrial reactive oxygen species-mediated signaling in endothelial cells. Am J Physiol Heart Circ Physiol 292: H2023-H2031, 2007. First published January 19, 2007; doi:10.1152/ajpheart.01283.2006.-Once thought of as toxic by-products of cellular metabolism, reactive oxygen species (ROS) have been implicated in a large variety of cell-signaling processes. Several enzymatic systems contribute to ROS production in vascular endothelial cells, including NA(D)PH oxidase, xanthine oxidase, uncoupled endothelial nitric oxide synthase, and the mitochondrial electron transport chain. The respiratory chain is the major source of ROS in most mammalian cells, but the role of mitochondriaderived ROS in vascular cell signaling has received little attention. A new paradigm has evolved in recent years postulating that, in addition to producing ATP, mitochondria also play a key role in cell signaling and regulate a variety of cellular functions. This review focuses on the emerging role of mitochondrial ROS as signaling molecules in vascular endothelial cells. Specifically, we discuss some recent findings that indicate that mitochondrial ROS regulate vascular endothelial function, focusing on major sites of ROS production in endothelial mitochondria, factors modulating mitochondrial ROS production, the physiological and clinical implications of endothelial mitochondrial ROS, and methodological considerations in the study of mitochondrial contribution to vascular ROS generation. mitochondria; endothelium REACTIVE OXYGEN SPECIES (ROS), such as superoxide (O 2

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...