Objective-Ca2ϩ -influx through transient receptor potential (TRP) channels was proposed to be important in endothelial function, although the precise role of specific TRP channels is unknown. Here, we investigated the role of the putatively mechanosensitive TRPV4 channel in the mechanisms of endothelium-dependent vasodilatation. Methods and Results-Expression and function of TRPV4 was investigated in rat carotid artery endothelial cells (RCAECs) by using in situ patch-clamp techniques, single-cell RT-PCR, Ca 2ϩ measurements, and pressure myography in carotid artery (CA) and Arteria gracilis. In RCAECs in situ, TRPV4 currents were activated by the selective TRPV4 opener 4␣-phorbol-12,13-didecanoate (4␣PDD), arachidonic acid, moderate warmth, and mechanically by hypotonic cell swelling. Single-cell RT-PCR in endothelial cells demonstrated mRNA expression of TRPV4. In FURA-2 Ca 2ϩ measurements, 4␣PDD increased [Ca 2ϩ ] i by Ϸ140 nmol/L above basal levels. In pressure myograph experiments in CAs and A gracilis, 4␣PDD caused robust endothelium-dependent and strictly endothelium-dependent vasodilatations by Ϸ80% (K D 0.3 mol/L), which were suppressed by the TRPV4 blocker ruthenium red (RuR). Shear stress-induced vasodilatation was similarly blocked by RuR and also by the phospholipase A 2 inhibitor arachidonyl trifluoromethyl ketone (AACOCF 3 ). 4␣PDD produced endothelium-derived hyperpolarizing factor (EDHF)-type responses in A gracilis but not in rat carotid artery. Shear stress did not produce EDHF-type vasodilatation in either vessel type. Conclusions-Ca2ϩ entry through endothelial TRPV4 channels triggers NO-and EDHF-dependent vasodilatation. Moreover, TRPV4 appears to be mechanistically important in endothelial mechanosensing of shear stress. Key Words: endothelium-dependent vasodilatation Ⅲ transient receptor potential Ⅲ TRPV4 Ⅲ calcium Ⅲ shear stress Ⅲ nitric oxide Ⅲ 4␣PDD Ⅲ rat carotid artery C a 2ϩ -influx in response to mechanical or humoral stimulation plays a significant role in a variety of endothelial functions and especially in the Ca 2ϩ -dependent synthesis of endothelium-derived vasodilators such as NO, prostacyclin, or the endothelium-derived hyperpolarizing factor (EDHF). 1

Abstract-The endothelium plays a key role in the control of vascular tone and alteration in endothelial cell function contributes to several cardiovascular disease states. Endothelium-dependent dilation is mediated by NO, prostacyclin, and an endothelium-derived hyperpolarizing factor (EDHF). EDHF signaling is thought to be initiated by activation of endothelial Ca 2ϩ

BackgroundIn blood vessels, the endothelium is a crucial signal transduction interface in control of vascular tone and blood pressure to ensure energy and oxygen supply according to the organs' needs. In response to vasoactive factors and to shear stress elicited by blood flow, the endothelium secretes vasodilating or vasocontracting autacoids, which adjust the contractile state of the smooth muscle. In endothelial sensing of shear stress, the osmo- and mechanosensitive Ca2+-permeable TRPV4 channel has been proposed to be candidate mechanosensor. Using TRPV4−/− mice, we now investigated whether the absence of endothelial TRPV4 alters shear-stress-induced arterial vasodilation.Methodology/Principal FindingsIn TRPV4−/− mice, loss of the TRPV4 protein was confirmed by Western blot, immunohistochemistry and by in situ-patch–clamp techniques in carotid artery endothelial cells (CAEC). Endothelium-dependent vasodilation was determined by pressure myography in carotid arteries (CA) from TRPV4−/− mice and wild-type littermates (WT). In WT CAEC, TRPV4 currents could be elicited by TRPV4 activators 4α-phorbol-12,13-didecanoate (4αPDD), arachidonic acid (AA), and by hypotonic cell swelling (HTS). In striking contrast, in TRPV4−/− mice, 4αPDD did not produce currents and currents elicited by AA and HTS were significantly reduced. 4αPDD caused a robust and endothelium-dependent vasodilation in WT mice, again conspicuously absent in TRPV4−/− mice. Shear stress-induced vasodilation could readily be evoked in WT, but was completely eliminated in TRPV4−/− mice. In addition, flow/reperfusion-induced vasodilation was significantly reduced in TRPV4−/− vs. WT mice. Vasodilation in response to acetylcholine, vasoconstriction in response to phenylephrine, and passive mechanical compliance did not differ between genotypes, greatly underscoring the specificity of the above trpv4-dependent phenotype for physiologically relevant shear stress.Conclusions/SignificanceGenetically encoded loss-of-function of trpv4 results in a loss of shear stress-induced vasodilation, a response pattern critically dependent on endothelial TRPV4 expression. Thus, Ca2+-influx through endothelial TRPV4 channels is a molecular mechanism contributing significantly to endothelial mechanotransduction.

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SummaryIn the yeast Saccharomyces cerevisiae , genes involved in phospholipid biosynthesis are activated by ICRE (inositol/choline-responsive element) upstream motifs and the corresponding heterodimeric binding factor, Ino2 + + + + Ino4. Both Ino2 and Ino4 contain basic helix-loop-helix (bHLH) domains required for ICRE binding, whereas transcriptional activation is mediated exclusively by Ino2. In this work, we describe a molecular analysis of functional minimal domains responsible for specific DNA recognition and transcriptional activation (TAD1 and TAD2). We also define the importance of individual amino acids within the more important activation domain TAD1. Random mutagenesis at five amino acid positions showed the importance of acidic as well as hydrophobic residues within this minimal TAD. We also investigated the contribution of known general transcription factors and co-activators for Ino2-dependent gene activation. Although an ada5 single mutant and a gal11 paf1 double mutant were severely affected, a partial reduction in activation was found for gcn5 and srb2 . Ino2 interacts physically with the basal transcription factor Sua7 (TFIIB of yeast). Interestingly, interaction is mediated by the HLH dimerization domain of Ino2 and by two non-overlapping domains within Sua7. Thus, Sua7 may compete with Ino4 for binding to the Ino2 activator, creating the possibility of positive and negative influence of Sua7 on ICREdependent gene expression.

Modulation of Ca2+‐activated K+ channels (KCa) has been implicated in the control of proliferation in vascular smooth muscle cells (VSMC) and other cell types. In the present study, we investigated the underlying signal transduction mechanisms leading to mitogen‐induced alterations in the expression pattern of intermediate‐conductance KCa in VSMC. Regulation of expression of IKCa/rKCa3.1 and BKCa/rKCa1.1 in A7r5 cells, a cell line derived from rat aortic VSMC, was investigated by patch‐clamp technique, quantitative RT–PCR, immunoblotting procedures, and siRNA strategy. PDGF stimulation for 2 and 48 h induced an 11‐ and 3.5‐fold increase in rKCa3.1 transcript levels resulting in a four‐ and seven‐fold increase in IKCa currents after 4 and 48 h, respectively. Upregulation of rKCa3.1 transcript levels and channel function required phosphorylation of extracellular signal‐regulated kinases (ERK1/2) and Ca2+ mobilization, but not activation of p38‐MAP kinase, c‐Jun NH(2)‐terminal kinase, protein kinase C, calcium‐calmodulin kinase II and Src kinases. In contrast to rKCa3.1, mRNA expression and functions of BKCa/rKCa1.1 were decreased by half following mitogenic stimulation. Downregulation of rKCa1.1 did not require ERK1/2 phosphorylation or Ca2+ mobilization. In an in vitro‐proliferation assay, knockdown of rKCa3.1 expression by siRNA completely abolished functional IKCa channels and mitogenesis. Mitogen‐induced upregulation of rKCa3.1 expression is mediated via activation of the Raf/MEK‐ and ERK‐signaling cascade in a Ca2+‐dependent manner. Upregulation of rKCa3.1 promotes VSMC proliferation and may thus represent a pharmacological target in cardiovascular disease states characterized by abnormal cell proliferation. British Journal of Pharmacology (2006) 148, 909–917. doi:

Structural genes of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae are coordinately regulated by a UAS element, designated ICRE (inositol/cholineresponsive element). Opi1 is a negative regulator responsible for repression of ICRE-dependent genes in the presence of an excess of inositol and choline. Gene regulation by phospholipid precursors has been also reported for the pathogenic yeast Candida albicans. Screening of a data base containing raw sequences of the C. albicans genome project allowed us to identify an open reading frame exhibiting weak similarity to Opi1. Expression of the putative CaOPI1 in an opi1 mutant of S. cerevisiae could restore repression of an ICRE-dependent reporter gene. Similar to OPI1, overexpression of CaOPI1 strongly inhibited derepression of ICRE-driven genes leading to inositol-requiring transformants. Previous work has shown that Opi1 mediates gene repression by interaction with the pleiotropic repressor Sin3. The genome of C. albicans also encodes a protein similar to Sin3 (CaSin3). By two-hybrid analyses and in vitro studies for protein-protein interaction we were able to show that CaOpi1 binds to ScSin3. ScOpi1 could also interact with CaSin3, while CaOpi1 failed to bind to CaSin3. Despite of some conservation of regulatory mechanisms between both yeasts, these results suggest that repression of phospholipid biosynthetic genes in C. albicans is mediated by a mechanism which does not involve recruitment of CaSin3 by CaOpi1.

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