Alveolar hypoxia may impair sodium-dependent alveolar fluid transport and induce pulmonary edema in rat and human lung, an effect that can be prevented by the inhalation of ␤ 2 -agonists. To investigate the mechanism of ␤ 2 -agonist-mediated stimulation of sodium transport under conditions of moderate hypoxia, we examined the effect of terbutaline on epithelial sodium channel (ENaC) expression and activity in cultured rat alveolar epithelial type II cells exposed to 3% O 2 for 24 h. Hypoxia reduced transepithelial sodium current and amiloridesensitive sodium channel activity without decreasing ENaC subunit mRNA or protein levels. The functional decrease was associated with reduced abundance of ENaC subunits (especially ␤ and ␥) in the apical membrane of hypoxic cells, as quantified by biotinylation. cAMP stimulation with terbutaline reversed the hypoxia-induced decrease in transepithelial sodium transport by stimulating sodium channel activity and markedly increased the abundance of ␤-and ␥-ENaC in the plasma membrane of hypoxic cells. The effect of terbutaline was prevented by brefeldin A, a blocker of anterograde transport. These novel results establish that hypoxiainduced inhibition of amiloride-sensitive sodium channel activity is mediated by decreased apical expression of ENaC subunits and that ␤ 2 -agonists reverse this effect by enhancing the insertion of ENaC subunits into the membrane of hypoxic alveolar epithelial cells.

Decrease in alveolar oxygen tension may induce acute lung injury with pulmonary edema. We investigated whether, in alveolar epithelial cells, expression and activity of epithelial sodium (Na) channels and Na,K-adenosine triphosphatase, the major components of transepithelial Na transport, were regulated by hypoxia. Exposure of cultured rat alveolar cells to 3% and 0% O2 for 18 h reduced Na channel activity estimated by amiloride-sensitive 22Na influx by 32% and 67%, respectively, whereas 5% O2 was without effect. The decrease in Na channel activity induced by 0% O2 was time-dependent, significant at 3 h of exposure and maximal at 12 and 18 h. It was associated with a time-dependent decline in the amount of mRNAs encoding the alpha-, beta-, and gamma-subunits of the rat epithelial Na channel (rENaC) and with a 42% decrease in alpha-rENaC protein synthesis as evaluated by immunoprecipitation after 18 h of exposure. The 0% O2 hypoxia also caused a time-dependent decrease in (1) ouabain-sensitive 86Rubidium influx in intact cells, (2) the maximal velocity of Na,K-ATPase on crude homogenates, and (3) alpha1- and beta1-Na,K-ATPase mRNA levels. Levels of rENaC and alpha1-Na,K-ATPase mRNA returned to control values within 48 h of reoxygenation, and this was associated with complete functional recovery. We conclude that hypoxia induced a downregulation of expression and activity of epithelial Na channels and Na,K-ATPase in alveolar cells. Subsequent decrease in Na reabsorption by alveolar epithelium could participate in the maintenance of hypoxia-induced alveolar edema.

Objectives-During inflammation, cell adhesion molecules are modulated or redistributed for leukocyte transmigration.Among molecules at the interendothelial junction, CD146 is involved in cell-cell cohesion and permeability, but its role in monocyte transmigration is unknown. Methods and Results-TNF enhanced CD146 expression at the junction and apical membrane of human umbilical veins endothelial cells (HUVECs) through CD146 synthesis and intracellular store redistribution. In addition, TNF increased the release of a soluble form (sCD146) through a metalloproteinase-dependent mechanism. The redistribution of CD146 to the junction led us to investigate its role in monocyte transmigration using THP1 and freshly isolated monocytes.Evidence that CD146 contributes to monocyte transmigration was provided by inhibition experiments using anti-CD146 antibodies and CD146 siRNA in HUVECs. In addition, sCD146 specifically bound both monocytes and HUVECs and dose-dependently increased monocyte transmigration. Assessment of sCD146 binding on immobilized CD146 failed to evidence any homophilic interaction. Together, our data suggest endothelial CD146 binds heterophilically with a yet unknown ligand on monocytes. Conclusions-Our results demonstrate that CD146 is regulated by the inflammatory cytokine TNF and that CD146 and sCD146 are both involved in monocyte transendothelial migration during inflammation. Key Words: endothelial cells Ⅲ inflammation Ⅲ cytokines Ⅲ human Ⅲ adhesion molecules T he endothelial junctions play a fundamental role in endothelial integrity, vascular permeability, and cellular traffic. 1 At least 2 types of cell-to-cell junctional structures have been identified in the endothelium: adherens junctions (AJ) and tight junctions (TJ). 2 These junctions are tightly regulated structures composed of several adhesion molecules interacting with cytoskeletal proteins. 3 Among the adhesive molecules, endothelial VE-cadherin 4 is localized in AJ and junctional adhesion molecule (JAM) 5 in TJ, whereas other molecules such as PECAM-1/CD31 (platelet endothelial cell adhesion molecule-1) and CD99 are not restricted to 1 type of junctional structure. 6 The inflammatory response is characterized by leukocyte infiltration from the circulation toward the tissues, after a multistep process in which proinflammatory endothelial activation results in increased vascular permeability and then in leukocyte adhesion and transmigration. 7 Endothelial activation is mediated by several inflammatory cytokines. Among them, TNF␣ increases the expression of cell adhesion molecules like ICAM-1 or VCAM-1 and induces the redistribution of junctional adhesion molecules such as PECAM-1, JAM, VE-cadherin, and CD99 which, in turn, promote the transendothelial migration of leukocytes. 8 We have previously shown that CD146 (S-Endo1 Ag) is a component of the endothelial junction localized outside defined junctional structures. 9 CD146, also referred to as MUC18, is a member of the immunoglobulin superfamily (IgSF) constitutively expressed in all typ...

The early phase of the stimulatory action of aldosterone on sodium reabsorption in tight epithelia involves hormone-regulated genes that remain to be identified. Using a subtractive hybridization technique on isolated renal cortical collecting ducts from rats injected with a physiological dose of aldosterone, we have identified an early response cDNA highly homologous to human and murine NDRG2 (N-Myc downstream regulated gene 2), which consists of four isoforms and belongs to a new family of differentiation-related genes. NDRG2 mRNA was expressed in classical aldosterone target epithelia, and in the kidney, it was specifically located in the collecting duct, the site of aldosterone-regulated sodium absorption. NDRG2 mRNA was increased within 45 min by aldosterone in the kidney and distal colon, whereas it was unaffected in the heart. In the RCCD2 collecting duct cell line, NDRG2 mRNA was enhanced as early as 15 min after aldosterone addition by transcription-dependent effects. NDRG2 was induced by aldosterone concentrations as low as 10 ؊9 M, and a maximal effect was observed at 10 ؊8 M. In contrast, the glucocorticoid dexamethasone was ineffective in NDRG2 expression, whereas the glucocorticoid-regulated gene sgk was induced. Taken together, these results indicate that NDRG2 regulation by aldosterone is an early mineralocorticoid-specific effect. Interestingly, NDRG2 is homologous to Drosophila MESK2, a component of the Ras pathway, suggesting that activation of the Ras cascade may play a significant role in mineralocorticoid signaling.

SummaryCD146 is an adhesion moleculep resent on endothelialc ells throughout the vascular tree.CD146 is also expressedbycirculating endothelialcells (CECs) widelyconsideredtobemature endothelialcells detachedfrominjured vessels.The discovery of circulating endothelialprogenitor cells(EPCs)originating from bone marrowp romptedu st oi nvestigate whetherC D146 circulating cells could also contains EPCs.Wetestedthis hypothesis using an approach combininge limination of CECs by an adhesion step, followedbyimmunomagnetic sorting of remaining CD146

Background information. The renal CCD (cortical collecting duct) plays a role in final volume and concentration of urine by a process that is regulated by the antidiuretic hormone, [arginine]vasopressin. This hormone induces an increase in water permeability due to the translocation of AQP2 (aquaporin 2) from the intracellular vesicles to the apical membrane of principal cells. During the transition from antidiuresis to diuresis, CCD cells are exposed to changes in environmental osmolality, and cell-volume regulation may be especially important for the maintenance of intracellular homoeostasis. Despite its importance, cell-volume regulation in CCD cells has not been widely investigated. Moreover, no studies have been carried out till date to evaluate the putative role of AQPs during this process in renal cells.Results. In the present study, we have studied the regulatory cell-volume responses to hypo-osmotic or hyperosmotic challenges in two CCD cell lines: one not expressing AQPs and the other stably transfected with AQP2. We have used a fluorescent probe technique in which the acquisition of single-cell kinetic data can be simultaneously recorded with the intracellular pH. Experiments with hyperosmotic mannitol media demonstrated that, independent of AQP2 expression, CCD cells shrink but fail to show regulatory volume increase, at least under the studied conditions. In contrast, under hypo-osmotic shocks, regulatory volume decrease occurs and the activation of these mechanisms is more rapid in AQP2 transfected cells. This regulatory response takes place in parallel with intracellular acidification, which is faster in cells expressing AQP2. The acidification and the initial regulatory volume decrease response were inhibited by glibenclamide and BaCl 2 only in AQP2 cells. Conclusions.These results suggest that increases in the osmotic water permeability due to the expression of AQP2 are critical for a rapid activation of regulatory volume decrease mechanisms, which would be linked to cystic fibrosis transmembrane conductance regulator and to barium-sensitive potassium channels. IntroductionThe mammalian collecting duct plays a central role in the final volume and concentration of urine. Twothirds of the hypo-osmotic fluid entering the collecting duct is reabsorbed into the CCD (cortical collecting duct) due to osmotic equilibration with the

CD146, an endothelial molecule involved in permeability and monocyte transmigration, has recently been reported to promote vessel growth. As CD146 is also detectable as a soluble form (sCD146), we hypothesized that sCD146 could stimulate angiogenesis. Experiments of Matrigel plugs in vivo showed that sCD146 displayed chemotactic activity on endogenous endothelial cells, and exogenously injected late endothelial progenitor cells (EPCs) . IntroductionCD146 is a component of the endothelial junction primarily expressed in endothelial cells. It is involved in the control of cell and tissue architecture, as demonstrated by the regulation of its expression during endothelium monolayer formation, its involvement in the control of paracellular permeability, and its colocalization with the actin cytoskeleton. 1 Besides its structural role, CD146 is also involved in cell signaling. 2,3 We have recently demonstrated that CD146 is involved in the regulation of monocyte transendothelial migration. 4 Recent findings indicate that CD146 displays angiogenic properties. In one study, the authors showed that an anti-CD146 antibody, mAb AA98, displayed antiangiogenic properties in chicken chorioallantoic membrane assays and inhibited tumor growth in different xenografted human tumor models in mice. In a model of human umbilical vein endothelial cells (HUVECs), it was also shown that silencing CD146 with specific siRNA inhibited the proliferation and migration of the cells. [5][6][7] Of interest, we have established that CD146 also exists in a soluble form (sCD146) as the result of metalloprotease-dependent shedding of membrane CD146. 4,8 sCD146 is detectable in the human serum, and its level is modulated in different pathologies, such as inflammatory bowel diseases, 9 pathologic pregnancies, 10 and chronic renal failure. 11 However, its exact role is still largely unknown.Postischemic neovascularization occurs as a result of 2 mechanisms: angiogenesis, which relies on mature endothelial cells already present at the ischemic site; and vasculogenesis, which involves the homing and endothelial differentiation of endothelial progenitor cells (EPCs) mobilized from the bone marrow. 12,13 Different angiogenic factors have been shown to trigger angiogenesis and/or vasculogenesis by directly or indirectly stimulating proliferation, differentiation, and migration of mature or precursor cells. Among these factors, the more effective are fibroblast growth factors (FGFs), vascular endothelial growth factor (VEGF), and angiopoietins (Ang). FGF-1 has been shown to stimulate the proliferation and differentiation of all cell types necessary for the constitution of an arterial vessel, including endothelial cells and smooth muscle cells. FGF-2 also promotes endothelial cell proliferation and organization of endothelial cells into capillary-like structures. 14 In vitro studies have clearly demonstrated that VEGF is a potent stimulator of angiogenesis, stimulating endothelial cell mitogenesis and migration, 15 and numerous clinical trials have been co...

Cancer extracellular vesicles (EVs) shuttle at distance and fertilize pre-metastatic niches facilitating subsequent seeding by tumor cells. However, the link between EV secretion mechanisms and their capacity to form pre-metastatic niches remains obscure. Using mouse models, we show that GTPases of the Ral family control, through the phospholipase D1, multi-vesicular bodies homeostasis and tune the biogenesis and secretion of pro-metastatic EVs. Importantly, EVs from RalA or RalB depleted cells have limited organotropic capacities in vivoand are less efficient in promoting metastasis. RalA and RalB reduce the EV levels of the adhesion molecule MCAM/CD146, which favors EV-mediated metastasis by allowing EVs targeting to the lungs. Finally, RalA, RalB, and MCAM/CD146, are factors of poor prognosis in breast cancer patients. Altogether, our study identifies RalGTPases as central molecules linking the mechanisms of EVs secretion and cargo loading to their capacity to disseminate and induce pre-metastatic niches in a CD146-dependent manner.