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NF-[kappa]B signaling is known to induce the expression of antiapoptotic and proinflammatory genes in endothelial cells (ECs). We have shown recently that [Ca.sup.2+] influx through canonical transient receptor potential (TRPC) channels activates NF-[kappa]B in ECs. Here we show that [Ca.sup.2+] influx signal prevents thrombin-induced apoptosis by inducing NF-[kappa]B-dependent A20 expression in ECs. Knockdown of TRPCI expressed in human umbilical vein ECs with small interfering RNA (siRNA) suppressed thrombin-induced [Ca.sup.2+] influx and NF-[kappa]B activation in ECs. Interestingly, we observed that thrombin induced >25% of cell death (apoptosis) in TRPC1-knockdown ECs whereas thrombin had no effect on control or control siRNA-transfected ECs. To understand the basis of EC survival, we performed gene microarray analysis using ECs. Thrombin stimulation increased only a set of NF-[kappa]B-regulated genes 3- to 14-fold over basal levels in ECs. Expression of the antiapoptotic gene A20 was the highest among these upregulated genes. Like TRPC1 knockdown, thrombin induced apoptosis in A20-knockdown ECs. To address the importance of [Ca.sup.2+] influx signal, we measured thrombin-induced A20 expression in control and TRPC1-knockdown ECs. Thrombin-induced p65/RelA binding to A20 promoter-specific NF-[kappa]B sequence and A20 protein expression were suppressed in TRPC1-knockdown ECs compared with control ECs. Furthermore, in TRPC1-knockdown ECs, thrombin induced the expression of proapoptotic proteins caspase-3 and BAX. Importantly, thrombin-induced apoptosis in TRPC1-knockdown ECs was prevented by adenovirus-mediated expression of A20. These results suggest that [Ca.sup.2+] influx via TRPC channels plays a critical role in the mechanism of cell survival signaling through A20 expression in ECs. transient receptor potential canonical channel-1; nuclear factor-[kappa]B doi:10.1152/ajpcell.00456.2009

Caveolin-1 (Cav-1) regulates agonist-induced [Ca.sup.2+] entry in endothelial cells; however, how Cav-1 regulates this process is poorly understood. Here, we describe that Cav-1 scaffold domain (N[H.sub.2]-terminal residues 82-101; CSD) interacts with transient receptor potential canonical channel 1 (TRPC1) and inositol 1,4,5-trisphosphate receptor 3 ([IP.sub.3]R3) to regulate [Ca.sup.2+] entry. We have shown previously that the TRPC1 COOH-terminal residues 781-789 bind to CSD. In the present study, we show that the TRPC1 COOH-terminal residues 781-789 truncated (TRPC1-C[DELTA]781-789) mutant expression abolished [Ca.sup.2+] store release-induced [Ca.sup.2+] influx in human dermal microvascular endothelial cell line (HMEC) and human embryonic kidney (HEK-293) cells. To understand the basis of loss of [Ca.sup.2+] influx, we determined TRPC1 binding to IP3R3. We observed that the wild-type (WT)-TRPC1 but not TRPC1-C[DELTA]781-789 effectively interacted with [IP.sub.3]R3. Similarly, WT-TRPC1 interacted with Cav-1, whereas TRPC1-C[DELTA]781-789 binding to Cav-1 was markedly suppressed. We also assessed the direct binding of Cav-1 with TRPC1 and observed that the WT-Cav-1 but not the Cav-1[DELTA]CSD effectively interacted with TRPC1. Since the interaction between TRPC1 and Cav-1[DELTA]CSD was reduced, we measured [Ca.sup.2+] store release-induced [Ca.sup.2+] influx in Cav-l[DELTA]CSD-transfected cells. Surprisingly, Cav-1[DELTA]CSD expression showed a gain-of-function in [Ca.sup.2+] entry in HMEC and HEK-293 cells. We observed a similar gain-of-function in [Ca.sup.2+] entry when Cav-1[DELTA]CSD was expressed in lung endothelial cells of Cav-1 knockout mice. Immunoprecipitation results revealed that WT-Cav-1 but not Cav-1[DELTA]CSD interacted with [IP.sub.3]R3. Furthermore, we observed using confocal imaging the colocalization of [IP.sub.3]R3 with WT-Cav-1 but not with Cav-1[DELTA]CSD on [Ca.sup.2+] store release in endothelial cells. These findings suggest that CSD interacts with TRPC1 and [IP.sub.3]R3 and thereby regulates [Ca.sup.2+] store release-induced [Ca.sup.2+] entry in endothelial cells. transient receptor potential channel 1; inositol 1,4,5-trisphosphate receptor; caveolin-1 knockout mice

We determined the effects of TNF-[alpha] on the expression of transient receptor potential channel (TRPC) homologues in human vascular endothelial cells and the consequences of TRPC expression on the endothelial permeability response. We observed that TNF-[alpha] exposure increased TRPC1 expression without significantly altering expression of other TRPC isoforms in human pulmonary artery endothelial cells (HPAEC). Because TRPC1 belongs to the store-operated cation channel family, we measured the [Ca.sup.2+] store depletion-mediated [Ca.sup.2+] influx in response to thrombin exposure. We observed that thrombininduced [Ca.sup.2+] influx in TNF-[alpha]-stimulated HPAEC was twofold greater than in control cells. To address the relationship between store-operated [Ca.sup.2+] influx and TRPC1 expression, we overexpressed TRPC1 by three- to fourfold in the human dermal microvascular endothelial cell line (HMEC) using the TRPC1 cDNA. Thrombininduced store [Ca.sup.2+] depletion in these cells caused approximately twofold greater increase in [Ca.sup.2+] influx than in control cells. Furthermore, the inositol 1,4,5-trisphosphate-sensitive store-operated cationic current was increased greater than twofold in TRPCl-transfected cells compared with control. To address the role of [Ca.sup.2+] influx via TRPC1 in signaling endothelial permeability, we measured actinstress fiber formation and transendothelial monolayer electrical resistance (TER) in the TRPC1 cDNA-transfected HMEC and TNF[alpha]-challenged HPAEC. Both thrombin-induced actin-stress fiber formation and a decrease in TER were augmented in TRPCl-overexpressing HMEC compared with control cells. TNF-[alpha]-induced increased TRPC1 expression in HPAEC also resulted in marked endothelial barrier dysfunction in response to thrombin. These findings indicate the expression level of TRPC1 in endothelial cells is a critical determinant of [Ca.sup.2+] influx and signaling of the increase in endothelial permeability. tumor necrosis factor-[alpha]; store-operated calcium ion influx; transient receptor potential channel 1; endothelial barrier dysfunction

We determined the concentration dependence of albumin binding, uptake, and transport in confluent monolayers of cultured rat lung microvascular endothelial cells (RLMVEC). Transport of [sup.125]I-albumin in RLMVEC monolayers occurred at a rate of 7.2 fmol * [min.sup.-1] * [10.sup.6] [cells.sup.-1]. Albumin transport was inhibited by cell surface depletion of the 60-kDa albumin-binding glycoprotein gp60 and by disruption of caveolae using methyl-[beta]-cyclodextrin. By contrast, gp60 activation (by means of gp60 cross-linking using primary and secondary antibodies) increased [sup.125]I-albumin uptake 2.3-fold. At 37[degrees]C, [sup.125]I-albumin uptake had a half time of 10 min and was competitively inhibited by unlabeled albumin (I[C.sub.50] = 1 [micro]M). Using a two-site model, we estimated by Scatchard analysis the affinity ([K.sub.D]) and maximal capacity ([B.sub.max]) of albumin uptake to be 0.87 [micro]M ([K.sub.D1]) and 0.47 pmol/[10.sup.6] cells ([B.sub.max1]) and 93.3 [micro]M ([K.sub.D2]) and 20.2 pmol/[10.sup.6] cells ([B.sub.max2]). At 4[degrees]C, we also observed two populations of specific binding sites, with high ([K.sub.D1] = 13.5 nM, 1% of the total) and low ([K.sub.D2] = 1.6 [micro]M) affinity. On the basis of these data, we propose a model in which the two binding affinities represent the clustered and unclustered gp60 forms. The model predicts that fluid phase albumin in caveolae accounts for the bulk of albumin internalized and transported in the endothelial monolayer. capillary permeability; vesicular transport; caveolae; albumin-binding glycoprotein gp60

Vogel, Stephen M., Richard D. Minshall, Milena Pilipovic, Chinnaswamy Tiruppathi, and Asrar B. Malik. Albumin uptake and transcytosis in endothelial cells in vivo induced by albumin-binding protein. Am J Physiol Lung Cell Mol Physiol 281: L1512-L1522, 2001.--The 60-kDa endothelial cell surface albumin-binding glycoprotein (gp60) is postulated to be a docking site for albumin that mediates the uptake of albumin and its transport in cultured microvessel endothelial cells. In the present study, we used an isolated Krebs-perfused rat lung preparation to address the in vivo role of gp60 in mediating albumin uptake and transport. Addition of primary anti-gp60 antibody to the perfusate followed by the secondary antibody to cross-link gp60 increased the vessel wall [sup.125.I]-albumin permeability-surface area (PS) product 2.5-fold without affecting the capillary filtration coefficient ([K.sub.f,c]; a measure of liquid permeability). In contrast, EDTA (5 mM), which induces interendothelial gap formation, produced parallel increases in both [K.sub.f,c] and [sup.125.I]-albumin PS product. Increasing perfusate albumin concentration to > 1 g/100 ml (E[C.sub.50] 1.2 g/100 ml) was sufficient to block [sup.125.I]-albumin PS product, indicating that the perfusate albumin competed with tracer albumin for transendothelial albumin transport. Cross-linking of gp60 in lungs perfused with saturating concentration of albumin resulted in a greater increase in [sup.125.I]-albumin PS product, indicating that gp60 function was capable of being modulated. These results show that activation of gp60 in pulmonary microvessels induces albumin uptake and its transport through a nonhydraulic pathway that fits with a model of albumin permeability via the transcellular pathway. rat lung; filipin; gp60; antibody cross-linking

Changes in cytosolic calcium activation and in human pulmonary artery endothelial cells (HPAEC) retraction response was measured to examine the kinetics of endothelial cell surface expression of proteolytically activated thrombin receptor (PAR-1) in HPAEC. The results indicated that endothelial cell surface PAR-1 can be quickly restored even when it is continuously exposed to a low concentration of thrombin. However, cellular PAR-1 pools are depleted by high thrombin concentrations and longer exposure times.

Immunofluorescence studies and cytosolic calcium determination were undertaken to determine the effects of protein kinase C (PKC) activation on the synthesis and activity of the proteolytically activated thrombin receptor 1 (PAR-1). Results indicate that PKC-catalyzed phosphorylation of PAR-1 leads to receptor inactivation. Further, PKC contributes to the loss of PAR-1 on the cell surface and to the heterologous desensitization of the receptor as well as to the inhibition of cellular signal transduction through calcium.

The thrombin receptor domains responsible for receptor activation in endothelial cells were studied using site-specific antibodies. Results of experiments on polyclonal antibodies directed against peptide sequences corresponding to postulated binding sites of thrombin suggested a critical role for the thrombin receptor's NH2-terminal extension and loop 2 sites. These binding sites influenced receptor activation in endothelial cells, which in turn lead to increased intracellular calcium and transendothelial permeability to albumin.

A decline in endothelial hydraulic conductivity (Lp), a measure of liquid permeability across endothelial and matrix barrier, is affected by albumin and lectin Ricinus communis agglutinin (RCA) in bovine pulmonary microvascular endothelial cell monolayers. This effect of albumin and RCA is consistent with the response associated with the binding of albumin and RCA to extracellular matrix (ECM). The permeability-reducing effect of these molecules is hindered by the administration of protamine sulfate, a polycation, thus suggesting the mechanism whereby albumin and RCA interact with ECM and render the endothelial layer to function as a barricade to liquid infiltration.

Japan F344 rats, deficient in dipeptidyl peptidase IV (DPP IV) were employed in examining DPP IV influence on intestinal hydrolysis and prolyl peptide uptake. Brush-border membrane hydrolysis was lesser in the Japan specimens. Control rats possessed greater resistance to weight loss than Japan F344 rats. In Japan rat's intestinal lumen, hydrolysis-resistant morphiceptin impact was higher. These Japan rat-specific reactions are attributed to the lack of DPP IV in the Japan rat-system.

The thrombin-induced increase in endothelial permeability requires the activation of protein kinase C (PKC) and the mobilization of Ca2+. The signaling pathway leading to endothelial permeability was investigated. The results showed that a synthetic thrombin, TRP-14, could induce the mobilization of Ca2+ but not the activation of PKC. These results indicate that thrombin-induced increase in endothelial permeability involves dual signals.

Expression of endothelial adhesivity to neutrophils was studied using human umbilical vein endothelium cells. These cells were challenged with human alpha-thrombin and the roles of P-selectin and intercellular adhesion molecule-1 (ICAM-1) in endothelial adhesivity were determined. Results indicated that P-selectin modulated initial endothelial adhesivity and ICAM-1 prolonged and stabilized this mechanism.

Because activation of the coagulation cascade and the generation of thrombin coexist with sepsis and the release of tumor necrosis factor (TNF)-[Alpha], we determined the effects of TNF-[Alpha] on the mechanism of thrombin-induced increase in endothelial permeability. We assessed [Ca.sup.2+] signaling in human umbilical vein endothelial cells. In human umbilical vein endothelial cells exposed to TNF-[Alpha] for 2 h, thrombin produced a rise in the intracellular [Ca.sup.2+] concentration ([[Ca.sup.2+]][sub.i]) lasting up to 10 min. In contrast, thrombin alone produced a rise in [[Ca.sup.2+]][sub.i] lasting for 3 min, whereas TNF-[Alpha] alone had no effect on [[Ca.sup.2+]][sub.i]. Thrombin-induced inositol 1,4,5-trisphosphate generation was not different between control and TNF-[Alpha]-exposed cells. In the absence of extracellular [Ca.sup.2+], thrombin produced similar increases in [[Ca.sup.2+]][sub.i] in both control and TNF-[Alpha]-exposed cells. In TNF-[Alpha]-exposed cells, the thrombin-induced [Ca.sup.2+] influx after intracellular [Ca.sup.2+] store depletion was significantly greater and prolonged compared with control cells. Increased [Ca.sup.2+] entry was associated with an approximately fourfold increase in Src activity and was sensitive to the Src kinase inhibitor PPI. After TNF-[Alpha] exposure, thrombin caused increased tyrosine phosphorylation of junctional proteins and actin stress fiber formation as well as augmented endothelial permeability. These results suggest that TNF-[Alpha] stimulation of endothelial cells results in amplification of the thrombin-induced [Ca.sup.2+] influx by an Src-dependent mechanism, thereby promoting loss of endothelial barrier function. store-operated calcium influx; Src tyrosine kinase

Requirement for [Ca.sup.2+] signaling in the mechanism of thrombin-induced increase in endothelial permeability. Am J Physiol Lung Cell Mol Physiol 280: L239-L247, 2001.--We compared the thrombin-activated responses in human umbilical vein endothelial cells (HUVECs) and a HUVEC-derived cell line, ECV304. Thrombin induced a 40-50% decrease in transendothelial monolayer electrical resistance and a twofold increase in [sup.125]I-albumin permeability in HUVECs, whereas it failed to alter the endothelial barrier function in ECV304 cells. Thrombin produced a brisk intracellular [Ca.sup.2+] concentration transient and phosphorylation of 20-kDa myosin light chain in HUVECs but not in ECV304 cells. Thrombin-induced phosphoinositide hydrolysis was comparable in ECV304 cells and HUVECs, indicating the activation of thrombin receptors in both cell types. [La.sup.3+] reduced both the thrombin-induced decrease in endothelial monolayer electrical resistance and the increase in [sup.125]I-albumin permeability in HUVECs. Because the absence of [Ca.sup.2+] signaling could explain the impairment in the permeability response in ECV304 cells, we studied the effect of increasing intracellular [Ca.sup.2+] concentration in ECV304 cells with thapsigargin. Exposure of ECV304 cells to thapsigargin caused decreased endothelial monolayer electrical resistance and increased [sup.125]I-albumin permeability. These results indicate that [Ca.sup.2+] influx and activation of [Ca.sup.2+]-dependent signaling pathways are important determinants of the thrombin-induced increase in endothelial permeability. human umbilical vein endothelial cells; ECV304 cells; cadherins; calcium signaling

We tested the hypothesis that the albumin-docking protein gp60, which is localized in caveolae, couples to the heterotrimeric GTP binding protein [G.sub.i], and thereby activates plasmalemmal vesicle formation and the directed migration of vesicles in endothelial cells (ECs). We used the water-soluble styryl pyridinium dye N-(3-triethylaminopropyl)-4-(p-dibutylaminostyryl) pyridinium dibromide (FM 1-43) to quantify vesicle trafficking by confocal and digital fluorescence microscopy. FM 1-43 and fluorescently labeled anti-gp60 antibody (Ab) were colocalized in endocytic vesicles within 5 min of gp60 activation. Vesicles migrated to the basolateral surface where they released FM 1-43, the fluid phase styryl probe. FM 1-43 fluorescence disappeared from the basolateral EC surface without the loss of anti-gp60 Ab fluorescence. Activation of cell-surface gp60 by cross-linking (using anti-gp60 Ab and secondary Ab) in EC grown on microporous filters increased transendothelial [.sup.125]I-albumin permeability without altering liquid permeability (hydraulic conductivity), thus, indicating the dissociation of hydraulic conductivity from the albumin permeability pathway. The findings that the sterol-binding agent, filipin, prevented gp60-activated vesicle formation and that caveolin-1 and gp60 were colocalized in vesicles suggest the caveolar origin of endocytic vesicles. Pertussis toxin pretreatment and expression of the dominant negative construct encoding an 11--amino acid [G[Alpha].sup.i] carboxyl-terminal peptide inhibited endothelial [.sup.125]I-albumin endocytosis and vesicle formation induced by gp60 activation. Expression of dominant negative Src (dn-Src) and overexpression of wild-type caveolin-1 also prevented gp60-activated endocytosis. Caveolin-1 overexpression resulted in the sequestration of [G[Alpha].sup.i] with the caveolin-1, whereas dn-Src inhibited [G[Alpha].sup.i] binding to caveolin-1. Thus, vesicle formation induced by gp60 and migration of vesicles to the basolateral membrane requires the interaction of gp60 with caveolin-1, followed by the activation of the downstream [G.sup.i]-coupled Src kinase signaling pathway. Key words: transcytosis * endocytosis * caveolae * microvascular endothelial cells * albumin permeability