A role for caveolin in transport of cholesterol from endoplasmic reticulum to plasma membrane

Caveolin is a 22-kDa membrane protein found associated with a coat material decorating the inner membrane surface of caveolae. A remarkable feature of this protein is its ability to migrate from caveolae directly to the endoplasmic reticulum (ER) when membrane cholesterol is oxidized. We now present evidence caveolin is involved in transporting newly synthesized cholesterol from the ER directly to caveolae. MA104 cells and normal human fibroblasts transported new cholesterol to caveolae with a half-time of approximately 10 min. The cholesterol then rapidly flowed from caveolae to non-caveolae membrane. Cholesterol moved out of caveolae even when the supply of fresh cholesterol from the ER was interrupted. Treatment of cells with 10 microg/ml progesterone blocked cholesterol movement from ER to caveolae. Simultaneously, caveolin accumulated in the lumen of the ER, suggesting cholesterol transport is linked to caveolin movement. Caveolae fractions from cells expressing caveolin were enriched in cholesterol 3-4-fold, while the same fractions from cells lacking caveolin were not enriched. Cholesterol transport to the cell surface was nearly 4 times more rapid in cells expressing caveolin than in matched cells lacking caveolin.     

Distribution of plasmalemmal Ca(2+)-pump and caveolin in the corneal epithelium during the wound healing process

PURPOSE: Caveolae are small plasmalemmal invaginations which are assumed to play various physiological functions. In the present study, distribution of two caveolae-specific proteins, the plasmalemmal Ca(2+)-pump and caveolin, was examined in the corneal epithelium in the normal state and after artificial wounding. METHODS: A central epithelial ablation was made in the mouse cornea by a razor blade. After various intervals, the corneas were excised, fixed, and rapidly frozen. The specimens were subjected to immunofluorescence microscopy and immunoelectron microscopy, using antibodies against the plasmalemmal Ca(2+)-pump or caveolin. RESULTS: In the normal corneal epithelium, both plasmalemmal Ca(2+)-pump and caveolin were observed along the cell surface by immunofluorescence microscopy, and were localized to caveolae by immunogold electron microscopy. In the regenerating epithelium, 12-18 h after injury, plasmalemmal Ca(2+)-pump was seen as many dots in the cytoplasm by immunofluorescence microscopy; in contrast, caveolin persisted along the cell surface. Immunoelectron microscopy revealed that the labeling for the plasmalemmal Ca(2+)-pump was located around membranous structures in the cytoplasm and was scarce along the plasma membrane, while caveolin remained in caveolae. The Ca(2+)-pump regained normal distribution when the wound was closed. By quantitation in electron micrographs, the number of caveolae per unit plasma membrane length was found to be decreased in the wounded corneal epithelium. CONCLUSIONS: The present results indicate that caveolae undergo compositional modification during the wound healing process of the corneal epithelium. Considering putative caveolar functions, the phenomenon may be related to possible fluctuations of the intracellular Ca(2+)-concentration in the regenerating epithelium.     

Probable regulation of factor VIIa-tissue factor and prothrombinase by factor Xa-TFPI and TFPI in vivo

Given that factor VIIa-tissue factor (TF) probably initiates coagulation in vivo, this study investigated the relationship between plasma concentrations of factor VIIa and prothrombin fragment 1 + 2 in plasma (the latter as an index of prothrombinase activity in vivo). The relationships between these two parameters and the concentrations of tissue factor pathway inhibitor (TFPI) and factor Xa-TFPI in plasma were also investigated. TFPI inactivates factor Xa in a reaction accelerated by heparin, whereas factor Xa-TFPI inactivates factor VIIa-TF and prothrombinase. Established enzyme-linked immunosorbent assays (ELISAs) were used to quantify TFPI and prothrombin fragment 1 + 2, whereas we developed an ELISA to quantify factor Xa-TFPI using affinity purified rabbit (anti-human TFPI)-IgG and chicken anti-(human factor Xa-TFPI)-IgY as the capture and detector antibodies, respectively. Plasma factor VIIa was quantified using truncated tissue factor. The concentrations of factor VIIa and prothrombin fragment 1 + 2 increased in parallel in the plasmas of up to 145 healthy adults assayed (P = 0.007), as did the concentrations of factor VIIa and TFPI (P = 0.0039), and prothrombin fragment 1 + 2 and TFPI (P = 0.013). In contrast, there was an inverse relationship between the concentrations of free factor Xa-TFPI and factor VIIa (P < 0.0001) and free factor Xa-TFPI and prothrombin fragment 1 + 2 (P = 0.0095). These results are consistent with factor Xa-TFPI regulating factor VIIa-tissue factor and prothrombinase in vivo.     

Sunlight exposure, hat use, and squamous cell skin cancer on the head and neck

TF (tissue factor) is a physiological inhibitor of blood coagulation in normal hemostasis and is a major initiator of clotting in thrombotic disease. TF functions as a protein cofactor for FVIIa. Coagulation at a site of injury is initiated by exposure of blood to cell-surface formation of TF/VIIa complex. The TF/VIIa complex then activates both factors IX and X leading to thrombin generation and fibrin formation. TFPI (tissue factor pathway inhibitor) appears to play a primary role in regulating TF-induced coagulation. Abnormal coagulation may contribute to the pathogenesis of many serious illnesses. In particular, induced expression of TF and TF-mediated coagulation occurs in atherosclerotic plaques, sepsis, malignancy, ARDS and glomerulonephritis. Several observations support the need for exogenous TFPI administration to effectively turn off the TF/VIIa complex in several clinical conditions with TF-induced coagulopathy. There are some reports about successful administration of rTFPI for antithrombotic therapy in humans.     

Studies of the mechanism for enhanced cell surface factor VIIa/tissue factor activation of factor X on fibroblast monolayers after their exposure to N-ethylmaleimide

Fibroblast monolayers constitutively expressing surface membrane tissue factor (TF) were treated with 0.1 mM N-ethylmaleimide (NEM) for 1 min to inhibit aminophospholipid translocase activity without inducing general cell damage. This resulted in increased anionic phospholipid in the outer leaflet of the cell surface membrane as measured by the binding of 125I-annexin V and by the ability of the monolayers to support the generation of prothrombinase. Specific binding of 125I-rVIIa to TF on NEM-treated monolayers was increased 3- to 4-fold over control monolayers after only brief exposure to 125I-rVIIa, but this difference progressively diminished with longer exposure times. A brief exposure of NEM-treated monolayers to rVIIa led to a maximum 3- to 4-fold enhancement of VIIa/TF catalytic activity towards factor X over control monolayers, but, in contrast to the binding studies, this 3- to 4-fold difference persisted despite increasing time of exposure to rVIIa. Adding prothrombin fragment 1 failed to diminish the enhanced VIIa/TF activation of factor X of NEM-treated monolayers. Moreover, adding annexin V, which was shown to abolish the ability of NEM to enhance factor X binding to the fibroblast monolayers, also failed to diminish the enhanced VIIa/TF activation of factor X. These data provide new evidence for a possible mechanism by which availability of anionic phospholipid in the outer layer of the cell membrane limits formation of functional VIIa/TF complexes on cell surfaces.     

Role of caveolae in cholesterol transport in arterial smooth muscle cells exposed to lipoproteins in vitro and in vivo

Arterial smooth muscle cells are able to shift between two major differentiated states with distinct morphologic and functional properties, a contractile phenotype and a synthetic phenotype. Recently, it was demonstrated that contractile smooth muscle cells have numerous caveolae and that these specialized regions of the plasma membrane, to a large extent, are lost when the cells are modified into a synthetic phenotype. At the same time, the levels of the cholesterol-binding membrane protein caveolin remained unchanged and caveolin was redistributed from the cell surface to the perinuclear cytoplasm. In the present investigation, electron microscopy was used to study how smooth muscle cells of different phenotypes react to exposure to low-density lipoprotein and other lipoproteins both in vitro and in vivo. Our findings indicate that contractile cells (present early in primary culture and in the media of normal arterial walls) do not accumulate lipids in the cytoplasm and release excess cholesterol by means of plasma membrane caveolae. Extracellularly, the expelled lipids were built into membranous configurations and piled up as myelin-like deposits. In synthetic cells (formed after a few days in primary culture and as a response to arterial injury), lipids gathered in cytoplasmic droplets and increased amounts of membranous inclusions appeared in endosomes and lysosomes. On the other hand, no signs of extracellular discharge of lipids were detected. The results suggest that contractile smooth muscle cells use caveolin and caveolae to free themselves of excess lipoprotein-derived cholesterol and so manage to maintain a balance in the influx and efflux of cholesterol. Synthetic smooth muscle cells show a Golgi-like immunostaining for caveolin but have an insufficient capacity to use this protein to transport cholesterol to the plasma membrane and out of the cell. Cholesterol will then rather be esterified and collect in lipid droplets, eventually leading to foam cell formation if the uptake of lipoprotein continues.     

Phosphatidylinositol 4-phosphate synthesis in immunoisolated caveolae-like vesicles and low buoyant density non-caveolar membranes

This study examined phosphatidylinositol 4-phosphate (PtdIns4P) synthesis in caveolae that have been suggested to be discrete signaling microdomains of the plasma membrane and are enriched in the marker protein caveolin. Caveolin-rich light membranes (CLMs) were isolated from A431 cells by detergent-free, discontinuous density-gradient centrifugation method. The CLM fraction was separated from the bulk of the cellular protein and was greatly enriched in PtdIns, PtdIns4P, and phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P2) and an adenosine-sensitive type II PtdIns 4-kinase activity. Preparation of CLMs by an OptiPrep-based cell fractionation procedure confirmed the co-localization of PtdIns 4-kinase and caveolin. Electron microscopy confirmed that an anti-caveolin antiserum immunopurified vesicles from CLMs that were within the size range described for caveolae in other systems. Co-immunoprecipitated PtdIns 4-kinase activity could utilize endogenous PtdIns, present within the caveolae-like vesicles, to produce PtdIns4P. The addition of recombinant phosphatidylinositol transfer protein increased PtdIns 4-kinase activity both in immunoisolated caveolae and CLMs. However, less than 1% of the total cellular PtdIns and PtdIns 4-kinase activity was present in caveolae-like vesicles, indicating that non-caveolar light membrane rafts are the main site for cellular PtdIns4P production.     

Reward magnitude, but not time of day, influences the trial-spacing effect in autoshaping with rats

An important regulator of the initiation of blood coagulation is the plasma glycoprotein, tissue factor pathway inhibitor (TFPI). TFPI inhibits factor Xa and factor VIIa/tissue factor complex, thereby dampens the proteolytic cascade of the tissue factor pathway. Plasma clot lysis is primarily mediated by the fibrinolytic enzyme, plasmin, which is generated through limited proteolysis of plasminogen by endogenous or exogenously administered plasminogen activators. In this study, the interaction of plasmin with recombinant E. coli-derived TFPI (rTFPI) was examined. Plasmin was found to cause a time and concentration dependent proteolysis of rTFPI, resulting in the decrease of anti-factor Xa (measured by chromogenic substrate assay) and anticoagulant (measured by tissue factor-induced clotting assay) activities. Amino-terminal sequencing of the proteolytic fragments revealed that plasmin cleaved rTFPI at K86-T87, R107-G108, R199-A200, K249-G250, and K256-R257. Western blot analysis showed that proteolysis of exogenously added rTFPI also occurred in plasma supplemented with urokinase, and this is accompanied by decrease of anticoagulant activity. These changes were abolished by addition of aprotinin, an inhibitor of plasmin. These data indicate that TFPI is susceptible to proteolysis when plasma fibrinolytic system is activated. The results taken together suggest that plasmin degradation of TFPI may contribute to rethrombosis after thrombolysis, and may contribute to the variability of the efficacy of TFPI in various thrombolysis/reocclusion studies reported previously.     

Selective inhibition of factor Xa is more efficient than factor VIIa-tissue factor complex blockade at facilitating coronary thrombolysis in the canine model

OBJECTIVES: We determined the effect of adjunctive inhibition of the extrinsic coagulation pathway by factor VIIa-tissue factor complex inhibitors, DEGR VIIa and tissue factor pathway inhibitor (TFPI), and the selective factor Xa inhibitor, tick anticoagulant peptide (TAP), after thrombolytic therapy with tissue-type plasminogen activator (t-PA) in a canine model of electrically induced coronary thrombosis. BACKGROUND: Ongoing thrombin generation is considered an important component of the heightened thrombin activity associated with thrombolytic therapy and may be responsible for reperfusion failure and reocclusion. METHODS: Forty-two dogs with electrically induced coronary thrombus undergoing thrombolysis with t-PA (1 mg/kg over 20 min) were randomly assigned to one of the following adjunctive regimens: TAP (30 micrograms/kg body weight per min for 90 min, n = 10); TFPI (100 to 150 micrograms/kg per min for 90 min, n = 10); DEGR VIIa (1- to 2-mg/kg bolus, n = 10) and saline control (n = 12). The dogs were observed for 120 min after thrombolysis for reocclusion. RESULTS: All three active study agents accelerated the time to reperfusion by an average of 12 min (all p < 0.05). Duration of reflow was greatest with TAP (117 +/- 8 min, p < 0.05 compared with saline control), whereas DEGR VIIa and TFPI did not prolong the duration of reflow. Reocclusion rates were similar among control, DEGR VIIa and TFPI groups (70%, 78% and 67%, respectively). Tick anticoagulant peptide reduced the occurrence of reocclusion (0%, p < 0.05 compared with saline control). CONCLUSIONS: In this experimental model, during systematic blockade of various extrinsic coagulation pathway proteins, we demonstrated that whereas acceleration of thrombolysis occurs with factor VIIa-tissue factor complex inhibition, optimal enhancement of thrombolysis was achieved through specific factor Xa blockade.     

Expression of tissue factor pathway inhibitor in vascular smooth muscle cells and its regulation by growth factors

Tissue factor pathway inhibitor (TFPI) in vivo is thought to be synthesized mainly by endothelial cells. To date, no significant regulator of TFPI synthesis has been described. Vascular smooth muscle cells (VSMC) express tissue factor in vitro and in vivo, which may contribute to vascular thrombosis. We hypothesized that VSMC might also express TFPI. To determine this, we examined growth-arrested coronary VSMC in culture and found that VSMC secreted an amount of TFPI similar to that seen in endothelial cells. Immunohistochemistry of normal human coronary arteries showed TFPI staining throughout the media and intima of the vessel with localization to VSMC and endothelial cells. To determine regulation of TFPI expression in VSMC, we examined the effects of serum stimulation on TFPI secretion and found that FBS induced a 5-fold increase in TFPI antigen and activity levels in conditioned medium at 48 hours (P<0.001) when compared with serum-free conditions. A similar stimulatory effect was seen with 10% pooled human serum. Moreover, epidermal growth factor and platelet-derived growth factor-B increased TFPI secretion by 4- to 5-fold and 2- to 3-fold, respectively (P<0.05), and these growth factors accounted for approximately 50% of the TFPI secretion effects of human serum. The serum effect was associated with a 3-fold increase in TFPI mRNA 24 hours after release from growth arrest and a 50% decrease in TFPI secretion after treatment with actinomycin D. Taken together, this study suggests that there is significant TFPI expression in VSMC in culture and in VSMC within the intima and media of the normal coronary artery wall. We present the first evidence for TFPI regulation by serum in VSMC and more specifically by its constituent growth factors, epidermal growth factor and platelet-derived growth factor-B.     

Localization of phospholipase D in detergent-insoluble, caveolin-rich membrane domains. Modulation by caveolin-1 expression and caveolin-182-101

The activation of cellular phospholipase D (PLD) is implicated in vesicular trafficking and signal transduction. Two mammalian PLD forms, designated PLD1 and PLD2, have been cloned, but their cellular localization and function are not fully understood. Here, we report that in HaCaT human keratinocytes, as well as other cell lines, PLD activity is highly enriched in low density, Triton X-100-insoluble membrane domains that contain the caveolar marker protein caveolin-1. Similar to other PLDs, the PLD activity in these membrane domains is stimulated by phosphatidylinositol 4, 5-bisphosphate and is inhibited by neomycin. Immunoblot analysis indicated that caveolin-rich membrane domains do not contain the PLD1 isoform. Stable transfection of mouse PLD2 in Chinese hamster ovary cells greatly increased PLD activity in these domains compared with PLD activity in control Chinese hamster ovary cells transfected with vector alone. PLD activity is enriched in low density Triton-insoluble membrane domains also in U937 promonocytes, even though these cells do not express caveolin-1. In U937 cells, also, PLD1 is largely excluded from low density Triton-insoluble membrane domains. Expression of recombinant caveolin-1 in v-Src-transformed NIH-3T3 cells resulted in up-regulation of PLD activity in the caveolin-containing membrane domains. The caveolin scaffolding peptide (caveolin-182-101) modulated the caveolar PLD activity, causing stimulation at concentration of 1-10 microM and inhibition at concentrations >10 microM. We conclude that a PLD activity, which is likely to represent PLD2, is enriched in low density Triton-insoluble membrane domains. The effects of caveolin-1 expression and of the caveolin scaffolding peptide suggest that in cells that express caveolin-1, PLD may be targeted to caveolae. The possible functions of PLD in the dynamics of caveolae and related domains and in signal transduction processes are discussed.     

Receptor-regulated translocation of endothelial nitric-oxide synthase

The endothelial nitric-oxide synthase (eNOS) is activated by transient increases in intracellular Ca2+ elicited by stimulation of diverse receptors, including bradykinin B2 receptors on endothelial cells. eNOS and B2 receptors are targeted to specialized signal-transducing domains in the plasma membrane termed plasmalemmal caveolae. Targeting to caveolae facilitates eNOS activation following receptor stimulation, but in resting cells, eNOS is tonically inhibited by its interactions with caveolin, the scaffolding protein in caveolae. We used a quantitative approach exploiting immunofluorescence microscopy to investigate regulation of the subcellular distribution of eNOS in endothelial cells by bradykinin and Ca2+. In resting cells, most of the eNOS is localized at the cell membrane. However, within 5 min following addition of bradykinin, nearly all the eNOS translocates to structures in the cell cytosol; following more protracted incubations with bradykinin, most of the cytosolic enzyme subsequently translocates back to the cell membrane. The bradykinin-induced internalization of eNOS is completely abrogated by the intracellular Ca2+ chelator BAPTA; conversely, Ca2+-mobilizing drugs and agonists promote eNOS translocation. These results establish that eNOS targeting to the membrane is labile and is subject to receptor-regulated Ca2+-dependent reversible translocation, providing another point for regulation of NO-dependent signaling in the vascular endothelium.     

Complex-dependent inhibition of factor VIIa by antithrombin III and heparin

The regulation of the factor VIIa-tissue factor complex is essential for control of the hemostatic response. However, the role of the inhibitor antithrombin III in the regulation of factor VIIa has remained in question. The inhibition of factor VIIa activity by antithrombin III and heparin in the presence and absence of tissue factor was evaluated using the fluorescent substrate m-LGR-nds. Our data show that the activity of recombinant human factor VIIa is inhibited by antithrombin III in the presence of heparin at a rate of 1.7 x 10(2) M-1 s-1. In the presence of tissue factor, the rate constant for this reaction increases to 5.6 x 10(3) M-1 s-1. A 1:1 stoichiometric complex between factor VIIa and antithrombin III, with an apparent molecular weight of 110,000, was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A heterogeneous mixture of factor VIIa products with molecular weights between 50,000 and 80,000, most likely representing proteolytically degraded factor VIIa-antithrombin III complexes, was also observed.     

Caveolin interacts with Trk A and p75(NTR) and regulates neurotrophin signaling pathways

Neurotrophins signal through Trk tyrosine kinase receptors and the low-affinity neurotrophin receptor p75(NTR). We have shown previously that activation of Trk A tyrosine kinase activity can inhibit p75(NTR)-dependent sphingomyelin hydrolysis, that caveolae are a localized site for p75(NTR) signaling, and that caveolin can directly interact with p75(NTR). The ability of caveolin to also interact with tyrosine kinase receptors and inhibit their activity led us to hypothesize that caveolin expression may modulate interactions between neurotrophin signaling pathways. PC12 cells were transfected with caveolin that was expressed efficiently and targeted to the appropriate membrane domains. Upon exposure to nerve growth factor (NGF), caveolin-PC12 cells were unable to develop extensive neuritic processes. Caveolin expression in PC12 cells was found to diminish the magnitude and duration of Trk A activation in vivo. This inhibition may be due to a direct interaction of caveolin with Trk A, because Trk A co-immunoprecipitated with caveolin from Cav-Trk A-PC12 cells, and a glutathione S-transferase-caveolin fusion protein bound to Trk A and inhibited NGF-induced autophosphorylation in vitro. Furthermore, the in vivo kinetics of the inhibition of Trk A tyrosine kinase activity by caveolin expression correlated with an increased ability of NGF to induce sphingomyelin hydrolysis through p75(NTR). In summary, our results suggest that the interaction of caveolin with neurotrophin receptors may have functional consequences in regulating signaling through p75(NTR) and Trk A in neuronal and glial cell populations.     

Glycine induces a novel form of long-term potentiation in the superficial layers of the superior colliculus

Diphtheria toxin is believed to enter sensitive mammalian cells via receptor-mediated endocytosis from clathrin-coated pits, while ricin can enter via both clathrin-dependent and clathrin-independent endocytosis. The present study has confirmed this by determining the toxin sensitivity of COS-7y cells which were transiently overexpressing a trans dominant negative mutant of dynamin, a GTPase required for the budding of clathrin-coated vesicles from the plasma membrane. Cells overexpressing wild-type dynamin showed normal receptor-mediated endocytosis of transferrin and remained sensitive to both diphtheria toxin and ricin. Cells overexpressing a mutant dynamin defective in GTP binding and hydrolysis were unable to endocytose transferrin and were protected against diphtheria toxin, but they remained completely sensitive to ricin intoxication. Treating non-transfected cells or cells overexpressing mutant dynamin with nystatin caused a redistribution of the caveolae membrane marker protein VIP21-caveolin from the cell surface to intracellular locations, but did not affect their sensitivity to ricin. The redistribution of caveolin seen after nystatin treatment may reflect the disappearance of caveolae. If this is the case, caveolae are not responsible for the endocytosis of ricin. An alternative clathrin-independent route may operate for ricin, since cellular uptake, intracellular transport, and translocation into the cytosol remain unaffected when clathrin-dependent endocytosis is effectively blocked.     

Human tissue factor pathway inhibitor fused to CD4 binds both FXa and TF/FVIIa at the cell surface

Tissue factor pathway inhibitor (TFPI) is one of the main regulators of the tissue factor (TF) pathway of coagulation. To tether human TFPI to the cell surface, full length or truncated TFPI lacking the third Kunitz domain were fused with domains three and four and the carboxy-terminal sequence of human CD4. Constructs were transfected into a mouse fibroblast cell line and individual clones were checked for expression using monoclonal antibodies directed against the first two TFPI Kunitz domains and against CD4. Specific human FXa binding was detected by flow cytometry using an anti-FX polyclonal antibody, and inhibition of FXa proteolytic activity was verified by chromogenic substrate assay using S-2765. In addition, TFPI-CD4-expressing cells, preincubated with FXa, specifically bound human TF-FVIIa complexes as revealed with an anti-human TF polyclonal antibody. No functional difference was observed between full length or truncated TFPI-CD4. These results demonstrate that functionally intact TFPI can be tethered to the cell surface. Genetic manipulation of, for example, endothelial cells leading to the stable expression of TFPI may inhibit the development of coronary artery heart disease following cardiac allotransplantation, and may inhibit thrombosis in the context of xenotransplantation.     

Localization and turnover of phosphatidylinositol 4,5-bisphosphate in caveolin-enriched membrane domains

Caveolae are small, plasma membrane invaginations that have been implicated in cell signaling. In A431 cells, approximately half of the total cellular phosphatidylinositol 4,5-bisphosphate (PtdIns 4, 5-P2) was found to be localized in low density, Triton-insoluble membrane domains enriched in caveolin. Treatment of cells with either epidermal growth factor or bradykinin for 5 min at 37 degrees C resulted in approximately a 50% decrease in this caveolar PtdIns 4,5-P2 with no change in the levels of plasma membrane PtdIns 4,5-P2. These data suggest that the PtdIns 4,5-P2 present in cells is largely compartmentalized and that the caveolar PtdIns 4,5-P2 is subject to hydrolysis by hormone-stimulated phospholipase C. As growth factor receptors, seven transmembrane domain receptors, heterotrimeric G proteins, and the inositol trisphosphate receptor have all been shown to be enriched in caveolae, these findings suggest that both the generation and response to inositol trisphosphate is highly compartmentalized within the cell.     

Rapid mechanotransduction in situ at the luminal cell surface of vascular endothelium and its caveolae

The vascular endothelium is uniquely positioned between the blood and tissue compartments to receive directly the fluid forces generated by the blood flowing through the vasculature. These forces invoke specific responses within endothelial cells and serve to modulate their intrinsic structure and function. The mechanisms by which hemodynamic forces are detected and converted by endothelia into a sequence of biological and even pathological responses are presently unknown. By purifying and subfractionating the luminal endothelial cell plasma membrane from tissue, we show, for the first time, that not only does mechanotransduction occur at the endothelial cell surface directly exposed to vascular flow in vivo but also increased flow in situ induces rapid tyrosine phosphorylation of luminal endothelial cell surface proteins located primarily in the plasmalemmal invaginations called caveolae. Increased flow induces the translocation of signaling molecules primarily to caveolae, ultimately activating the Ras-Raf-mitogen-activated protein kinase pathway. This signaling appears to require intact caveolae. Filipin-induced disassembly of caveolae inhibits both proximal signaling events at the cell surface and downstream activation of the mitogen-activated protein kinase pathway. With the molecular machinery required for mediating rapid flow-induced responses as seen in endothelium, caveolae may be flow-sensing organelles converting mechanical stimuli into chemical signals transmitted into the cell.