Loss of caveolin expression in type I pneumocytes as an indicator of subcellular alterations during lung fibrogenesis

 Caveolin is a major structural protein of caveolae, also known as plasmalemmal vesicles, which are particularly abundant in type I pneumocytes and capillary endothelial cells of lung parenchyma. Here we demonstrate that caveolin expression in the alveolar epithelium of rats and mini pigs is strikingly downregulated after irradiation-induced lung injury. Indirect immunoperoxidase staining with polyclonal anti-caveolin antibodies, confirmed by double fluorescence studies with type I cell-specific monoclonal anti-cytokeratin antibodies or lectins, revealed a dramatic loss of caveolin immunoreactivity in type I pneumocytes. In contrast, caveolin expression increased in endothelial cells. Immunoblotting of lung homogenates from normal and irradiated rats using specific anti-caveolin antibodies confirmed the presence of caveolin in normal tissue and its marked decrease of expression in fibrotic tissue. The loss of caveolin as an important structural protein of caveolae in alveolar epithelial cells may be an early indicator of serious type I cell injury during fibrogenesis. The increase of caveolin immunoreactivity in endothelia of blood vessels may indicate that different types of caveolae and/or different regulatory mechanisms of caveolin expression exist. Accepted: 28 May 1997     

Caveolae and calcium handling, a review and a hypothesis

Caveolae are associated with molecules crucial for calcium handling. This review considers the roles of caveolae in calcium handling for smooth muscle and interstitial cells of Cajal (ICC). Structural studies showed that the plasma membrane calcium pump (PMCA), a sodium-calcium exchanger (NCX1), and a myogenic nNOS appear to be colocalized with caveolin 1, the main constituent of these caveolae. Voltage dependent calcium channels (VDCC) are associated but not co-localized with caveolin 1, as are proteins of the peripheral sarcoplasmic reticulum (SR) such as calreticulin. Only the nNOS is absent from caveolin 1 knockout animals. Functional studies in calcium free media suggest that a source of calcium in tonic smooth muscles exists, partly sequestered from extracellular EGTA. This source supported sustained contractions to carbachol using VDCC and dependent on activity of the SERCA pump. This source is postulated to be caveolae, near peripheral SR. New evidence, presented here, suggests that a similar source exists in phasic smooth muscle of the intestine and its ICC. These results suggest that caveolae and peripheral SR are a functional unit recycling calcium through VDCC and controlling its local concentration. Calcium handling molecules associated with caveolae in smooth muscle and ICC were identified and their possible functions also reviewed.     

Changes in membrane cholesterol affect caveolin-1 localization and ICC-pacing in mouse jejunum

Pacing of mouse is dependent on the spontaneous activity of interstitial cells of Cajal in the myenteric plexus (ICC-MP). These ICC, as well as intestinal smooth muscle, contain small membrane invaginations called caveolae. Caveolae are signaling centers formed by insertions of caveolin proteins in the inner aspect of the plasma membrane. Caveolins bind signaling proteins and thereby negatively modulate their signaling. We disrupted caveolae by treating intestinal segments with methyl beta-clodextrin (CD) to remove cholesterol or with water-soluble cholesterol (WSC) to load cholesterol. Both of these treatments reduced pacing frequencies, and these effects were reversed by the other agent. These treatments also inhibited paced contractions, but complete reversal was not observed. To evaluate the specificity of the effects of CD and WSC, additional studies were made of their effects on responses to carbamoyl choline and to stimulation of cholinergic nerves. Neither of these treatments affected these sets of responses compared with their respective time controls. Immunochemical and ultrastructural studies showed that caveolin 1 was present in smooth muscle membranes and ICC-MP. CD depleted both caveolin 1 and caveolae, whereas WSC increased the amount of caveolin 1 immunoreactivity and altered its distribution but failed to increase the number of caveolae. The effects of each agent were reversed in major part by the other. We conclude that signaling through caveolae may play a role in pacing by ICC but does not affect responses to acetylcholine from nerves or when added exogenously.     

Recruitment of endothelial caveolae into mechanotransduction pathways by flow conditioning in vitro

The luminal surface of rat lung microvascular endothelial cells in situ is sensitive to changing hemodynamic parameters. Acute mechanosignaling events initiated in response to flow changes in perfused lung microvessels are localized within specialized invaginated microdomains called caveolae. Here we report that chronic exposure to shear stress alters caveolin expression and distribution, increases caveolae density, and leads to enhanced mechanosensitivity to subsequent changes in hemodynamic forces within cultured endothelial cells. Flow-preconditioned cells expressed a fivefold increase in caveolin (and other caveolar-residing proteins) at the luminal surface compared with no-flow controls. The density of morphologically identifiable caveolae was enhanced sixfold at the luminal cell surface of flow-conditioned cells. Laminar shear stress applied to static endothelial cultures (flow step of 5 dyn/cm2), enhanced the tyrosine phosphorylation of luminal surface proteins by 1.7-fold, including caveolin-1 by 1.3-fold, increased Ser1179 phosphorylation of endothelial nitric oxide synthase (eNOS) by 2.6-fold, and induced a 1.4-fold activation of mitogen-activated protein kinases (ERK1/2) over no-flow controls. The same shear step applied to endothelial cells preconditioned under 10 dyn/cm2 of laminar shear stress for 6 h and induced a sevenfold increase of total phosphotyrosine signal at the luminal endothelial cell surface enhanced caveolin-1 tyrosine phosphorylation 5.8-fold and eNOS phosphorylation by 3.3-fold over static control values. In addition, phosphorylated caveolin-1 and eNOS proteins were preferentially localized to caveolar microdomains. In contrast, ERK1/2 activation was not detected in conditioned cells after acute shear challenge. These data suggest that cultured endothelial cells respond to a sustained flow environment by directing caveolae to the cell surface where they serve to mediate, at least in part, mechanotransduction responses.     

Albumin catabolism in diabetic rats

The kinetics of albumin catabolism were studied in normal rats and rats with streptozotocin induced diabetes (blood glucose greater than 500 mg%). Whether determined from the clearance of 125I-albumin from plasma or from the whole body, after 10 days of severe, uncontrolled diabetes there was a 30-35% decrease in the catabolic rate for albumin in the diabetic rats compared to normals. There was also about a 35% contraction of the relative extravascular distribution volume for albumin in the diabetic rats, and about a 25% decrease in the total body mass of albumin. However, the concentration of albumin in the circulation was the same in normal and diabetic animals. We conclude that when the rate of albumin synthesis is substantially depressed in diabetes, the rat maintains normal plasma albumin concentration both by decreasing albumin's fractional catabolic rate and by shifting albumin from the extravascular to the vascular compartment.     

Caveolin and its cellular and subcellular immunolocalisation in lung alveolar epithelium: implications for alveolar epithelial type I cell function

Caveolae are flask-shaped invaginations of the plasmalemma which pinch off to form discrete vesicles within the cell cytoplasm. Biochemically, caveolae may be distinguished by the presence of a protein, caveolin, that is the principal component of filaments constituting their striated cytoplasmic coat. Squamous alveolar epithelial type I (ATI) cells, comprising approximately 95% of the surface area of lung alveolar epithelium, possess numerous plasmalemmal invaginations and cytoplasmic vesicles ultrastructurally indicative of caveolae. However, an ultrastructural appearance does not universally imply the biochemical presence of caveolin. This immunocytochemical study has utilised a novel application of confocal laser scanning and electron microscopy unequivocally to localise caveolin-1 to ATI cells. Further, cytoplasmic vesicles and flask-shaped membrane invaginations in the ATI cell were morphologically identified whose membranes were decorated with anti-caveolin-1 immunogold label. Coexistent with this, however, in both ATI and capillary endothelial cells could be seen membrane invaginations morphologically characteristic of caveolae, but which lacked associated caveolin immunogold label. This could reflect a true biochemical heterogeneity in populations of morphologically similar plasmalemmal invaginations or an antigen threshold requirement for labelling. The cuboidal alveolar epithelial type II cell (ATII) also displayed specific label for caveolin-1 but with no ultrastructural evidence for the formation of caveolae. The biochemical association of caveolin with ATI cell vesicles has broad implications for the assignment and further study of ATI cell function.     

Vesicle formation and trafficking in endothelial cells and regulation of endothelial barrier function

Endothelial barrier function is regulated in part by the transcellular transport of albumin and other macromolecules via endothelial caveolae (i.e., this process is defined as transcytosis). Using pulmonary microvascular endothelial cells, we have identified the specific interactions between a cell surface albumin-docking protein gp60 and caveolin-1 as well as components of the signaling machinery, heterotrimeric G protein (G(i))- and Src-family tyrosine kinase. Ligation of gp60 on the apical membrane induces the release of caveolae from the apical membrane and activation of endocytosis. The formed vesicles contain the gp60-bound albumin and also albumin and other solutes present in the fluid phase. Vesicles are transported in a polarized manner to the basolateral membrane, releasing their contents by exocytosis into the subendothelial space. The signaling functions of G(i) and Src are important in the release of caveolae from the plasma membrane. The Src-induced phosphorylation of caveolin-1 is crucial in regulating interactions of caveolin-1 with other components of the signaling machinery such as G(i), and key signaling entry of caveolae into the cytoplasm and endocytosis of albumin and other solutes. This review addresses the basis of transcytosis in endothelial cells, its central role as a determinant of endothelial barrier function, and signaling mechanisms involved in regulating fission of caveolae and trafficking of the formed vesicles from the luminal to abluminal side of the endothelial barrier.     

Diabetes reduces aortic endothelial gap junctions in ApoE-deficient mice: simvastatin exacerbates the reduction.

We examined the endothelial gap junctions in diabetic hyperlipidemic mice. Male apolipoprotein E (apoE)-deficient mice were made diabetic by streptozotocin. Three weeks later, the animals were treated with simvastatin for 2 weeks. The expression of aortic gap junctions in the non-diabetic (n=10), untreated diabetic (n=10), and simvastatin-treated diabetic animals (n=6) was analyzed. There was a >4-fold increase in serum cholesterol level and >50% increase in plaque areas in the diabetic mice, regardless of simvastatin treatment. Western blotting of aortae showed reduced expression of connexin37 (Cx37) and Cx40 in the diabetic mice, which were further decreased in the simvastatin-treated diabetic mice. Immunoconfocal microscopy showed that endothelial gap junctions made of Cx37 and Cx40 were both reduced in the untreated diabetic mice compared with the non-diabetic mice (decrease: Cx37, 41%; Cx40, 42%; both p<0.01). The reduction was greater in the simvastatin-treated mice (decrease in treated diabetic vs non-diabetic: Cx37, 61%; Cx40, 79%; both p<0.01; decrease in treated diabetic vs untreated diabetic: Cx37, 34%; Cx40, 63%; both p<0.01). Cx37 and Cx40 were decreased in the endothelium of plaque surface. Cx43 appeared in the medial layer and inner layer of the intima. All three connexins were rarely expressed in monocytes/macrophages inside the plaques. In conclusion, in apoE-deficient mice, streptozotocin-induced diabetes is associated with downregulation of endothelial Cx37 and Cx40 gap junctions. Short-term treatment with simvastatin exacerbates the downregulation.     

Estrogen downregulates the number of caveolae and the level of caveolin in uterine smooth muscle

Estrogen and progesterone, while regulating uterine functions, also regulate the number of caveolae and the level of caveolin. Large numbers of caveolae, as well as elevated expression of caveolin-1 and caveolin-2 isoforms in the myometrium of ovariectomised (OVX) rats were detected. 17beta-estradiol (E2) has a downregulating effect: the treatment of OVX rats with E2 (5 microg/animal) reduced the formation of caveolae by approx. 90%. Western blots clearly demonstrated the reduction of membrane caveolin-1 and -2 content. Progesterone treatment (2.5 mg/animal) alone did not cause any substantial change, but prevented the effect of estrogen. Control experiments showed that the quantity of Na+/K+-ATPase, a plasma membrane protein excluded from caveolae, was not downregulated by E2. The administration of the pure estrogen receptor (ERalpha) antagonist ICI 182,780 (1 mg/animal) not only compensated for the inhibitory effect of E2, but further increased the level of caveolin-1 in the myometrium of OVX rats and facilitated the formation of caveolae by approximately 70%. In contrast, the partial antagonist tamoxifen (1 mg/animal) mimicked the effect of estrogen. The amount of caveolin also changed during pregnancy. During the first half of pregnancy the expression of caveolin was suppressed, but it gradually increased until delivery. Our results indicate that the formation and number of caveolae are influenced by the physiological state of the uterus in a hormone dependent manner.     

Caveolin-1 expression and caveolae biogenesis during cell transdifferentiation in lung alveolar epithelial primary cultures.

Caveolae are omega-shaped invaginations of the plasmalemma possessing a cytoplasmic membrane protein coat of caveolin. Caveolae are present in the in vivo alveolar epithelial type I (ATI) lung cell, but absent in its progenitor, the alveolar epithelial type II (ATII) cell. In primary culture ATII cells grown on a plastic substratum acquire with time an ATI-"like" phenotype. We demonstrate that freshly isolated rat ATII cells lack caveolae and expression of caveolin-1 (a critical caveolae structural protein). As the ATII cells acquire an ATI-like phenotype in primary culture caveolin-1 expression increases, with caveolin-1 signal at 192 h postseeding up to 50-fold greater than at 60 h; caveolae were morphologically evident only after 132 h. When maintaining the differentiated ATII phenotype with time, i.e., culture upon collagen with an apical interface of air, a temporal increase in caveolin-1 expression was not observed, with only very faint signals evident even at 192 h postseeding; at no time did these cultures display caveolae. In late primary ATII cultures caveolin-1 expression and caveolae biogenesis occur as a function of in vitro transformation from the ATII to the ATI-like phenotype. The results have broad implications for the in vitro study of the role of caveolae and caveolin in alveolar epithelial cell biology.     

Evaluation of glycated insulin in diabetic animals using immunocytochemistry and radioimmunoassay

Glycated insulin was evaluated in plasma and biological tissues of diabetic animal models by immunocytochemistry (ICC) and a novel radioimmunoassay. Glycated insulin circulated at 0.10 +/- 0.04 ng/ml and 2.20 +/- 0.14 ng/ml in lean and diabetic obese (ob/ob) mice, corresponding to 12.5 and 9.8% total plasma insulin, respectively. The concentration of glycated insulin was elevated 22-fold in obese mice compared to controls (P < 0.001). In the pancreas, glycated insulin was 48 +/- 10 and 83 +/- 4 ng/g wt (P < 0.05) in lean and obese mice, respectively, representing approximately 2% total insulin in the diabetic pancreas (4.60 +/- 0.17 microg/g wt). ICC revealed fluorescent positively stained cells in pancreatic islets from hydrocortisone (HC)-treated diabetic rats. Fasting of HC-treated rats, resulted in 3-fold and 15-fold reductions in plasma glycated insulin (P < 0.01) and insulin (P < 0.001), respectively. Following a 30 min feeding period in these insulin resistant rats, plasma glucose, insulin, and glycated insulin increased (P < 0.001) rapidly with 1.4-, 1.6-, and 2.9-fold elevations, respectively. Injection of HC-treated rats with insulin (50 U/kg) resulted in a rapid 33% decrease of plasma glucose (P < 0.001) and a marked 4-fold increase in plasma insulin (P < 0.01), whereas glycated insulin concentrations remained unchanged. Since glycation of insulin impairs biological activity, physiologically regulated secretion of glycated insulin into the circulation in diabetic animal models suggests a role in the pathogenesis of diabetes.     

Glomerular function in spontaneously diabetic rats.

This study was undertaken to determine whether hyperfiltration exists at the single nephron level and whether albumin excretion is increased early in the course of diabetes in Biobreeding rats. Diabetic rats were studied at 8-12 weeks after the onset of diabetes. Control animals were age-matched, diabetes-resistant rats. Urinary and tubular fluid albumin concentrations were measured by polyacrylamide gel electrophoresis. Clearance and micropuncture techniques were used to determine whole kidney and single nephron glomerular filtration rate, renal blood flow, and glomerular capillary pressure. The urinary albumin excretion rate (1.3 +/- 0.1 mg/24 hr) and the tubular fluid albumin concentration (4.7 +/- 0.7 mg/dl) in the diabetic group were significantly elevated when compared with urinary albumin excretion (0.9 +/- 0.1 mg/24 hr) and tubular fluid albumin concentration (2.5 +/- 0.5 mg/dl) in the control group. There were no significant differences in glomerular hemodynamics (whole kidney or single nephron glomerular filtration rate or glomerular capillary pressure) between diabetic and control rats. The kidney weight and kidney weight to body weight ratio were significantly higher in diabetic rats when compared with control rats. Early diabetes in Biobreeding rats is characterized by mild albuminuria and increased kidney size, but not glomerular hyperfiltration.     

The biology of caveolae: lessons from caveolin knockout mice and implications for human disease

Caveolae, plasma membrane invaginations that serve as membrane organizing centers, are found in most cell types, but are enriched in adipocytes, endothelial cells, and myocytes. Three members of the caveolin family (Cav-1, -2, and -3) are essential for the formation of caveolae. Specialized motifs in the caveolin proteins function to recruit lipids and proteins to caveolae for participation in intracellular trafficking of cellular components and operation in signal transduction. Mutations in the gene encoding CAV-1 are associated with the development and progression of breast cancers, whereas mutations in the CAV-3 gene result in Rippling Muscle Disease and a form of Limb-Girdle Muscular Dystrophy. The generation of caveolin-null mice has confirmed the essential role of these proteins in caveolae biogenesis and in the pathophysiology of diverse tissues. Caveolin-null mice provide new animal models for studying the pathogenesis of a number of human diseases, including cancer, diabetes, atherosclerosis, restrictive lung disease and pulmonary fibrosis, cardiomyopathy, muscular dystrophy, and bladder dysfunction.     

Interactions of copper with glycated proteins: possible involvement in the etiology of diabetic neuropathy

Humans and animals with diabetes frequently develop peripheral vascular dysfunction and peripheral neuropathies. There is accumulating evidence that impaired peripheral nerve function may derive from diminished endoneural blood flow. The decrements in nerve blood flow may, in turn, be due to diminished endothelium-dependent vasodilation. Although a number of possible causes of this defective vasodilation have been suggested, none has been definitely proven. Regardless of the precise cause, the impaired vasodilatory activity may reflect diminished availability of endothelium-derived relaxing factor (EDRF), variously thought to be nitric oxide or thiol adducts of nitric oxide. Other investigators have reported that administration of transition metal chelators to diabetic rats corrects EDRF-mediated arterial relaxation and restores both neural blood flow and nerve conduction velocity, suggesting the involvement of transition metals. Our investigations center about the hypothesis that glycated proteins bind transition metals such as copper and iron, and that such 'glycochelates' accumulate within the vasculature in diabetes and catalytically inactivate EDRF. In partial support of this hypothesis: (1) Glycated albumin binds approximately 3-fold greater amounts of both copper and iron. (2) Copper bound to glycated albumin remains redox active (e.g. capable of supporting the oxidation of ascorbic acid). (3) Copper and copper-containing glycochelates cause the rapid decomposition of one putative form of EDRF, nitrosocysteine. (4) The amount of exchangeable (i.e. chelatable) copper in the plasma of diabetic rats is approximately twice that in normal rat plasma. (5) Similarly, tail tendons of diabetic animals have about twice as much bound copper as do tendons of normal rats. (6) Implants bearing adsorbed glycated albumin placed in the peritonea of normal mice for 48 h accumulate approximately 5 times as much bound copper as do implants coated with control albumin. Overall, these observations support--but do not conclusively prove - the hypothesis that transition metals such as copper, bound to glycated proteins, may blunt normal EDRF-dependent relaxation of diabetic arteries and provide a rationale for the use of transition metal chelators in the therapy of diabetic vasculopathy and neuropathy.     

Caveolae-deficient endothelial cells show defects in the uptake and transport of albumin in vivo.

The role of endothelial cell caveolae in the uptake and transport of macromolecules from the blood-space to the tissue-space remains controversial. To address this issue directly, we employed caveolin-1 gene knock-out mice that lack caveolin-1 protein expression and caveolae organelles. Here, we show that endothelial cell caveolae are required for the efficient uptake and transport of a known caveolar ligand, i.e. albumin, in vivo. Caveolin-1-null mice were perfused with 5-nm gold-conjugated albumin, and its uptake was followed by transmission electron microscopy. Our results indicate that gold-conjugated albumin is not endocytosed by Cav-1-deficient lung endothelial cells and remains in the blood vessel lumen; in contrast, gold-conjugated albumin was concentrated and internalized by lung endothelial cell caveolae in wild-type mice, as expected. To quantitate this defect in uptake, we next studied the endocytosis of radioiodinated albumin using aortic ring segments from wild-type and Cav-1-null mice. Interestingly, little or no uptake of radioiodinated albumin was observed in the aortic segments from Cav-1-deficient mice, whereas aortic segments from wild-type mice showed robust uptake that was time- and temperature-dependent and competed by unlabeled albumin. We conclude that endothelial cell caveolae are required for the efficient uptake and transport of albumin from the blood to the interstitium.     

Intracellular retention of caveolin 1 in presenilin-deficient cells

Mutations in genes encoding presenilins (PS1 and PS2) are responsible for the majority of early onset familial Alzheimer's disease. PS, a critical component of gamma-secretase, is responsible for the intramembranous cleavage of amyloid precursor protein and Notch. Other physiological functions have been assigned to PS without any clear identification of the mechanisms underlying these multiple biological roles. The early embryonic lethality of PS1 and PS2 double knock-out (PS1/2 null) mice prevents the evaluation of physiological roles of PS. To investigate new functions for presenilins, we performed a proteomic approach by using cells derived from PS1/2 null blastocysts and wild type controls. We identified a presenilin-dependent cell-surface binding of albumin. Binding of albumin depends on intact caveolae on the cellular surface. Abnormal caveolin 1 localization in PS1/2 null cells was associated with a loss of caveolae and an absence of caveolin 1 expression within lipid rafts. Expressing PS1 or PS2 but not the intracellular form of Notch1 in PS1/2 null cells restored normal caveolin 1 localization, demonstrating that presenilins are required for the subcellular trafficking of caveolin 1 independently from Notch activity. Despite an expression of both caveolin 1 and PS1 within lipid raft-enriched fractions after sucrose density centrifugation in wild type cells, no direct interaction between these two proteins was detected, implying that presenilins affect caveolin 1 trafficking in an indirect manner. We conclude that presenilins are required for caveolae formation by controlling transport of intracellular caveolin 1 to the plasma membrane.     

The shape of caveolae is omega-like after glutaraldehyde fixation and cup-like after cryofixation

Caveolae were defined as flask- or omega-shaped plasma membrane invaginations, abundant in adipocytes, fibroblasts, endothelial and smooth muscle cells. The major protein component of caveolar membranes is an integral membrane protein named caveolin. We compared the freeze-fracture behavior of caveolae in glutaraldehyde-fixed and cryofixed mouse fibroblast cells and found distinct differences. In glutaraldehyde-fixed cells almost all caveolae were cross-fractured through their pore and only very few caveolar membranes were membrane-fractured. We found the reverse situation in rapid frozen cells without any chemical fixation where most of the caveolae were membrane-fractured, showing different degrees of invagination from nearly flat to deeply invaginated. In ultrathin sections of glutaraldehyde-fixed heart endothelial cells, caveolae exhibit the well known omega-like shape. In high-pressure frozen, freeze-substituted and low temperature embedded heart endothelial cells, the caveolae frequently exhibit a cup-like shape without any constriction or pore. The cup-like caveolar shape could also be shown by tilt series analysis of freeze-fracture replicas obtained from cryofixed cells. Freeze-fracture immunolabeling of caveolin-1 revealed a lateral belt-like caveolin alignment. These findings point out that the constricted “neck” region of caveolae in most cases is an effect that is caused and intensified by the glutaraldehyde fixation. Our data indicate that caveolae in vivo show all degrees of invagination from nearly flat via cup-like depressed to in a few cases omega-like.     

Caveolin regulation of endothelial function

Caveolae are the sites in the cell membrane responsible for concentrating an array of signaling molecules critical for cell function. Recent studies have begun to identify the functions of caveolin-1, the 22-kDa caveolar protein that oligomerizes and inserts into the cytoplasmic face of the plasma membrane. Caveolin-1 appears to regulate caveolar internalization by stabilizing caveolae at the plasma membrane rather than controlling the shape of the membrane invagination. Because caveolin-1 is a scaffolding protein, it has also been hypothesized to function as a "master regulator" of signaling molecules in caveolae. Deletion of the caveolin-1 gene in mice resulted in cardiac hypertrophy and lung fibrosis, indicating its importance in cardiac and lung development. In the endothelium, caveolin-1 regulates nitric oxide signaling by binding to and inhibiting endothelial nitric oxide synthase (eNOS). Increased cytosolic Ca2+ or activation of the kinase Akt leads to eNOS activation and its dissociation from caveolin-1. Caveolae have also been proposed as the vesicle carriers responsible for transcellular transport (transcytosis) in endothelial cells. Transcytosis, the primary means of albumin transport across continuous endothelia, occurs by fission of caveolae from the membrane. This event is regulated by tyrosine phosphorylation of caveolin-1 and dynamin. As Ca2+ influx channels and pumps are localized in caveolae, caveolin-1 is also an important determinant of Ca2+ signaling in endothelial cells. Many of these findings were presented in San Diego, CA, at the 2003 Experimental Biology symposium "Caveolin Regulation of Endothelial Function" and are reviewed in this summary.     

Tyrosine phosphorylation-dependence of caveolae-mediated endocytosis

Caveolae are flask-shaped plasma membrane invaginations that mediate endocytosis and transcytosis of plasma macromolecules, such as albumin, insulin and low-density lipoprotein (LDL), as well as certain viruses, bacteria and bacterial toxins. Caveolae-mediated transcytosis of macromolecules is critical for maintaining vascular homeostasis by regulating the oncotic pressure gradient and tissue delivery of drugs, vitamins, lipids and ions. Entrapment of cargo within caveolae induces activation of signalling cascades leading to caveolae fission and internalization. Activation of Src tyrosine kinase is an early and essential step that triggers detachment of loaded caveolae from the plasma membrane. In this review, we examine how Src-mediated phosphorylation regulates caveolae-mediated transport by orchestrating the localization and activity of essential proteins of the endocytic machinery to regulate caveolae formation and fission.     

Induction of caveolin during adipogenesis and association of GLUT4 with caveolin-rich vesicles

Caveolae, also termed plasmalemmal vesicles, are small, flask-shaped, non-clathrin-coated invaginations of the plasma membrane. Caveolin is a principal component of the filaments that make up the striated coat of caveolae. Using caveolin as a marker protein for the organelle, we found that adipose tissue is the single most abundant source of caveolae identified thus far. Caveolin mRNA and protein are strongly induced during differentiation of 3T3-L1 fibroblasts to adipocytes; during adipogenesis there is also a dramatic increase in the complexity of the protein composition of caveolin-rich membrane domains. About 10- 15% of the insulin-responsive glucose transporter GLUT4 is found in this caveolin-rich fraction, and immuno-isolated vesicles containing GLUT4 also contain caveolin. However, in non-stimulated adipocytes the majority of caveolin fractionates with the plasma membrane, while most GLUT4 associates with low-density microsomes. Upon addition of insulin to 3T3-L1 adipocytes, there is a significant increase in the amount of GLUT4 associated with caveolin-rich membrane domains, an increase in the amount of caveolin associated with the plasma membrane, and a decrease in the amount of caveolin associated with low-density microsomes. Caveolin does not undergo a change in phosphorylation upon stimulation of 3T3-L1 adipocytes with insulin. However, after treatment with insulin it is associated with a 32-kD phosphorylated protein. Caveolae thus may play an important role in the vesicular transport of GLUT4 to or from the plasma membrane. 3T3-L1 adipocytes offer an attractive system to study the function of caveolae in several cellular trafficking and signaling events.