Clustered folate receptors deliver 5-methyltetrahydrofolate to cytoplasm of MA104 cells.

Previously, a high affinity, glycosylphosphatidylinositol-anchored receptor for folate and a caveolae internalization cycle have been found necessary for potocytosis of 5-methyltetrahydrofolate in MA104. We now show by cell fractionation that folate receptors also must be clustered in caveolae for potocytosis. An enriched fraction of caveolae from control cells retained 65-70% of the [3H]folic acid bound to cells in culture. Exposure of cells to the cholesterol-binding drug, filipin, which is known to uncluster receptors, shifted approximately 50% of the bound [3H]folic acid from the caveolae fraction to the noncaveolae membrane fraction and markedly inhibited internalization of [3H]folic acid. An mAb directed against the folate receptor also shifted approximately 50% of the caveolae-associated [3H]folic acid to noncaveolae membrane, indicating the antibody perturbs the normal receptor distribution. Concordantly, the mAb inhibited the delivery of 5-methyl[3H]tetrahydrofolate to the cytoplasm. Receptor bound 5-methyl[3H]tetrahydrofolate moved directly from caveolae to the cytoplasm and was not blocked by phenylarsine oxide, an inhibitor of receptor-mediated endocytosis. These results suggest cell fractionation can be used to study the uptake of molecules by caveolae.

Hormonal regulation of caveolae internalization.

Caveolae undergo a cyclic transition from a flat segment of membrane to a vesicle that then returns to the cell surface. Here we present evidence that this cycle depends on a population of protein kinase C-alpha (PKC-alpha) molecules that reside in the caveolae membrane where they phosphorylate a 90-kD protein. This cycle can be interrupted by treatment of the cells with phorbol-12,13-dibutyrate or agents that raise the concentration of diacylglycerol in the cell. Each of these conditions displaces PKC-alpha from caveolae, inhibits the phosphorylation of the 90-kD protein, and prevents internalization. Caveolae also contain a protein phosphatase that dephosphorylates the 90-kD once PKC-alpha is gone. A similar dissociation of PKC-alpha from caveolae and inhibition of invagination was observed when cells were treated with histamine. This effect was blocked by pyrilamine but not cimetidine, indicating the involvement of histamine H1 receptors. These findings suggest that the caveolae internalization cycle is hormonally regulated.

Caveolin moves from caveolae to the Golgi apparatus in response to cholesterol oxidation.

Caveolae are a membrane specialization used to internalize molecules by potocytosis. Caveolin, an integral membrane protein, is associated with the striated coat present on the cytoplasmic surface of the caveolae membrane. We now report that oxidation of caveolar cholesterol with cholesterol oxidase rapidly displaces the caveolin from the plasma membrane to intracellular vesicles that colocalize with Golgi apparatus markers. After the enzyme is removed from the medium, caveolin returns to caveolae. When untreated cells are gently homogenized, caveolin on the plasma membrane is accessible to both anti-caveolin IgG and trypsin. After cholesterol oxidase treatment, however, Golgi-associated caveolin is inaccessible to both of these molecules. Brefeldin A, which inhibits ER to Golgi trafficking, blocks the appearance of caveolin in the Golgi apparatus but does not prevent caveolin from leaving the plasma membrane. Indirect immunogold localization experiments show that in the presence of cholesterol oxidase caveolin leaves the plasma membrane and becomes associated with endoplasmic reticulum and Golgi compartments. Surprisingly, the loss of caveolin from the plasma membrane does not affect the number or morphology of the caveolae.

Caveolin cycles between plasma membrane caveolae and the Golgi complex by microtubule-dependent and microtubule-independent steps.

Caveolin is a protein associated with the characteristic coats that decorate the cytoplasmic face of plasma membrane caveolae. Recently it was found that exposure of human fibroblasts to cholesterol oxidase (CO) rapidly induces caveolin to redistribute to the ER and then to the Golgi complex, and that subsequent removal of CO allows caveolin to return to the plasma membrane (Smart, E. J., Y.-S. Ying, P. A. Conrad, R. G. W. Anderson, J. Cell Biol. 1994, 127:1185-1197). We now present evidence that caveolin normally undergoes microtubule-dependent cycling between the plasma membrane and the Golgi. In cells that were treated briefly with nocodazole and then with a mixture of nocodazole plus CO, caveolin relocated from the plasma membrane to the ER and then to the ER/Golgi intermediate compartment (ERGIC), but subsequent movement to the Golgi was not observed. Even in the absence of CO, nocodazole caused caveolin to accumulate in the ERGIC. Nocodazole did not retard the movement of caveolin from the Golgi to the plasma membrane after removal of CO. Incubation of cells at 15 degrees followed by elevation of the temperature to 37 degrees caused caveolin to accumulate first in the ERGIC and then in the Golgi, before finally reestablishing its normal steady state distribution predominantly in plasma membrane caveolae. In cells released from a 15 degrees block, movement of caveolin from the Golgi to the plasma membrane was not inhibited by nocodazole. Taken together, these results imply that caveolin cycles constitutively between the plasma membrane and the Golgi by a multi-step process, one of which, ERGIC-to-Golgi transport, requires microtubules. This novel, bidirectional pathway may indicate roles for microtubules in the maintenance of caveolae, and for caveolin in shuttling fatty acids and cholesterol between the plasma membrane and the ER/Golgi system.

Protein kinase C activators inhibit receptor-mediated potocytosis by preventing internalization of caveolae.

Potocytosis is an endocytic pathway that utilizes glycosylphosphatidylinositol-anchored membrane proteins and caveolae to concentrate and internalize small molecules. We now report that activators of protein kinase C are potent inhibitors of potocytosis. Activators such as phorbol-12-myristate-13-acetate (PMA) inhibit the internalization of receptors for 5-methyltetrahydrofolate but allow the internal receptor pool to return to the cell surface. PMA does not affect the clustering of the folate receptor but instead markedly reduces the number of caveolae. Exposure to PMA totally blocks the intracellular accumulation of 5-methyltetrahydrofolate without affecting receptor-independent uptake or the formation of polyglutamylated species of 5-methyltetrahydrofolate in the cytoplasm. These data suggest that PMA inhibits uptake by inactivating caveolae internalization.

Isolation and characterization of a Chlamydomonas reinhardtii mutant lacking the gamma-subunit of chloroplast coupling factor 1 (CF1).

Several nonphotoautotrophic mutants of Chlamydomonas reinhardtii were generated by transforming strain nit1-305 (cw 15) with exogenous DNA. An enrichment for potential photophosphorylation mutants was performed on medium containing arsenate, and acetate-requiring auxotrophs were then identified by replica plating. Strains containing a potential mutation in the nuclear DNA encoding the chloroplast coupling factor 1 (CF1) gamma-subunit (the atpC gene) were first identified serologically with a monospecific antiserum directed against the CF1 gamma-subunit polypeptide. Of several mutants isolated, one, designated T1-54, was characterized at the protein, DNA, and RNA levels. Mutant strain T1-54 lacks anti-CF1 gamma-subunit cross-reacting material, exhibits polymorphism at the atpC locus compared with the parental strain, and lacks the mRNA transcript for the CF1 gamma-subunit. The data are consistent with there being an insertion of exogenous DNA, a deletion of DNA, or both at the 5' end of the gene encoding the CF1 gamma-subunit.

Macrophage scavenger receptors and foam cell formation.

Scavenger receptors bind and internalize modified low-density lipoprotein (LDL) and high-density lipoprotein (HDL). Because the expression of scavenger receptors is not down-regulated by cholesterol, macrophages (Mphi) expressing scavenger receptors can internalize substantial quantities of cholesteryl ester from oxidized LDL and HDL, leading to foam cell formation. Mphi express several different classes of the growing scavenger receptor family on their cell surface and their relative contribution to Mphi cholesterol physiology and atherogenesis is the subject of intense investigation. We focus on the potential role of two scavenger receptors, macrosialin and SR-BI/II in Mphi cholesterol metabolism. Macrosialin is a predominantly Mphi-specific oxidized LDL-binding protein and an atherogenic diet markedly up-regulates its hepatic expression in atherosclerosis-susceptible and atherosclerosis-resistant mouse strains. The HDL receptor, SR-BI and its splicing variant SR-BII, colocalize with caveolin in caveolae in Mphi. Caveolae are initial acceptor sites for cholesteryl esters and these findings indicate a possible role for caveolae and SR-BI in Mphi-selective lipid uptake and in regulating Mphi cholesterol flux in the vascular wall.

Cardiac myocyte adenosine receptors and caveolae.

The purine nucleoside adenosine exerts numerous effects in the mammalian heart, the most well-recognized being regulation of coronary blood flow and cardiac conduction. These effects are mediated via activation of G protein linked adenosine receptor subtypes, A(2a) and A(1) receptors, located primarily on vascular cells and cardiac myocytes, respectively. Although adenosine A(1) receptors are also expressed in ventricular myocytes, adenosine exerts no significant direct effects in these cells. A recent report from our laboratory indicates that ventricular myocyte A(1) receptors are concentrated in caveolin enriched plasma membrane microdomains referred to as caveolae. This review focuses on these recent findings and their relevance to subcellular compartmentalization of A(1) receptor signaling in ventricular myocardium.

Influence of caveolin, cholesterol, and lipoproteins on nitric oxide synthase: implications for vascular disease.

Caveolin-1 traffics cholesterol between the endoplasmic reticulum and cell surface caveolae in a non-vesicle chaperone complex which contains heat shock protein 56, cyclophilin 40, and cyclophilin A. Recent studies demonstrate that endothelial nitric oxide synthase (eNOS), caveolin, hetero-trimeric G-protein coupled receptors, and a calcium channel form an activation complex that is associated with cholesterol-rich caveolae. Oxidized LDL depletes caveolae of cholesterol and prevents agonist stimulation of eNOS by disrupting the activation complex. HDL antagonizes the effects of oxLDL by donating cholesterol to caveolae, thereby preserving the structure and function of caveolae. These findings and others provide a possible mechanistic basis for some of the molecular changes observed in vascular disease.

Class B scavenger receptors, caveolae and cholesterol homeostasis.

Class B scavenger receptors are predominantly localized to cholesterol and sphingomyelin-enriched domains within the plasma membrane, called caveolae. Caveolae and their associated protein, caveolin, have been implicated in cholesterol trafficking and in the regulation of cellular cholesterol homeostasis. Recent studies indicate that scavenger receptor, class B, type I (SR-BI) mediates cholesterol flux between cells and lipoproteins. Caveolae appear to be the sites within the plasma membrane where such exchange occurs, suggesting that the regulation of caveolae and caveolins may be pivotal to the net flux of cholesterol between cells and lipoproteins when they are bound to SR-BI.

The role of caveolae and caveolin in vesicle-dependent and vesicle-independent trafficking.

Caveolae can mediate endocytosis, transcytosis, and potocytosis. Our understanding of these processes as well as the elucidation of the molecular machinery involved has greatly expanded. In addition, caveolin, a 22 kDa protein often associated with caveolae, can promote the trafficking of sterol through the cytoplasm independent of vesicles. Caveolin also influences the formation, morphology, and function of caveolae. The ability of caveolae and caveolin to mediate macromolecular transport directly impacts a variety of physiological and pathophysiological processes.

The prevalence of malnutrition in elderly hip fracture patients.

AIM: To determine the prevalence of protein and energy malnutrition in elderly patients with a fracture of the proximal femur, in New Zealand. METHODS: Consecutive elderly patients (65 years and over) admitted to Christchurch Hospital with a fracture of the proximal femur over a four-month period were recruited. Nutritional indices were measured within three days of admission. These included triceps skinfold thicknesses, mid upper arm circumference, serum albumin and pre-albumin. RESULTS: Forty-two per cent of patients had at least two, and nine per cent had three, indicators of protein and energy malnutrition present on admission. There was no significant difference in the prevalence of malnutrition between young old (<80 years) and old old (80 years and over) patients. Patients residing in an institution had lower mean protein reserves, as indicated by lower corrected arm muscle area (p=0.003) and pre-albumin levels (p=0.09), than those living in the community. A drink, rather than a pudding or biscuit, was the preferred protein and energy supplement form. Ensure Plus (lactose-free) and Fortisip (lactose-free) were the most preferred drink supplements. CONCLUSION: Protein and energy malnutrition is common in elderly New Zealanders who fracture their femur. The prevalence is comparable to overseas data. These patients prefer nutritional supplementation given as a drink.

Complementation of a Chlamydomonas reinhardtii mutant defective in the nuclear gene encoding the chloroplast coupling factor 1 (CF1) gamma-subunit (atpC).

Chlamydomonas reinhardtii strain atpC1 is a mutant defective in the nuclear gene that encodes the CF1 ATP synthase gamma-subunit polypeptide. Photoautotrophic growth was restored to atpC1 after it was transformed with wild-type DNA. Transformed strains were acetate-independent and arsenate-sensitive, similar in phenotype to the progenitor wild-type strain from which atpC1 was generated. Three transformed strains were examined in detail. Southern blot analyses demonstrated that the transformants were complements and not revertants. The transforming DNA integrated into the nuclear genome in a nonhomologous manner and at a low copy number. Northern blot analyses showed that the gamma-subunit mRNA in the complemented strains was expressed at the same relative level as that of wild-type. Western blots of total protein showed that whereas atpC1 was unable to synthesize any CF1 gamma-subunit, all three complemented strains could. Furthermore, the Western blot analyses demonstrated that the mutation in atpC1 had a pleiotropic effect on the accumulation of the CF1 beta-subunit which was relieved upon complementation. Cell extracts from atpC1 did not have any CF1-dependent catalytic activity, whereas extracts from all of the complemented strains and the wild-type strain had identical activities.

Palmitoylation of caveolin-1 is required for cholesterol binding, chaperone complex formation, and rapid transport of cholesterol to caveolae.

We previously demonstrated that a caveolin-chaperone complex transports newly synthesized cholesterol from the endoplasmic reticulum through the cytoplasm to caveolae. Caveolin-1 has a 33-amino acid hydrophobic domain and three sites of palmitoylation in proximity to the hydrophobic domain. In the present study, we hypothesized that palmitoylation of caveolin-1 is necessary for binding of cholesterol, formation of a caveolin-chaperone transport complex, and rapid, direct transport of cholesterol to caveolae. To test this hypothesis, four caveolin-1 constructs were generated that substituted an alanine for a cysteine at position 133, 143, or 156 or all three sites (triple mutant). These mutated caveolins and wild type caveolin-1 were stably expressed in the lymphoid cell line, L1210-JF, which does not express caveolin-1, does not form a caveolin-chaperone complex, and does not transport newly synthesized cholesterol to caveolae. All of the caveolins were expressed and the proteins localized to plasma membrane caveolae. Wild type caveolin-1 and mutant 133 assembled into complete transport complexes and rapidly (10-20 min) transported cholesterol to caveolae. Caveolin mutants 143 and 156 did not assemble into complete transport complexes, weakly associated with cholesterol, and transported small amounts of cholesterol to caveolae. The triple mutant did not assemble into complete transport complexes and did not associate with cholesterol. We conclude that palmitoylation of caveolin-1 at positions 143 and 156 is required for cholesterol binding and transport complex formation.

Characterization of a cytosolic heat-shock protein-caveolin chaperone complex. Involvement in cholesterol trafficking.

Caveolin is a 22-kDa protein that appears to play a critical role in regulating the cholesterol concentration of caveolae. Even though caveolin is thought to be a membrane protein, several reports suggest that this peculiar protein can traffic independently of membrane vesicles. We now present evidence that a cytosolic pool of caveolin is part of a heat-shock protein-immunophilin chaperone complex consisting of caveolin, heat-shock protein 56, cyclophilin 40, cyclophilin A, and cholesterol. Treatment of NIH 3T3 cells with 1 microM cyclosporin A or 100 nM rapamycin disrupted the putative transport complex and prevented rapid (10-20 min) transport of cholesterol to caveolae. The lymphoid cell line, L1210-JF, does not express caveolin, does not form an immunophilin-caveolin complex, and does not transport newly synthesized cholesterol to caveolae. Transfection of caveolin cDNA into L1210-JF cells allowed the assembly of a transport complex identical to that found in NIH 3T3 cells. In addition, newly synthesized cholesterol in transfected cells was rapidly (10-20 min) and specifically transported to caveolae. These data strongly suggest that a caveolin-chaperone complex is a mechanism by which newly synthesized cholesterol is transported from the endoplasmic reticulum through the cytoplasm to caveolae.

17beta-Estradiol promotes the up-regulation of SR-BII in HepG2 cells and in rat livers.

The scavenger receptor class B type I (SR-BI) binds to HDL and mediates the selective uptake of cholesterol esters from HDL to cells. SR-BII is an alternatively spliced product of the SR-BI gene that only differs in the C-terminal cytoplasmic domain. Previous studies with male mice demonstrated that SR-BII comprises about 12% of the total SR-BI/SR-BII present in liver. In the current studies we used a liver cell line, HepG2, and a rat estrogen replacement model to examine the effects of estrogen on the expression of SR-BII. HepG2 cells express SR-BI but not SR-BII. SR-BI/SR-BII - blocking antibodies demonstrated that HepG2 cells selectively internalize cholesterol esters in a SR-BI - dependent manner. Incubation of HepG2 cells with 10 pM of 17beta-estradiol for 12 h eliminated the expression of SR-BI and promoted the up-regulation of SR-BII. Radiolabeled HDL-binding studies demonstrated that 17beta-estradiol increased the number of HDL binding sites by 3-fold in HepG2 cells. However, 17beta-estradiol - treated cell internalized approximately 25% less cholesterol ester than vehicle-only-treated cells. The livers obtained from male rats and ovariectomized female rats contained SR-BI and a small amount of SR-BII. In contrast, the livers obtained from intact female rats and ovariectomized female rats receiving estrogen replacement contained SR-BII and a small amount of SR-BI. The amount of SR-BI and SR-BII in adrenal tissue was not affected by any of the experimental treatments. We conclude that estrogen up-regulates SR-BII in HepG2 cells and rat liver.

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.

High density lipoprotein prevents oxidized low density lipoprotein-induced inhibition of endothelial nitric-oxide synthase localization and activation in caveolae.

Oxidized LDL (oxLDL) depletes caveolae of cholesterol, resulting in the displacement of endothelial nitric-oxide synthase (eNOS) from caveolae and impaired eNOS activation. In the present study, we determined if the class B scavenger receptors, CD36 and SR-BI, are involved in regulating nitric-oxide synthase localization and function. We demonstrate that CD36 and SR-BI are expressed in endothelial cells, co-fractionate with caveolae, and co-immunoprecipitate with caveolin-1. Co-incubation of cells with 10 microgram/ml high density lipoprotein (HDL) prevented oxLDL-induced translocation of eNOS from caveolae and restored acetylcholine-induced nitric-oxide synthase stimulation. Acetylcholine caused eNOS activation in cells incubated with 10 microgram/ml oxLDL (10-15 thiobarbituric acid-reactive substances) and blocking antibodies to CD36, whereas cells treated with only oxLDL were unresponsive. Furthermore, CD36-blocking antibodies prevented oxLDL-induced redistribution of eNOS. SR-BI-blocking antibodies were used to demonstrate that the effects of HDL are mediate by SR-BI. HDL binding to SR-BI maintained the concentration of caveola-associated cholesterol by promoting the uptake of cholesterol esters, thereby preventing oxLDL-induced depletion of caveola cholesterol. We conclude that CD36 mediates the effects of oxLDL on caveola composition and eNOS activation. Furthermore, HDL prevents oxLDL from decreasing the capacity for eNOS activation by preserving the cholesterol concentration in caveolae and, thereby maintaining the subcellular location of eNOS.

Activated cardiac adenosine A(1) receptors translocate out of caveolae.

The cardiac affects of the purine nucleoside, adenosine, are well known. Adenosine increases coronary blood flow, exerts direct negative chronotropic and dromotropic effects, and exerts indirect anti-adrenergic effects. These effects of adenosine are mediated via the activation of specific G protein-coupled receptors. There is increasing evidence that caveolae play a role in the compartmentalization of receptors and second messengers in the vicinity of the plasma membrane. Several reports demonstrate that G protein-coupled receptors redistribute to caveolae in response to receptor occupation. In this study, we tested the hypothesis that adenosine A(1) receptors would translocate to caveolae in the presence of agonists. Surprisingly, in unstimulated rat cardiac ventricular myocytes, 67 +/- 5% of adenosine A(1) receptors were isolated with caveolae. However, incubation with the adenosine A(1) receptor agonist 2-chlorocyclopentyladenosine induced the rapid translocation of the A(1) receptors from caveolae into non-caveolae plasma membrane, an effect that was blocked by the adenosine A(1) receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine. An adenosine A(2a) receptor agonist did not alter the localization of A(1) receptors to caveolae. These data suggest that the translocation of A(1) receptors out of caveolae and away from compartmentalized signaling molecules may explain why activation of ventricular myocyte A(1) receptors are associated with few direct effects.