Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation.

The clathrin-coated pit lattice is held onto the plasma membrane by an integral membrane protein that binds the clathrin AP-2 subunit with high affinity. In vitro studies have suggested that this protein controls the assembly of the pit because membrane bound AP-2 is required for lattice assembly. If so, the AP-2 binding site must be a resident protein of the coated pit and recycle with other receptors that enter cells through this pathway. Proper recycling, however, would require the switching off of AP-2 binding to allow the binding site to travel through the endocytic pathway unencumbered. Evidence for this hypothesis has been revealed by the cationic amphiphilic class of drugs (CAD), which have previously been found to inhibit receptor recycling. Incubation of human fibroblasts in the presence of these drugs caused clathrin lattices to assemble on endosomal membranes and at the same time prevented coated pit assembly at the cell surface. These effects suggest that CADs reverse an on/off switch that controls AP-2 binding to membranes. We conclude that cells have a mechanism for switching on and off AP-2 binding during the endocytic cycle.

Lowering the cholesterol content of MA104 cells inhibits receptor-mediated transport of folate.

The folate receptor is clustered on the surface of MA104 cells in association with caveolae. This relationship is thought to be essential for the proper internalization and recycling of the receptor during the delivery of 5-methyltetrahydrofolate to the cytoplasm of folate-depleted cells. Both the clustered organization of the receptor and the integrity of caveolae are disrupted when cells are deprived of cholesterol. We now show that cholesterol depletion of MA104 cells markedly reduces the rate of 5-methyltetrahydrofolate internalization and causes a 70% decline in the number of receptors present in the internal, recycling compartment. This effect is consistent with morphologic data showing that cholesterol-depleted MA104 cells have a reduced number of caveolae as well as fewer receptors per caveolae.

Cholesterol controls the clustering of the glycophospholipid-anchored membrane receptor for 5-methyltetrahydrofolate.

The folate receptor is a glycosyl-phosphatidylinositol (GPI)-anchored membrane protein that mediates the delivery of 5-methyltetrahydrofolate to the cytoplasm of MA104 cells. Ordinarily the receptor is sequestered into numerous discrete clusters that are associated with an uncoated pit membrane specialization called a caveola. By using two different methodological approaches, we found that the maintenance of both receptor clusters and caveolae depends upon the presence of cholesterol in the membrane. These results suggest that cholesterol plays a critical role in maintaining the caveola membrane domain and modulates the interaction of GPI-anchored membrane proteins via their phospholipid anchors.

The glycophospholipid-linked folate receptor internalizes folate without entering the clathrin-coated pit endocytic pathway.

The folate receptor, also known as the membrane folate-binding protein, is maximally expressed on the surface of folate-depleted tissue culture cells and mediates the high affinity accumulation of 5-methyltetrahydrofolic acid in the cytoplasm of these cells. Recent evidence suggests that this receptor recycles during folate internalization and that it is anchored in the membrane by a glycosyl-phosphatidylinositol linkage. Using quantitative immunocytochemistry, we now show that (a) this receptor is highly clustered on the cell surface; (b) these clusters are preferentially associated with uncoated membrane invaginations rather than clathrin-coated pits; and (c) the receptor is not present in endosomes or lysosomes. This receptor appears to physically move in and out of the cell using a novel uncoated pit pathway that does not merge with the clathrin-coated pit endocytic machinery.

Purification and characterization of smooth muscle cell caveolae.

Plasmalemmal caveolae are a membrane specialization that mediates transcytosis across endothelial cells and the uptake of small molecules and ions by both epithelial and connective tissue cells. Recent findings suggest that caveolae may, in addition, be involved in signal transduction. To better understand the molecular composition of this membrane specialization, we have developed a biochemical method for purifying caveolae from chicken smooth muscle cells. Biochemical and morphological markers indicate that we can obtain approximately 1.5 mg of protein in the caveolae fraction from approximately 100 g of chicken gizzard. Gel electrophoresis shows that there are more than 30 proteins enriched in caveolae relative to the plasma membrane. Among these proteins are: caveolin, a structural molecule of the caveolae coat; multiple, glycosylphosphatidylinositol-anchored membrane proteins; both G alpha and G beta subunits of heterotrimeric GTP-binding protein; and the Ras-related GTP-binding protein, Rap1A/B. The method we have developed will facilitate future studies on the structure and function of caveolae.

Sequestration of GPI-anchored proteins in caveolae triggered by cross-linking.

Glycosyl-phosphatidylinositol (GPI)-anchored proteins have been reported to reside in clusters collected over small membrane invaginations called caveolae. The detection of different GPI-anchored proteins with fluorescently labeled monoclonal antibodies showed that these proteins are not constitutively concentrated in caveolae; they enter these structures independently after cross-linking with polyclonal secondary antibodies. Analysis of the cell surface distribution of the GPI-anchored folate receptor by electron microscopy confirms these observations. Thus, multimerization of GPI-anchored proteins regulates their sequestration in caveolae, but in the absence of agents that promote clustering they are diffusely distributed over the plasma membrane.

Complementary DNA for the folate binding protein correctly predicts anchoring to the membrane by glycosyl-phosphatidylinositol.

Membrane bound and soluble forms of a high-affinity folate binding protein have been found in kidney, placenta, serum, milk, and in several cell lines. The two forms have similar binding characteristics for folates, are immunologically cross-reactive and based upon limited amino acid sequence data, are nearly identical. Based upon pulse-chase experiments, a precursor-product relationship has been suggested. The membrane form has been shown to mediate the transport of folate in cells grown in physiological concentrations of folate. A function for the soluble form has not yet been identified. We constructed a cDNA library from a human carcinoma cell line, Caco-2, which expresses the membrane form abundantly. The library was screened and a near full-length cDNA for the folate binder was isolated. Transfection of COS cells with the cDNA inserted in an expression vector resulted in marked overexpression of a membrane-associated folate binder as assessed by direct binding of radiolabeled folate and by indirect immunofluorescence. The deduced amino acid sequence is not consistent with a typical membrane spanning domain but rather with a signal for anchoring via a glycosyl-phosphatidylinositol linkage. Release of the binder with a phosphatidylinositol-specific phospholipase C strongly supports this hypothesis.

Characterization of cholinergic muscarinic receptors in cow tracheal muscle membranes. Effect of maturation.

The parasympathetic nervous system is important in the control of basal airway muscle tone and caliber. We characterized muscarinic cholinergic receptors in isolated tracheal membranes from cows of three age groups (immature, less than 2 weeks; transition, 3-5 months; and mature, greater than 5 years) using l-[3H]quinuclidinyl benzilate (l-[3H]QNB) as the radioligand. There were significant decreases in the densities of l-[3H]QNB binding sites with maturation (Bmax: 2344 +/- 169 vs 1381 +/- 85 vs 1116 +/- 80 fmol/mg protein for tissues from immature, transition and mature cows respectively). No change in the dissociation constant was observed with maturation (Kd: 0.38 +/- 0.09 vs 0.55 +/- 0.06 vs 0.50 +/- 0.07 nM for tissues from immature, transition and mature animals respectively). The association and dissociation rate constants did not vary between tissues from immature and mature animals. The specific activity of the enzyme, acetylcholinesterase, was correlated with the density of l-[3H]QNB binding sites present in the tracheal homogenates; that is, with maturation, there were significant decreases in acetylcholinesterase activity [0.28 +/- 0.01 vs 0.16 +/- 0.02 vs 0.08 +/- 0.01 mol X l-1 X min-1 X (mg protein)-1 for tissues from immature, transition and mature animals respectively]. All competition binding studies using muscarinic antagonists exhibited single site binding and did not show any differences in drug affinities between the age groups. In contrast, multiple binding sites were observed with carbachol, methacholine and muscarine, and there were significant decreases in receptor affinities for the muscarinic agonists. No changes in the proportion of high and low affinity sites were found. These results indicate that with maturation there are alterations in the properties of muscarinic receptors in tracheal smooth muscle.

Airway responsiveness and prostaglandin generation in scorbutic guinea pigs.

Airway responsiveness to histamine aerosol and lung prostaglandin generation were investigated in normal, partially vitamin C deficient and scorbutic guinea pigs. The ascorbic acid content of the lung expressed as microgram/100 mg wet weight lung parenchyma decreased from 22.1 +/- 1.8 (mean +/- SE) in the control group to 9.0 +/- 1.4 and 1.8 +/- 0.4 in tissues from partially ascorbic acid deficient and scorbutic animals, respectively. Guinea pigs on low and ascorbic acid deficient diets developed significant airway hyperresponsiveness to histamine aerosol after 3 and 4 weeks. Indomethacin (30 mg/Kg, i.p.) further increased the airway hyperresponsiveness in scorbutic animals but was without effect in control animals. Prostaglandin generation from different parts of the lung was significantly changed by the diets. However, airway hyperresponsiveness was not directly attributable to altered prostanoid generation. Scorbutic conditions did not alter the electrophysiological characteristics of airway smooth muscle namely, resting membrane potential and electrogenic sodium pump activity. In summary, ascorbic acid deficiency causes airway hyperresponsiveness to histamine in guinea pigs. This alteration seems not to be related to an altered prostaglandin generation by the lung or to the electrophysiological properties of airway smooth muscle.

Effects of ascorbic acid deficiency on the in vitro biosynthesis of cyclooxygenase metabolites in guinea pig lungs.

Endogenous levels of ascorbic acid may play a role in regulating the biosynthesis of cyclooxygenase metabolites in lungs of male guinea pigs. The in vitro biosyntheses of prostaglandins, prostacyclin and thromboxane were examined using isolated microsomal membranes from control and ascorbic acid deficient guinea pigs, under conditions in which the substrate concentration ( [3H]-arachidonic acid) was varied from 10-100 microM. Maintenance of guinea pigs for two weeks on an ascorbic acid deficient diet did not alter lung/body weight ratios, nor protein content of the lungs. Lung microsomes from ascorbic acid deficient guinea pigs demonstrated a greater biosynthesis of total cyclooxygenase metabolites at low substrate concentrations. A significant increase in the PGF2 alpha synthesis was observed in the scorbutic microsomes at 20 microM arachidonic acid. At higher substrate concentrations the production of PGF2 alpha was significantly reduced in ascorbic acid deficient animals. By contrast, biosynthesis of thromboxane or prostacyclin in treated animals was not significantly different from control microsomes. At a substrate concentration of 100 microM, there was equivalent synthesis of total cyclooxygenase metabolites in control and vitamin C deficient animals. The changes in prostaglandin biosynthesis were not due to an interaction of ascorbic acid with glutathione levels in the lung. These results support the hypothesis that ascorbic acid may modulate cyclooxygenase activity in the lung in a substrate dependent nature.

Membrane properties of bovine airway smooth muscle cells: effects of maturation.

Airway smooth muscle cells of bovine trachealis muscle from immature (2 weeks old), developing (5 months old) and mature animals (greater than 5 years old) have resting membrane potentials of -63.5 +/- 0.4 mV, -62.2 +/- 0.8 mV and -60.3 +/- 0.7 mV, respectively. These resting potentials were not significantly different. The contribution of the electrogenic sodium pump to the resting membrane potential of airway smooth muscle cells decreases significantly with age being 38.3% +/- 0.9, 30.6% +/- 0.8 and 21.3% +/- 0.9 in tissues from immature, developing and mature cows, respectively. The contribution of the electrogenic sodium pump to resting membrane potential was not influenced by the degree of stretch (applied load) of the airway smooth muscle cells. However, resting membrane potential was reduced with increasing tone especially when loads between 5 to 10 g were applied. The densities of Na+/K+ pump sites, as measured by [3H] ouabain binding, decreased significantly as a consequence of maturation and were 4.12 +/- 5.23, 3.08 +/- 0.26 and 1.94 +/- 0.38 pmoles/mg protein ouabain binding in tissues from immature, developing and mature animals respectively. The affinity of the pump sites for the cardiac glycoside, ouabain, did not change during maturation. We conclude that maturation alters both the number of Na+/K+ pump sites and the contribution of the electrogenic sodium pump to the membrane potential of airway smooth muscle cells. These age related changes may contribute to the reduced airway reactivity to both bronchoconstrictor and bronchodilator agents previously observed both in vitro and in vivo.

Ascorbic acid promotes prostanoid release in human lung parenchyma.

Ascorbic acid reduces airway reactivity to inhaled bronchoconstrictor agents in man and guinea pigs. The precise mechanism(s) responsible for this effect are unknown, but in both species an acute indomethacin treatment reverses the action of the ascorbic acid. To determine if ascorbic acid promotes prostanoid synthesis and/or inhibits degradation, human lung parenchymal slices (100-200 mg) were incubated for 60 minutes in oxygenated Tyrode's solution alone or with sodium ascorbate (0.001 M-1 M) and/or methacholine (1 microM-100 microM) and/or indomethacin (0.17 microM-17 microM). Aliquots of the incubation medium were assayed by radioimmunoassay for PGE2, PGF2 alpha, thromboxane B2 and 6-keto-PGF1 alpha. Ascorbic acid increased the accumulation of all four prostanoids in the incubation medium, especially thromboxane B2 and 6-keto-PGF1 alpha. This stimulatory effect of ascorbic acid was concentration-dependent and was inhibited by indomethacin. We conclude that ascorbic acid can alter prostanoid generation by human lung tissue and this effect may, in part, explain its antibronchoconstrictor activity in man.

Folate receptor allows cells to grow in low concentrations of 5-methyltetrahydrofolate.

The folate receptor mediates the uptake of 5-methyltetrahydrofolate in certain cultured cells by a process called potocytosis. When these cells are grown in physiological concentrations of folate, the receptor increases the efficiency of vitamin uptake by 30-fold. We now show that PAM 212 cells, a mouse keratinocyte cell line, are unable to grow in 1 nM 5-methyltetrahydrofolate unless they express a functional folate receptor. These results suggest that under certain conditions, tissue cells in the body may depend on the folate receptor to obtain enough 5-methyltetrahydrofolate for growth.

Caveolin, a protein component of caveolae membrane coats.

Caveolae have been implicated in the transcytosis of macromolecules across endothelial cells and in the receptor-mediated uptake of 5-methyltetrahydrofolate. Structural studies indicate that caveolae are decorated on their cytoplasmic surface by a unique array of filaments or strands that form striated coatings. To understand how these nonclathrin-coated pits function, we performed structural analysis of the striated coat and searched for the molecular component(s) of the coat material. The coat cannot be removed by washing with high salt; however, exposure of membranes to cholesterol-binding drugs caused invaginated caveolae to flatten and the striated coat to disassemble. Antibodies directed against a 22 kd substrate for v-src tyrosine kinase in virus-transformed chick embryo fibroblasts decorated the filaments, suggesting that this molecule is a component of the coat. We have named the molecule caveolin. Caveolae represent a third type of coated membrane specialization that is involved in molecular transport.