| For medical practitioners and the general public - Cholesterol Journal Article Catalog. |
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| Medicine: a cholesterol connection in RNAi Rossi, J. J. (2004), Nature 432(7014): 155-6. |
| Medium-chain triacylglycerols may not raise cholesterol Hayes, K. C. (2000), Am J Clin Nutr 72(6): 1583-4. |
| Meet the cholesterol busters Healy, B. (2004), US News World Rep 136(10): 54. |
| Melatonin improved the disturbances in hepatic prooxidant and antioxidant balance and hepatotoxicity induced by a high cholesterol diet in C57BL/6J mice Balkan, J., G. Sener, et al. (2004), Int J Vitam Nutr Res 74(5): 349-54. Abstract: We examined the effect of melatonin in prooxidant and antioxidant state in the liver of C57BL/6J mice fed on a high cholesterol (HC) diet. Mice were fed with normal mice chow containing 1.5% cholesterol and 0.5% cholic acid for 4 months without and with melatonin (10 mg/L in drinking water) treatment. HC diet was observed to increase malondialdehyde (MDA) and diene conjugate (DC) levels in the liver. This diet lowered glutathione (GSH), alpha-tocopherol, and total ascorbic acid levels as well as glutathione peroxidase (GSH-Px) and glutathione transferase (GST) activities in the liver, but hepatic superoxide dismutase (SOD) activity remained unchanged. Although melatonin treatment did not affect these parameters in mice fed a normal diet, it reduced hepatic MDA and DC levels in mice fed an HC diet. Hepatic alpha-tocopherol and ascorbic acid levels increased, but hepatic GSH levels remained unchanged in the melatonin-treated HC group as compared to the HC group. Melatonin treatment was found to increase liver GSH-Px and GST activities in mice fed an HC diet. However, SOD activity did not alter in the liver of hypercholesterolemic mice following melatonin treatment. In addition, the histopathological lesions observed in the cholesterol-plus-melatonin group were less severe than those seen in the cholesterol group. According to these observations, we can say that melatonin treatment has an ameliorating effect on the disturbances in prooxidant and antioxidant balance and histopathological lesions in the liver of mice following cholesterol feeding. |
| Melatonin inhibits LDL receptor activity and cholesterol synthesis in freshly isolated human mononuclear leukocytes Muller-Wieland, D., B. Behnke, et al. (1994), Biochem Biophys Res Commun 203(1): 416-21. Abstract: There is some indirect evidence that the pineal hormone melatonin can suppress plasma levels of cholesterol in hypercholesterolemic rats. We have examined the effects of the hormone on cellular cholesterol metabolism in freshly isolated human mononuclear leukocytes. Incubation of cells for up to 20 h in a lipid-free medium resulted in an increase in the rate of cholesterol synthesis from 14Cacetate and the high affinity accumulation and degradation of 125Ilabeled low density lipoprotein (LDL). Addition of melatonin in increasing concentrations to the incubation medium at zero time inhibited cholesterol synthesis and the specific accumulation and degradation of 125Ilabeled LDL; at a concentration of 100 microM, the inhibitions were 38%, 42%, and 48%, respectively. Similar results were obtained using 14Cmevalonate as precursor. Fatty acid synthesis was not altered under these conditions. In contrast to cholesterol, the synthesis of the first cyclic compound lanosterol was not affected by the pineal hormone. These results implicate that melatonin inhibits this pathway between lanosterol and cholesterol. The action of melatonin on LDL receptor activity appeared to be mediated by a decrease in the number of LDL receptors and not by a change in binding affinity. Pharmacological characterization of the potential melatonin receptor site using several analogs like tryptamine, 5-hydroxytryptamine,N-acetyl-5-hydroxytryptamine, 5-methoxytryptamine, and 6-chloromelatonin indicated that the 5-methoxy group is indispensible for the hormone action on cholesterol synthesis. The data provide evidence that melatonin can modulate cholesterol metabolism in human cells. |
| Melatonin reduces cholesterol accumulation and prooxidant state induced by high cholesterol diet in the plasma, the liver and probably in the aorta of C57BL/6J mice Sener, G., J. Balkan, et al. (2004), J Pineal Res 36(3): 212-6. Abstract: We examined the hypolipidemic and antioxidative effects of melatonin in plasma, liver and aorta of C57BL/6J mice fed on a high cholesterol (HC) diet. Mice were fed normal mice chow containing 1.5% cholesterol and 0.5% cholic acid for 4 months with or without melatonin (10 mg/L in drinking water) treatment. HC diet was observed to increase cholesterol, triglyceride and diene conjugate (DC) levels in plasma and liver. There was a tendency towards an increase in cholesterol level in the aorta following HC diet. In addition, aortic DC levels were higher than those of control group. No fatty streaks or plaques developed in the aorta of mice following HC diet, but in some sections, derangement of the endothelial layer was detected. Melatonin treatment was found to reduce plasma, liver cholesterol and DC levels as well as liver triglyceride levels in hypercholesterolemic mice. Aortic cholesterol and DC levels were also reduced in hypercholesterolemic mice when given melatonin, although not statistically significant. There were no differences in aortic histopathological findings of mice fed on a HC diet with and without melatonin treatment. In conclusion, our results indicate that melatonin reduces HC diet-induced cholesterol accumulation and prooxidant state in the plasma, liver and probably in the aorta. |
| Membrane binding and substrate access merge in cytochrome P450 7A1, a key enzyme in degradation of cholesterol Nakayama, K., A. Puchkaev, et al. (2001), J Biol Chem 276(33): 31459-65. Abstract: To study membrane topology and mechanism for substrate specificity, we truncated residues 2-24 in microsomal cytochrome P450 7A1 (P450 7A1) and introduced conservative and nonconservative substitutions at positions 214-227. Heterologous expression in Escherichia coli was followed by investigation of the subcellular distribution of the mutant P450s and determination of the kinetic and substrate binding parameters for cholesterol. The results indicate that a hydrophobic region, comprising residues 214-227, forms a secondary site of attachment to the membrane in P450 7A1 in addition to the NH(2)-terminal signal-anchor sequence. There are two groups of residues at this enzyme-membrane interface. The first are those whose mutation results in more cytosolic P450 (Val-214, His-225, and Met-226). The second group are those whose mutation leads to more membrane-bound P450 (Phe-215, Leu-218, Ile-224, and Phe-227). In addition, the V214A, V214L, V214T, F215A, F215L, F215Y, L218I, L218V, V219T, and M226A mutants showed a 5-12-fold increased K(m) for cholesterol. The k(cat) of the V214A, V214L, V219T, and M226A mutants was increased up to 1.8-fold, and that of the V214T, F215A, F215L, F215Y, L218I, and L218V mutants was decreased 3-10.5-fold. Based on analysis of these mutations we suggest that cholesterol enters P450 7A1 through the membrane, and Val-214, Phe-215, and Leu-218 are the residues located near the point of cholesterol entry. The results provide an understanding of both the P450 7A1-membrane interactions and the mechanism for substrate specificity. |
| Membrane bound CETP mediates the transfer of free cholesterol between lipoproteins and membranes Ruiz-Noriega, M., I. Silva-Cardenas, et al. (1994), Biochem Biophys Res Commun 202(3): 1322-8. Abstract: Cholesteryl ester transfer protein (CETP) has been shown to transfer cholesteryl esters among plasma lipoproteins. However, when reconstituted into phosphatidylcholine liposomes, the 74,000 protein mediates above non-specific values the transfer of HDL bound 3Hcholesterol into the artificial membrane system. Employing the known cDNA sequence of CETP, we synthesized a series of oligonucleotides with specific sequences for different regions of CETP and RNA isolated from tissues known to be producers of CETP (liver), and tissues not design to synthesize and secrete CETP (ovary, lung, intestine and heart). Hybridization experiments showed that independently of the type of tissue tested CETP sequences were found. It is suggested that a membrane form of CETP might have important repercussions locally. |
| Membrane cholesterol alters gallbladder muscle contractility in prairie dogs Yu, P., Q. Chen, et al. (1996), Am J Physiol 271(1 Pt 1): G56-61. Abstract: The cause and effect relationship between membrane cholesterol and gallbladder muscle contractility was examined by altering membrane cholesterol to phospholipid mole ratio using cholesterol-rich or cholesterol-free liposomes. Gallbladder single muscle cells, from prairie dogs that were fed either a regular or high-cholesterol (1.2%) diet, were isolated enzymatically with collagenase. Plasma membranes of gallbladder muscle were purified in sucrose gradient. Cholesterol was measured using the cholesterol oxidase method. Phospholipids were measured with the method of G.R. Bartlett (J. Biol. Chem. 234: 466-468, 1959). The results of this experiment are 1) after high-cholesterol feeding, cholesterol contents and cholesterol/ phospholipid mole ratio in plasma membranes of gallbladder muscle increased 90%, and muscle cell contraction in response to cholecystokinin octapeptide decreased 58%; 2) similar changes were observed when normal gallbladder muscle cells were incubated with cholesterol-rich liposomes for 2 h; and 3) the changes induced either in vivo or in vitro were reversed when muscle cells were subsequently incubated with cholesterol-free liposomes for 2-6 h. We conclude that gallbladder muscle may incorporate excess cholesterol into its plasma membrane when exposed to a cholesterol-rich environment, that excess membrane cholesterol impairs muscle contractility, and that these changes appear to be reversible. |
| Membrane cholesterol and insulin receptor in erythrocytes Maehara, K. (1991), Fukuoka Igaku Zasshi 82(11): 586-602. Abstract: The author studied influence of membrane cholesterol on insulin receptor behavior using human erythrocytes after modification of their membrane cholesterol contents. Insulin binding to the erythrocytes containing higher membrane cholesterol which were obtained from alcoholic liver cirrhosis (ALC) and familial lecithin: cholesterol acyl transferase (LCAT) deficiency patients was also examined. 1) Influence of modified membrane cholesterol contents on insulin binding to erythrocytes (in vitro experiments): The cholesterol content of human erythrocyte membranes was modified by incubating the cells with phosphatidylcholine/cholesterol and phosphatidylcholine vesicles. Then insulin binding to the erythrocytes was measured as a function of membrane cholesterol content. Low membrane cholesterol decreased insulin binding at 10 degrees C and 15 degrees C, but increased insulin binding at 25 degrees C and 37 degrees C. Scatchard plot analysis showed that low membrane cholesterol decreased a number of the receptor without alteration of affinity. Phosphatidylcholine spin probe showed a dual effect of cholesterol on membrane fluidity, fluidizing below but rigidifying membranes above 20 degrees C. It is suggested that membrane cholesterol affects insulin receptor behavior through alteration of membrane fluidity, depending on the phase state of the membranes. 2) Insulin binding and membrane lipid composition of erythrocytes from ALC and LCAT deficiency patients: The erythrocytes from both ALC and LCAT deficiency patients are known to have higher content of membrane cholesterol. Insulin binding and membrane lipid composition of erythrocytes in both diseases were measured to see the influence of cholesterol on insulin binding in sites. a) Erythrocytes from ALC patients: In the erythrocytes from ALC patients, cholesterol to phospholipid molar ratio was significantly increased as compared with normal subjects. In phospholipid acyl chains, contents of polyunsaturated fatty acid significantly decreased in ALC patients. Insulin binding to erythrocytes was significantly increased in the patients of ALC. Scatchard plot analysis (at 15 degrees C) showed significant increase of the receptor number in erythrocytes from the ALC patients as compared to the normal subjects, while the affinity did not differ between the groups. The increased cholesterol content in the erythrocyte membranes was suggested to be responsible for the increased receptor number and insulin binding to the erythrocytes from ALC patients. b) Erythrocytes from LCAT deficiency patients: Membrane cholesterol to phospholipid molar ratio was increased in two patients of LCAT deficiency studied. In phospholipid classes, increase of phosphatidylcholine and decreases of both phosphatidylethanolamine and sphingomyelin were observed in the erythrocytes from both patients.(ABSTRACT TRUNCATED AT 400 WORDS) |
| Membrane cholesterol and the regulation of signal transduction Fielding, C. J. and P. E. Fielding (2004), Biochem Soc Trans 32(Pt 1): 65-9. Abstract: The plasma membrane of mammalian cells consists of microdomains differing in lipid and protein composition. Two distinct classes of cholesterol/sphingolipid microdomain (caveolae and lipid rafts) are assembly points for transmembrane signalling complexes. Recent evidence suggests that transient changes in cholesterol content may be important in regulating signal transduction. |
| Membrane cholesterol but not putative receptors mediates anandamide-induced hepatocyte apoptosis Biswas, K. K., K. P. Sarker, et al. (2003), Hepatology 38(5): 1167-77. Abstract: The endogenous cannabinoid anandamide, a lipid mediator, induces various physiologic events such as vascular relaxation, inhibition of gap-junctions formation, tumor proliferation, neurologic analgesia, and apoptosis. Although increased concentration of anandamide in plasma has been implicated in pathophysiologic states including endotoxin-induced hypotension, the effects of anandamide on hepatocytes still remain unclear. In this study, we present evidence that plasma anandamide concentration is highly increased in severe hepatitis and cirrhosis patients. In addition, concentrations of anandamide within the pathophysiologic range potently induced apoptosis of hepatoma cell line (Hep G2) and primary hepatocytes, suggesting a possible link between increased anandamide level and hepatocyte damage. Anandamide-induced cell death was preceded by G0/G1 cell-cycle arrest, activation of proapoptotic signaling (i.e., p38 MAPK and JNK), and inhibition of antiapoptotic signaling (i.e., PKB/Akt) pathways. Moreover, anandamide increased susceptibility to oxidative stress-induced hepatocyte damage. In this context, methyl-beta-cyclodextrin (MCD), a membrane cholesterol depletor, or mevastatin, an HMG-CoA reductase inhibitor, or N-acetyl cysteine, an antioxidant, potently inhibited the anandamide-induced proapoptotic events and cell death, whereas putative cannabinoid receptor antagonists did not exhibit an inhibitory effect on anandamide-induced cell death. Furthermore, binding assay using polymyxin beads revealed that anandamide could interact with cholesterol. In conclusion, our data suggest that cholesterol present in the cell membrane determines the fate of hepatocytes exposed to anandamide, possibly functioning as an anandamide receptor. |
| Membrane cholesterol content accounts for developmental differences in surface B cell receptor compartmentalization and signaling Karnell, F. G., R. J. Brezski, et al. (2005), J Biol Chem 280(27): 25621-8. Abstract: Recent studies argue for an important role for cholesterol in maintaining plasma membrane heterogeneity and influencing a variety of cellular processes, including signaling, adhesion, and permeability. Here, we document that tolerance-sensitive transitional immature B cells maintain significantly lower membrane unesterified cholesterol levels than mature-stage splenic B cells. In addition, the relatively low level of cholesterol in transitional immature B cells impairs compartmentalization of their B cell receptor (BCR) into cholesterol-enriched domains following BCR aggregation and reduces their ability to sustain certain aspects of BCR signaling as compared with mature B cells. These studies establish an unexpected difference in the lipid composition of peripheral transitional immature and mature B cells and point to a determining role for development-associated differences in cholesterol content for the differential responses of these B cells to BCR engagement. |
| Membrane cholesterol content modulates activation of BK channels in colonic epithelia Lam, R. S., A. R. Shaw, et al. (2004), Biochim Biophys Acta 1667(2): 241-8. Abstract: Changes in the level of membrane cholesterol regulate a variety of signaling processes including those mediated by acylated signaling molecules that localize to lipid rafts. Recently several types of ion channels have been shown to have cholesterol-dependent activity and to localize to lipid rafts. In this study, we have investigated the role of cholesterol in the regulation of ion transport in colonic epithelial cells. We observed that methyl-beta-cyclodextrin (MbetaCD), a cholesterol-sequestering molecule, activated transepithelial short circuit current (Isc), but only from the basolateral side. Similar results were obtained with a cholesterol-binding agent, filipin, and with the sphingomyelin-degrading enzyme, sphingomyelinase. Experiments with DeltaF508CFTR mutant mice indicated that raft disruption affected CFTR-mediated anion secretion, while pharmacological studies showed that this effect was due to activation of basolateral large conductance Ca2+-activated K+ (BK) channels. Sucrose density gradient centrifugation studies demonstrated that BK channels were normally present in the high-density fraction containing the detergent-insoluble cytoskeleton, and that following treatment with MbetaCD, BK channels redistributed into detergent-soluble fractions. Our evidence therefore implicates novel high-density cholesterol-enriched plasma membrane microdomains in the modulation of BK channel activation and anion secretion in colonic epithelia. |
| Membrane cholesterol content modulates activation of volume-regulated anion current in bovine endothelial cells Levitan, I., A. E. Christian, et al. (2000), J Gen Physiol 115(4): 405-16. Abstract: Activation of volume-regulated anion current (VRAC) plays a key role in the maintenance of cellular volume homeostasis. The mechanisms, however, that regulate VRAC activity are not fully understood. We have examined whether VRAC activation is modulated by the cholesterol content of the membrane bilayer. The cholesterol content of bovine aortic endothelial cells was increased by two independent methods: (a) exposure to a methyl-beta-cyclodextrin saturated with cholesterol, or (b) exposure to cholesterol-enriched lipid dispersions. Enrichment of bovine aortic endothelial cells with cholesterol resulted in a suppression of VRAC activation in response to a mild osmotic gradient, but not to a strong osmotic gradient. Depletion of membrane cholesterol by exposing the cells to methyl-beta-cyclodextrin not complexed with cholesterol resulted in an enhancement of VRAC activation when the cells were challenged with a mild osmotic gradient. VRAC activity in cells challenged with a strong osmotic gradient were unaffected by depletion of membrane cholesterol. These observations show that changes in membrane cholesterol content shift VRAC sensitivity to osmotic gradients. Changes in VRAC activation were not accompanied by changes in anion permeability ratios, indicating that channel selectivity was not affected by the changes in membrane cholesterol. This suggests that membrane cholesterol content affects the equilibrium between the closed and open states of VRAC channel rather than the basic pore properties of the channel. We hypothesize that changes in membrane cholesterol modulate VRAC activity by affecting the membrane deformation energy associated with channel opening. |
| Membrane cholesterol content of cholesterol/phospholipid vesicles determines the susceptibility to both damage and protection by bile salts: implications for bile physiology van de Heijning, B. J., A. M. van den Broek, et al. (1997), Eur J Gastroenterol Hepatol 9(5): 473-9. Abstract: OBJECTIVES: To investigate the effect of membrane lipid composition on the susceptibility to bile salt damage and protection. DESIGN: Artificial model cholesterol/phospholipid (c/p) membranes (vesicles) with a varying cholesterol (0-15 mM) and phospholipid content (3-30 mM), and with a c/p ratio ranging up to 1.70, were prepared by sonication. We examined the effect of incubation with increasing concentrations of either tauroursodeoxycholate (TUDC), taurocholate (TC) or taurodeoxycholate (TDC) alone, or with proportionally varying mixtures of TUDC and TDC. METHOD: Vesicle integrity was assessed by the change in optical absorbance at 340 nm. RESULTS: Absorption of the bile salt-vesicle mixture decreased, with increasing bile salt concentration and hydrophobicity: TUDC less than TC less than TDC. Moreover, bile salt-induced damage also depended on membrane composition: vesicles containing more than 5 mM cholesterol and with a c/p ratio greater than 0.8 were less likely to be solubilized by 30 mM bile salt. Similarly, only in cholesterol-rich vesicles (c/p > 0.5) was a protective effect of TUDC against membrane disruption by TDC revealed upon incubation with various TUDC/TDC mixtures. CONCLUSION: Apart from the bile salt concentration and hydrophobicity, the cholesterol content of vesicles is pivotal, both in the bile salt-induced solubilization of cholesterol/phospholipid vesicles and in the potency of TUDC to prevent this. |
| Membrane cholesterol dynamics: cholesterol domains and kinetic pools Schroeder, F., J. R. Jefferson, et al. (1991), Proc Soc Exp Biol Med 196(3): 235-52. Abstract: Nonreceptor mediated cholesterol uptake and reverse cholesterol transport in cells occur through cellular membranes. Thus, elucidation of cholesterol dynamics in membranes is essential to understanding cellular cholesterol accumulation and loss. To this end, it has become increasingly evident that cholesterol is not randomly distributed in either model or biologic membranes. Instead, membrane cholesterol appears to be organized into structural and kinetic domains or pools. Cholesterol-rich and poor domains can even be observed histochemically and physically isolated from epithelial cell surface membranes. The physiologic importance of these domains is 2-fold: (i) Select membrane proteins (receptors, transporters, etc.) are localized in either cholesterol-rich or cholesterol-poor domains. Consequently, the structure and properties of the domains rather than of the bulk lipid may selectively affect the function of proteins residing therein. (ii) Kinetic evidence suggests that cholesterol transport through and between membranes may occur through specific domains or pools. Regulation of the size and properties of such domains may be controlling factors of cholesterol transport or accumulation in cells. Recent technologic advances in the use of fluorescent sterols have allowed examination of cholesterol domain structure in model and biologic membranes. These techniques have been applied to examine the role of high-density lipoprotein, cholesterol lowering drugs, and intracellular lipid transfer proteins in membrane sterol domain structure and sterol movement between membranes. |
| Membrane cholesterol interferes with neuronal apoptosis induced by soluble oligomers but not fibrils of amyloid-beta peptide Sponne, I., A. Fifre, et al. (2004), Faseb J 18(7): 836-8. Abstract: Neuronal cell death in Alzheimer's disease (AD) is partly induced by the interaction of the amyloid-beta peptide (Abeta) with the plasma membrane of target cells. Accordingly, recent studies have suggested that cholesterol, an important component of membranes that controls their physical properties and functions, plays a critical role in neurodegenerative diseases. We report here that the enrichment of the neuronal plasma membrane with cholesterol protects cortical neurons from apoptosis induced by soluble oligomers of the Abeta(1-40) peptide. Conversely, cholesterol depletion using cyclodextrin renders cells more vulnerable to the cytotoxic effects of the Abeta-soluble oligomers. Increasing the cholesterol content of small unilamellar liposomes also decreases Abeta-dependent liposome fusion. We clearly demonstrate that cholesterol protection is specific to the soluble conformation of Abeta, because we observed no protective effects on cortical neurons treated by amyloid fibrils of the Abeta(1-40) peptide. This may provide a new opportunity for the development of an effective AD therapy as well as elucidate the impact of the cholesterol level during AD development. |
| Membrane cholesterol modulates dihydropyridine receptor function in mice fetal skeletal muscle cells Pouvreau, S., C. Berthier, et al. (2004), J Physiol 555(Pt 2): 365-81. Abstract: Caveolae and transverse (T-) tubules are membrane structures enriched in cholesterol and glycosphingolipids. They play an important role in receptor signalling and myogenesis. The T-system is also highly enriched in dihydropyridine receptors (DHPRs), which control excitation-contraction (E-C) coupling. Recent results have shown that a depletion of membrane cholesterol alters caveolae and T-tubules, yet detailed functional studies of DHPR expression are lacking. Here we studied electrophysiological and morphological effects of methyl-beta-cyclodextrin (MbetaCD), a cholesterol-sequestering drug, on freshly isolated fetal skeletal muscle cells. Exposure of fetal myofibres to 1-3 mM MbetaCD for 1 h at 37 degrees C led to a significant reduction in caveolae and T-tubule areas and to a decrease in cell membrane electrical capacitance. In whole-cell voltage-clamp experiments, the L-type Ca(2+) current amplitude was significantly reduced, and its voltage dependence was shifted approximately 15 mV towards more positive potentials. Activation and inactivation kinetics were slower in treated cells than in control cells and stimulation by a saturating concentration of Bay K 8644 was enhanced. In addition, intramembrane charge movement and Ca(2+) transients evoked by a depolarization were reduced without a shift of the midpoint, indicating a weakening of E-C coupling. In contrast, T-type Ca(2+) current was not affected by MbetaCD treatment. Most of the L-type Ca(2+) conductance reduction and E-C coupling weakening could be explained by a decrease of the number of DHPRs due to the disruption of caveolae and T-tubules. However, the effects on L-type channel gating kinetics suggest that membrane cholesterol content modulates DHPR function. Moreover, the significant shift of the voltage dependence of L-type current without any change in the voltage dependence of charge movement and Ca(2+) transients suggests that cholesterol differentially regulates the two functions of the DHPR. |
| Membrane cholesterol modulates galanin-GalR2 interaction Pang, L., M. Graziano, et al. (1999), Biochemistry 38(37): 12003-11. Abstract: The neuropeptide galanin mediates a number of diverse physiological and pathophysiological actions via interaction with membrane-bound receptors. The role that membrane cholesterol plays in modulating the interaction between galanin and one of the three cloned galanin receptor subtypes (GalR2) expressed in Chinese hamster ovary (CHO) cells was examined. Reduction of membrane cholesterol by treatment with methyl-beta-cyclodextrin (CD) or by culturing cells in lipoprotein-deficient serum markedly decreased galanin binding to the receptor. Addition of cholesterol back to CD-treated, cholesterol-depleted membranes restored galanin binding to control levels. Hill analysis suggests that the GalR2 binds multiple molecules of cholesterol (n >/= 3) in a positively cooperative manner. This interaction appears to be cholesterol-specific as only cholesterol and a limited number of cholesterol analogues were able to rescue galanin binding. The inability of some of these analogues to rescue the binding activity also suggests that binding of galanin to GalR2 is independent of membrane fluidity as, like cholesterol, cholesterol analogues generally rigidize membranes. In addition, treatment of the membranes with other modulators of membrane fluidity, e.g. ethanol, did not affect galanin binding to the GalR2. In contrast, treatment of membranes, with filipin, a molecule that clusters cholesterol within the membranes, or with cholesterol oxidase resulted in markedly reduced galanin binding. Incubation of membranes with 100 microM GTP-gamma-S did not alter the IC(50) for CD in the prebinding assay treatment suggesting that the effect of cholesterol was independent of G protein interaction. Preincubation of intact cells with CD also drastically impaired the ability of galanin to activate intracellular inositol phosphate accumulation in GalR2-transfected CHO cells. These data detail a new mechanism for the regulation of galanin receptor signaling which may link altered functions of GalRs with abnormal cholesterol metabolism. |