[Phospholipid content of plasma membranes and phospholipase C activity in epithelial cells of the large intestine in colitis-associated carcinogenesis in rats].
2
Citation
0
Reference
20
Related Paper
Citation Trend
Abstract:
The decrease of major cytoplasmic membrane phospholipids (phosphatidylcholine and phosphatidylethanolamine) content was established in mucosal epithelial cell under colon inflammation pathology--ulcerative colitis. It was shown that aforementioned changes were associated with the increase of phospholipids' hydrolyzing enzyme--phospholipase C activity and intracellular Ca2+ concentration enlargement. Carcinogenesis stimulation under inflammation was accompanied by phospholipase C activity increase when quantity of investigated phospholipids (phosphatidylcholine, phosphatidylinozytol, phosphatidylserine) separately decreased and cytoplasmic Ca2+ value normalization was established.Keywords:
Phosphatidylethanolamine
Phospholipase A1
Cite
In this paper we compared several lipid characteristics of the homogenate and the corresponding plasma membrane in undifferentiated and differentiated HT29 human colon cancer cells, using normal human colonic cells as a reference. Electron microscopy showed that HT29 cells were morphologically undifferentiated when cultured in the presence of either glucose or inosine without glucose at early confluency. On the contrary, HT29 cells cultured at late confluency in a glucose-free medium containing inosine or grown in nude mice exhibited an enterocytic differentiation with the presence of tight junctions and an apical brush border. The cell homogenate and the plasma membrane were prepared from each cell type. The study of specific marker enzymes showed the same degree of purity in all plasma membranes, with a highly marked increase of brush border-associated hydrolases (N-aminopeptidase and alkaline phosphatase) only in the organelles isolated from differentiated HT29 and colonic cells. Respective similar increases in the amount of free cholesterol and phospholipid and in the free cholesterol:phospholipid molar ratio were found in the plasma membrane as compared with the homogenate in all HT29 cell types. This ratio, due to an increased phospholipid content in both homogenate and plasma membrane, was lowered in colonic cells. No differences in the phospholipid profile were found between the homogenates of all cell types and the plasma membrane of undifferentiated HT29 cells, with the exception of a decrease of cardiolipin in this organelle. On the contrary, the plasma membrane phospholipid composition was different from that of the corresponding homogenate in differentiated HT29 and colonic cells. The most striking changes were a highly increased sphingomyelin amount and concomitant decreases in phosphatidylethanolamine, phosphatidylserine, and cardiolipin. Moreover, differences in the percentage of phosphatidylcholine plus sphingomyelin as well as in phosphatidylcholine:sphingomyelin, phosphatidylethanolamine, and/or phosphatidylcholine molar ratios were also found. The monounsaturated:polyunsaturated fatty acid ratio in phosphatidylethanolamine was similar in differentiated HT29 and colonic cells and lower than in undifferentiated HT29 cells. A decrease in this latter ratio in phosphatidylcholine was also observed in colonic cells and HT29 cells grown in nude mice. These changes were essentially due to opposite variations in the percentage of palmitoleic acid and those of linoleic and/or arachidonic acids in both phospholipids. Thus, these data indicate that undifferentiated HT29 cells were characterized by the absence of a specific phospholipid composition in their plasma membrane, which is suggested to be related to altered phospholipid sorting. The plasma membrane phospholipid profile reversed essentially to the normal pattern when HT29 cells recovered the ability to differentiate.
Brush border
Organelle
Cite
Citations (41)
The incorporation of [14C]oleic and [14C]linoleic acid into phospholipids and neutral lipids was compared in two recently immortalized airway epithelial cell lines. In addition, the effects of adrenergic stimulation on phospholipid turnover was examined. Both cell lines readily incorporated the fatty acids into all phospholipid and neutral lipid fractions. Isoproterenol (1 microM) induced Ca2+ transients in both cell lines, indicating a functional beta-adrenergic response. Epinephrine (10 microM; 15 min) stimulation of cells prelabeled with [14C]linoleic acid increased the percentage of label in phosphatidylcholine in one cell line. Lipid metabolism can now be extensively studied in human airway epithelia.
Cite
Citations (3)
Phosphatidylethanolamine
Cite
Citations (9)
Galactosamine
Cite
Citations (12)
Peroxidation induced by ascorbate on phospholipids of isolated rat liver microsomes were accompanied by losses in glucose-6-phosphatase activity (EC 3.1.3.9.). The existence of marked differences in the degradation rate for each phospholipid suggests a relationship between the alteration of phosphatidylcholine containing one saturated and one unsaturated fatty acid and the decrease in activity of glucose-6-phosphatase; the inactivation of this enzyme was unrelated to the alteration of other phospholipids. These results support the idea that glucose-6-phosphatase and molecules of phosphatidylcholine having one saturated and one unsaturated fatty acid are in close apposition within the microsomal membrane.
Glucose 6-phosphatase
Microsoma
Cite
Citations (1)
Choline kinase
Choline
Phosphotransferases
Cite
Citations (5)
Primary (astronomy)
Cite
Citations (7)
Pancreatic secretion is required for efficient cholesterol absorption by the intestine, but the factors responsible for this effect have not been clearly defined. To identify factors involved and to investigate their role in cholesterol uptake, we studied the effect of Viokase®, a porcine pancreatic extract, on cholesterol uptake into human intestinal Caco-2 cells. Viokase is capable of facilitating cholesterol uptake into these cells such that the level of uptake is 5-fold higher in the presence of solubilized Viokase. This stimulation is time-dependent and is dependent on the presence of bile salt. However, bile salt-stimulated pancreatic cholesterol esterase, which has been proposed to mediate cholesterol uptake, is not fully responsible. The major cholesterol transport activity was purified and identified as pancreatic phospholipase A2. Anti-phospholipase A2 antibodies abolished virtually all of the phospholipase A2 and cholesterol transport activity of solubilized Viokase. We demonstrate that both phospholipase A2 and cholesterol esterase increase cholesterol uptake by hydrolyzing the phosphatidylcholine that is used to prepare the cholesterol-containing micelles. In the absence of cholesterol esterase or phospholipase A2, uptake of cholesterol from micelles containing phosphatidylcholine is not as efficient as uptake from micelles containing phospholipase A2-hydrolytic products. These results indicate that phospholipase A2 may mediate cholesterol absorption by altering the physical-chemical state of cholesterol within the intestine. Pancreatic secretion is required for efficient cholesterol absorption by the intestine, but the factors responsible for this effect have not been clearly defined. To identify factors involved and to investigate their role in cholesterol uptake, we studied the effect of Viokase®, a porcine pancreatic extract, on cholesterol uptake into human intestinal Caco-2 cells. Viokase is capable of facilitating cholesterol uptake into these cells such that the level of uptake is 5-fold higher in the presence of solubilized Viokase. This stimulation is time-dependent and is dependent on the presence of bile salt. However, bile salt-stimulated pancreatic cholesterol esterase, which has been proposed to mediate cholesterol uptake, is not fully responsible. The major cholesterol transport activity was purified and identified as pancreatic phospholipase A2. Anti-phospholipase A2 antibodies abolished virtually all of the phospholipase A2 and cholesterol transport activity of solubilized Viokase. We demonstrate that both phospholipase A2 and cholesterol esterase increase cholesterol uptake by hydrolyzing the phosphatidylcholine that is used to prepare the cholesterol-containing micelles. In the absence of cholesterol esterase or phospholipase A2, uptake of cholesterol from micelles containing phosphatidylcholine is not as efficient as uptake from micelles containing phospholipase A2-hydrolytic products. These results indicate that phospholipase A2 may mediate cholesterol absorption by altering the physical-chemical state of cholesterol within the intestine. The serum cholesterol level is determined mainly by cholesterol synthesis in the liver and clearance of cholesterol-containing lipoproteins and also by the amount of cholesterol absorbed from the intestine. This is demonstrated by the fact that inhibition of cholesterol absorption can decrease serum cholesterol, specifically low density lipoprotein cholesterol (1Gylling H. Miettinen T.A. Atherosclerosis. 1995; 117: 305-308Abstract Full Text PDF PubMed Scopus (70) Google Scholar, 2Schaefer E.J. Levy R.I. Shepherd J. Packard C.J. Miller N.E. Pharmacological Control of Hyperlipidemia. Prous, Barcelona1986: 119-132Google Scholar). The intestinal cholesterol pool has two sources; typically one-third comes from the diet, and the remainder is endogenous cholesterol from bile (3Wilson M.D. Rudel L.L. J. Lipid Res. 1994; 35: 943-955Abstract Full Text PDF PubMed Google Scholar, 4Grundy S.M. Annu. Rev. Nutr. 1983; 3: 71-96Crossref PubMed Scopus (212) Google Scholar). Cholesterol absorption is not complete and varies widely among individuals; in humans, the percent of cholesterol load absorbed in the intestine has been estimated to vary from 15 to 75% (4Grundy S.M. Annu. Rev. Nutr. 1983; 3: 71-96Crossref PubMed Scopus (212) Google Scholar), and individuals respond differently to changes in dietary cholesterol (5Katan M.B. Beynen A.C. Am. J. Epidemiol. 1987; 125: 387-399Crossref PubMed Scopus (128) Google Scholar). This variation suggests that metabolic or genetic factors regulate absorption. Although cholesterol absorption has been widely studied, the multiple factors involved are not fully understood. However, the absolute requirement for bile is established; bile salts are necessary for solubilization of cholesterol from the oil phase into micelles, from which it is available for absorption (6Siperstein M.D. Chaikoff I.L. Reinhardt W.O. J. Biol. Chem. 1952; 198: 111-114Abstract Full Text PDF PubMed Google Scholar, 7Borja C.R. Vahouny G.V. Treadwell C.R. Am. J. Physiol. 1964; 206: 223-228Crossref PubMed Scopus (54) Google Scholar). Pancreatic secretions also appear to be required. Many studies have shown that giving pancreatic enzymes as a dietary supplement increases fat absorption in patients with pancreatic insufficiency, and one report has specifically demonstrated that enzyme supplementation increases cholesterol absorption in these patients (8Vuoristo M. Väänänen H. Miettinen T. Gastroenterology. 1992; 102: 647-655Abstract Full Text PDF PubMed Google Scholar). Pancreatectomized dogs and humans have low plasma cholesterol, which can be increased by feeding raw pancreas or pancreatin, a pancreatic extract preparation (9Chaikoff I.L. Kaplan A. J. Biol. Chem. 1935; 112: 155-165Abstract Full Text PDF Google Scholar,10Bell C.C.J. Swell L. Proc. Soc. Exp. Biol. Med. 1968; 128: 575-577Crossref PubMed Scopus (12) Google Scholar). Of the pancreatic proteins, cholesterol esterase (CEase), 1The abbreviations used are: CEase, cholesterol esterase; PC, phosphatidylcholine; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis. also known as bile salt-stimulated lipase and carboxyl ester lipase, has received most attention as having a potential role in cholesterol absorption. CEase has a wide substrate specificity, hydrolyzing tri-, di-, and monoglycerides and phospholipids in vitro (11Rudd E.A. Brockman H.L. Borgström B. Brockman H.L. Lipases. Elsevier Science Publishers, Amsterdam1984: 185-204Google Scholar). It also hydrolyzes cholesterol esters, which form a small part of dietary cholesterol and cannot be absorbed without prior hydrolysis to free cholesterol (12Vahouny G.V. Treadwell C.R. Proc. Soc. Exp. Biol. Med. 1964; 116: 496-498Crossref PubMed Scopus (26) Google Scholar). Its role in absorption of free cholesterol has been under debate for many years with conflicting evidence regarding its importance in vivo (13Gallo L.L. Clark S.B. Myers S. Vahouny G.V. J. Lipid Res. 1984; 25: 604-612Abstract Full Text PDF PubMed Google Scholar, 14Watt S.M. Simmonds W.J. J. Lipid Res. 1981; 22: 157-165Abstract Full Text PDF PubMed Google Scholar). In vitro, human intestinal Caco-2 cells have been used as a model for cholesterol uptake into the intestinal mucosa. Lopez-Candales et al.(15Lopez-Candales A. Bosner M.S. Spilburg C.A. Lange L.G. Biochemistry. 1993; 32: 12085-12089Crossref PubMed Scopus (75) Google Scholar) reported that CEase stimulated cholesterol uptake from egg phosphatidylcholine (PC) vesicles by Caco-2 cells, whereas Huang and Hui (16Huang Y. Hui D.Y. J. Lipid Res. 1990; 31: 2029-2037Abstract Full Text PDF PubMed Google Scholar) found no stimulation using a similar system. However, the latter study was performed at suboptimal concentrations of bile salt (15Lopez-Candales A. Bosner M.S. Spilburg C.A. Lange L.G. Biochemistry. 1993; 32: 12085-12089Crossref PubMed Scopus (75) Google Scholar). Shamir et al. (17Shamir R. Johnson W.J. Zolfaghari R. Lee H.S. Fisher E.A. Biochemistry. 1995; 34: 6351-6358Crossref PubMed Scopus (57) Google Scholar) also found no indication that CEase increased unesterified cholesterol uptake from egg PC or monoolein vesicles. Disruption of the CEase gene in mice confirmed the role of CEase in hydrolysis of cholesterol ester but found no evidence of a role for CEase in the absorption of free cholesterol (18Howles P.N. Carter C.P. Hui D.Y. J. Biol. Chem. 1996; 271: 7196-7202Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). The demonstrated importance of pancreatic proteins, combined with increasing data against a role for CEase in unesterified cholesterol uptake, motivated this study to investigate the presence in pancreas of proteins other than CEase which facilitate absorption of free cholesterol. A commercially available porcine pancreatic extract, Viokase®, was used to study cholesterol uptake into Caco-2 cells. Viokase has been used to increase lipid absorption in patients with pancreatic insufficiency (19Marotta F. O'Keefe S.J.D. Marks I.N. Girdwood A. Young G. Dig. Dis. Sci. 1989; 34: 456-461Crossref PubMed Scopus (35) Google Scholar). We describe the identification of the major cholesterol transport activity in the extract as pancreatic phospholipase A2 and investigate the mechanism by which this enzyme facilitates cholesterol uptake in this model system. Colonic adenocarcinoma Caco-2 cells were from the American Type Culture Collection. 10–20% polyacrylamide Tricine gels were from NOVEX, Novel Experimental Technology. Fast Stain was purchased from Zoion Biotechnology. Viokase brand of pancrelipase USP was obtained from A. H. Robins (0.7 g contained 16,800 USP units of lipase, 70,000 USP units of protease, and 70,000 USP units of amylase). Thin layer chromatography (TLC) plates used were UniplateTMSilica Gel G from Analtech Inc. Porcine pancreatic phospholipase A2, egg yolk 3-sn-phosphatidylcholine (egg PC), 1-palmitoyl, sn-glycero-3-PC, l- andd-dipalmitoyl PC, bovine serum albumin, Sephadex G-100, S-Sepharose, and heparin-agarose were all from Sigma Chemical Co. Tissue culture supplies were from Life Technologies, Inc. Bovine pancreatic CEase was purified to homogeneity, and anti-bovine CEase antibody was prepared in rabbits as described previously (20Spilburg C.A. Cox D.G. Wang X. Bernat B.A. Bosner M.S. Lange L.G. Biochemistry. 1995; 34: 15532-15538Crossref PubMed Scopus (20) Google Scholar, 21Cox D.G. Leung C.K.T. Kyger E.M. Spilburg C.A. Lange L.G. Biochemistry. 1990; 29: 3842-3848Crossref PubMed Scopus (18) Google Scholar). Cholesterol was purchased from Steraloids Inc. 1-Palmitoyl-2-oleoyl,sn-glycero-3-phosphocholine was obtained from Avanti Polar Lipids. [1,2-3H]Cholesterol (53.8 Ci/mmol) was obtained from American Radiolabeled Chemical, Inc.l-3-Phosphatidylcholine, 1-palmitoyl-2-[1-14C]oleoyl (50–62 mCi/mmol) was obtained from Amersham Corp. The phospholipase assay kit was purchased from Cayman Chemical Company. Viokase (10 g) was solubilized in 100 ml of ice-cold PBS containing 0.2 mmphenylmethylsulfonyl fluoride, 1 μm leupeptin, 1.25 μm pepstatin, and 0.2 mm EDTA and stirred on ice for 30 min. The mixture was centrifuged at 9,500 ×g for 10 min at 4 °C and the supernatant collected for use. The yield was usually 20 mg/ml protein. If not used immediately, aliquots were stored frozen at −80 °C. Because of the high concentrations of protease in Viokase, protease inhibitors were required to maximize the recovery of cholesterol esterase activity during the solubilization step. Caco-2 cells were maintained in minimal essential medium with Earle's salts containing 20% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mml-glutamine at 37 °C with 5.0% CO2. For transport assays, cells were plated into 24-well dishes at a density of 1 × 105 cells/cm2. Cells were used 4–6 days after plating, which was 2–4 days after reaching confluence. Vesicles were prepared by drying 0.25 ml of egg PC (150 mm in hexane), 0.0174 ml of cholesterol (2.6 mm in chloroform), and 30 μCi of [3H]cholesterol under N2. To the dried lipids, 2.5 ml of 0.1 m Tris-HCl, pH 7.2, was added and the mixture sonicated (Branson sonicator, setting 3) for 45 min at 4 °C. The preparation was centrifuged at 9,500 × g for 30 min at 4 °C, and the vesicles were decanted and stored at 4 °C. When other phospholipids were used, the same method was employed, and the final concentration of phospholipid was maintained unless otherwise stated. Preparation of cells for the assay was carried out with the cells on ice. Growth media were discarded and cells washed with 1.0 ml of Earle's balanced salt solution containing 0.02 m Hepes, pH 7.4, and the wash discarded. Each well received 0.25 ml of buffer (final concentrations 1 × minimal essential medium with Earle's salts, 1.0% bovine serum albumin, 2 mmtaurochenodeoxycholate) containing 0.06 μCi of cholesterol/egg PC vesicles (0.3 mm PC) and the indicated amount of enzyme or control buffer (25 mm acetate buffer, pH 5.1). Cells were incubated at 37 °C for the specified time, usually 1.5 h, then placed on ice. Incubation media were removed, cells washed twice with 1.0 ml each of ice-cold PBS containing 1 mg/ml bovine serum albumin, then twice with ice-cold PBS. Cells were solubilized by incubation with 0.2 ml of 1.0 n NaOH at room temperature for 10 min. Aliquots were assayed for protein concentration and for radioactivity by scintillation counting after neutralization with an equal volume of 1.0 n HCl. The effects of anti-phospholipase A2 antibodies on [3H]cholesterol uptake were assessed by preincubating the assay mixtures described above with 33.0 μg/ml solubilized Viokase, 50 μg/ml soybean trypsin inhibitor, 100 μm leupeptin, and the concentrations of antibodies listed in the figures. After 15 min at 4 °C, the [3H]cholesterol vesicles were added, the mixtures placed on the cells, and the uptake of [3H]cholesterol was measured as described above. In some experiments, Caco-2 cells were pretreated with 37 nm phospholipase A2 or 100 nmCEase, or buffer alone in a final volume of 0.25 ml of uptake buffer (final concentrations 1 × minimal essential medium with Earle's salts, 1.0% bovine serum albumin, 2 mmtaurochenodeoxycholate, and 0.3 mm egg PC). After 1.5 h at 37 °C, the cells were washed extensively, and the uptake of radiolabeled cholesterol was determined by the cholesterol transport assay in the presence and absence of 37 nmphospholipase A2 or 100 nm CEase as described above. Release of [14C]oleic acid over a 5- or 10-min period from vesicles containing cholesteryl [1-14C]oleate and egg yolk PC was measured in 100 mm Tris-HCl, pH 7.5, and 8 mmtaurocholate, as described previously (21Cox D.G. Leung C.K.T. Kyger E.M. Spilburg C.A. Lange L.G. Biochemistry. 1990; 29: 3842-3848Crossref PubMed Scopus (18) Google Scholar). Polyacrylamide 10–20% gradient gels were run in SDS running buffer (24 mm Tris, 192 mm glycine, 0.1% SDS). For recovery of protein, two samples of approximately 12 μg each were electrophoresed and half of the gel stained with Fast Stain. From the lane loaded on the unstained half of the gel, 0.5- or 1.0-cm slices were excised and placed in separate tubes. The slices were mashed with a pipette tip, and 60 μl of 25 mmacetate buffer, pH 5.1, was added. The slices were incubated at 4 °C overnight and then centrifuged at 2,400 × g for 5 min. The protein-containing supernatant was used in the cholesterol transport assay. Immunoblotting of phospholipase A2 and Viokase with anti-phospholipase A2 antibodies was carried out as described previously (20Spilburg C.A. Cox D.G. Wang X. Bernat B.A. Bosner M.S. Lange L.G. Biochemistry. 1995; 34: 15532-15538Crossref PubMed Scopus (20) Google Scholar). A sample of 12.5 ml (20 mg/ml) of Viokase solubilized in PBS containing protease inhibitors was applied to a 500-ml Sephadex G-100 column (100 × 2.5 cm) and eluted with approximately 600 ml of PBS at a flow rate of 12 ml/h. Protein eluted in three well separated peaks, and fractions were tested for cholesterol transport activity. All activity was found in the first peak, just after the void volume, which was estimated to contain less than 5% of total protein. Active fractions were pooled, dialyzed in 25 mm acetate buffer, pH 5.1, and approximately 3 mg of total protein was applied to an S-Sepharose column (25-ml bed volume). The column was washed with 25 mm acetate buffer, pH 5.1, then washed with a gradient of buffer containing 0–1,000 mmNaCl (total wash volume 200 ml) at a flow rate of 10 ml/h. Four peaks of protein eluted with approximately 60, 180, 270, and 370 mm NaCl. Fractions were tested for cholesterol transport activity, and the peaks eluting with 180 and 370 mm NaCl were found to have transport activity. The active fractions eluting with 180 or 370 mm NaCl were pooled separately (pool 1 and pool 2) and contained 400 and 250 μg of protein, respectively. Both pools were dialyzed in 25 mm acetate buffer, pH 5.1. Each of the two pools was then applied separately to a 10-ml bed volume heparin-agarose column in 25 mm acetate buffer, pH 5.1, at a flow rate of 15 ml/h and washed with buffer at the same rate. For pool 1, 380 μg of protein was loaded, and the activity did not bind to the heparin column. From the 20-ml flow-through a 10-μl sample was active in the transport assay. Hereafter, this protein is referred to as Hep-P1. With pool 2, 225 μg of protein was applied to the heparin column. The activity bound to the heparin column and was eluted with a step wash of buffer containing 200 mm NaCl. Antibodies to Hep-P1 were raised in rabbits by Josman Laboratories, and the IgG fraction of immune and nonimmune serum was prepared by ammonium sulfate precipitation and chromatography on DEAE Affi-Gel Blue according to standard procedures (22Cooper H.M. Paterson Y. Ausubel F.A. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.L. Struhl K. Current Protocols in Molecular Biology. Green Publishing and Wiley-Interscience, New York1990: 11.13.1-11.13.4Google Scholar). The antibodies reacted with both the major and minor isoforms of porcine phospholipase A2. In some experiments, the active fractions from the Sephadex G-100 column were extracted by shaking for 3 min with 2 volumes of ice-cold diisopropylether:n-butyl alcohol (1.5:1.0, v:v). After phase separation by centrifugation, the solvent phase was recovered and dried under N2 at 40 °C. Residue was dissolved in 20 μl of chloroform and was subjected to TLC as described below. Cholesterol transport assays were performed as described above. After the specified time, incubation medium was removed from each well and placed on ice. Chloroform:methanol (5.0 ml of 2:1, v:v) and 1.0 ml of acid H2O (H2O containing 0.1% H2SO4 and 1% chloroform:methanol, 2:1, v:v) was added and the mixture shaken well. The layers were separated by low speed centrifugation at 4 °C for 10 min. The lower organic layer was recovered and dried under N2 at 40 °C. Residue was dissolved in 20 μl of chloroform and was chromatographed using chloroform:methanol:acetic acid:water (65:35:8:4) until the solvent front was halfway up the plate. The plate was dried and then placed in a second chamber containing hexane:ether:acetic acid (86:16:1) until the solvent front was at the top of the plate. The plate was dried, and the lipids were stained with I2 vapor. Vesicles containing 1-palmitoyl-2-[1-14C]oleoyl PC were prepared by mixing 2.0 μCi of radiolabeled phospholipid with 12.5 mg of unlabeled 1-palmitoyl-2-oleoyl PC and drying the mixture under N2. A volume of 2.5 ml of 150 mm Tris-HCl, pH 7.5, was added and the mixture was sonicated, centrifuged, and the supernatant stored at 4 °C. The specific activity of the vesicles was approximately 4 × 105 dpm/mg phospholipid. Reaction mixtures contained a final volume of 0.25 ml of Hanks' buffered salt solution supplemented with 2.0 mm taurochenodeoxycholate, 1.0% bovine serum albumin, 1.0 mm CaCl2, pH 7.4, 150 μg of 1-palmitoyl-2-[1-14C]oleoyl PC vesicles, and the indicated amount of enzyme diluted in PBS. After incubation for 30 min at 37 °C, each sample was extracted for total lipids, as described above, after the addition of 20 μg each of lyso-PC and oleic acid as carriers. The lipids were separated by TLC using the two solvent system described above, the lipid-containing areas were visualized with I2 vapor, scraped, and radioactivity was determined by scintillation counting. The effects of anti-phospholipase A2 antibodies on 1-palmitoyl-2-[1-14C]oleoyl PC hydrolysis were assessed by preincubating the assay mixtures described above with 12.0 μg/ml solubilized Viokase, 50 μg/ml soybean trypsin inhibitor, 100 μm leupeptin, and the concentrations of antibodies listed in the figures. After 15 min at 4 °C, the radiolabeled phospholipid vesicles were added, and hydrolysis of 1-palmitoyl-2-[1-14C]oleoyl PC was measured as described above. Cell protein was measured using the Bio-Rad protein assay kit with bovine serum albumin as the standard. Phospholipids were measured by the method of Bartlett (23Bartlett G.R. J. Biol. Chem. 1959; 234: 466-468Abstract Full Text PDF PubMed Google Scholar). Amino-terminal protein sequence was obtained at the Protein and Nucleic Acid Facility of Stanford University School of Medicine. Statistical analyses were performed using the computer program InStat 2.01 and an unpaired, two-tailed Mann-Whitney test. In the Caco-2 model system used, with cholesterol presented in mixed micelles of egg PC and bile salt, there is a background level of uptake of cholesterol using buffer alone. Stimulation of cholesterol uptake above this background level is referred to as cholesterol transport activity. The ability of Viokase to stimulate uptake of cholesterol into Caco-2 cells was examined by performing the cholesterol transport assay in the presence of increasing amounts of solubilized Viokase. Uptake was stimulated in a concentration-dependent manner, reaching a plateau at approximately 5-fold stimulation over buffer alone with 6 μg of Viokase protein (Fig. 1 A). The effect was dependent on presence of a narrow range of bile salt concentrations (Fig. 1 B) and was rapid, with stimulation of uptake seen within 15 min; at time points up to 4 h, facilitated transport was maintained at 5-fold over background (Fig. 1 C). Pancreatic CEase has been shown to stimulate cholesterol uptake in this model system (15Lopez-Candales A. Bosner M.S. Spilburg C.A. Lange L.G. Biochemistry. 1993; 32: 12085-12089Crossref PubMed Scopus (75) Google Scholar) and is expected to be present in Viokase. Therefore, a series of studies was performed to evaluate whether the transport activity was the result of CEase. The bile salt-dependent cholesteryl oleate hydrolytic activity of both purified bovine CEase and Viokase was assessed. On a mass basis, purified bovine pancreatic CEase was found to be about 1,000-fold more active than the impure Viokase (Fig. 2 A). To test whether this hydrolytic activity in Viokase corresponded to cholesterol transport activity, equivalent amounts of cholesteryl oleate hydrolytic units of both purified bovine CEase and Viokase were used to measure cholesterol uptake into Caco-2 cells. With increasing amounts of hydrolytic activity, Viokase stimulated cholesterol uptake whereas even at the highest amount of hydrolytic units used, equivalent to 0.2 nm enzyme, CEase showed no stimulation above background (Fig. 2 B). Thus, CEase, measured by cholesteryl oleate hydrolytic activity, does not account for all of the cholesterol transport activity of solubilized Viokase. Further evidence that cholesteryl oleate hydrolysis and cholesterol transport are distinct activities in the pancreatic extract was a difference in sensitivity to boiling. Solubilized Viokase and purified bovine pancreatic CEase were diluted into 25 mm acetate buffer, pH 5.1, and incubated at 100 °C for 10 min. This inactivated the cholesteryl oleate hydrolytic activity of both preparations and also the cholesterol transport activity of CEase, whereas there was little reduction of Viokase-facilitated cholesterol transport (TableI). These combined results suggested that cholesteryl oleate hydrolysis and cholesterol transport activity were exhibited by different proteins.Table IEffect of elevated temperature on cholesterol uptake and cholesteryl oleate hydrolytic activities of Viokase extract and purified CEaseAdditionTemperatureUptake of [3H]cholesterolHydrolysis of cholesteryl [14C]oleate°Cdpm/μg cell proteindpm/10 minNone24.447Viokase496.72,265Viokase10085.842CEase476.41,358CEase10022.449Viokase (160 μg of protein) or CEase (34 μg of protein) was incubated for 10 min at either 4 °C or 100 °C in 25 mm acetate buffer, pH 5.1. Precipitated protein was removed by low speed centrifugation, and aliquots, containing 3 μg of Viokase or 1.7 μg of CEase, were removed for measurement of [3H]cholesterol uptake in Caco-2 cells as described under "Experimental Procedures." Samples of Viokase or CEase were removed for measurement of hydrolysis of cholesteryl [14C]oleate as described under "Experimental Procedures." The data represent the average of duplicate determinations and are representative of two separate experiments. Open table in a new tab Viokase (160 μg of protein) or CEase (34 μg of protein) was incubated for 10 min at either 4 °C or 100 °C in 25 mm acetate buffer, pH 5.1. Precipitated protein was removed by low speed centrifugation, and aliquots, containing 3 μg of Viokase or 1.7 μg of CEase, were removed for measurement of [3H]cholesterol uptake in Caco-2 cells as described under "Experimental Procedures." Samples of Viokase or CEase were removed for measurement of hydrolysis of cholesteryl [14C]oleate as described under "Experimental Procedures." The data represent the average of duplicate determinations and are representative of two separate experiments. Sequential column chromatography was used to purify the cholesterol transport activity from Viokase. The first step was size exclusion chromatography on Sephadex G-100 in which the cholesterol transport activity eluted from the column just after the void volume. The active fractions were pooled and further purified by ion exchange chromatography on S-Sepharose. Two separate peaks of cholesterol transport activity eluted, one with 180 mm NaCl and the second with 370 mm NaCl. Only the peak eluting with 370 mm NaCl exhibited cholesteryl oleate hydrolysis activity. When the first peak was further purified by chromatography on heparin-agarose, cholesterol transport activity did not bind to the column. The active flow-through fractions were pooled and designated Hep-P1. In contrast, cholesterol transport activity from the second S-Sepharose peak bound to heparin-agarose and was eluted with 200 mm NaCl (designated Hep-P2). Hep-P1 and Hep-P2 were analyzed by immunoblotting with bovine CEase antibody, and immunoreactivity was demonstrated in Hep-P2 but not Hep-P1. Thus, the heparin-binding cholesterol transport activity was attributed to CEase (but see later in this section). Bovine CEase has previously been shown to bind to heparin-agarose (21Cox D.G. Leung C.K.T. Kyger E.M. Spilburg C.A. Lange L.G. Biochemistry. 1990; 29: 3842-3848Crossref PubMed Scopus (18) Google Scholar). When the Hep-P1 fraction was concentrated and a sample (12 μg) electrophoresed under denaturing conditions, only one major band, with an apparent molecular mass of 14 kDa, was observed (Fig.3 A). To confirm that this band represented the cholesterol transport activity, slices were excised from an unstained SDS-polyacrylamide gel, and protein recovered from each slice was used to measure cholesterol transport. Only the slice corresponding to the 14-kDa band stimulated uptake of cholesterol into Caco-2 cells (Fig. 3), demonstrating that the 14-kDa band was responsible for the observed cholesterol transport activity. A sample (approximately 100 pmol) of Hep-P1 was subjected to amino-terminal sequencing and was identified as the minor isoform of phospholipase A2. Phospholipase A2 is secreted from the pancreas as a zymogen, which is activated by proteolytic cleavage of the amino-terminal 7 amino acids. Pig pancreas has two isoforms of phospholipase A2 which differ by only 4 residues, of which 2 occur within the amino-terminal 17 residues of the mature protein (24Puijk W.C. Verheij H.M. Wietzes P. de Haas G.H. Biochim. Biophys. Acta. 1979; 580: 411-415Crossref PubMed Scopus (32) Google Scholar). The sequence obtained corresponded exactly to the 17 amino-terminal amino acids of the active, minor isoform, which is about 5% by weight of total pancreatic phospholipase A2(24Puijk W.C. Verheij H.M. Wietzes P. de Haas G.H. Biochim. Biophys. Acta. 1979; 580: 411-415Crossref PubMed Scopus (32) Google Scholar). To confirm the result that phospholipase A2 was responsible for cholesterol transport, SDS-PAGE and Western blotting with anti-porcine pancreatic phospholipase A2 antiserum was performed and revealed an immunoreactive band of 14 kDa from both Hep-P1 and phospholipase A2 purchased from Sigma. Phospholipase A2 (Sigma) was tested for cholesterol transport activity, and both this and Hep-P1 showed a 5-fold stimulation of cholesterol uptake with an EC50 of 6–10 nm. Sigma phospholipase A2 gives multiple bands on SDS-PAGE (data not shown). This material was further purified by hydrophobic chromatography on phenyl-Sepharose eluted with an inverse gradient of (NH4)2SO4, followed by affinity chromatography on heparin-agarose. Cholesterol transport activity was found in two separate peaks; the first was the flow-through, which represented unbound material, and the second was material that bound to the heparin column and eluted wi
Esterase
Cite
Citations (73)
It is established that in membranes of small intestine epithelial cells microvilli in rats, which were administered cholesterol for a long time, the content of this sterol and its esters as well as of phospholipids is different. Differences in the content of lipids are a reason of changes in the sucrose, alkaline phosphatase and leucilamino-peptidase activities. These changes are accompanied by an increase on the molar cholesterol esters-free cholesterol ratio in blood plasma and cholesterol-phospholipids ratio in the microvilli membranes. Beta-sitosterol administered to experimental animals incorporates into the intestine plasma membranes, blocks up the transfer sites, inhibiting cholesterol absorption, which evidently decreases the sterol content in blood serum.
Intestinal mucosa
Cite
Citations (1)
Lysophosphatidylcholine
Lysophosphatidylethanolamine
Phosphatidylethanolamine
Cite
Citations (4)