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Identification and expression of the human intestinal epithelial apical membrane calcium transporter
A70 AGA ABSTRACTS
GASTROENTEROLOGY Vol. 118, No.4
548
550
IDENTIFICATION AND EXPRESSION OF THE HUMAN INTESTINAL EPITHELIAL APICAL MEMBRANE CALCIUM TRANSPORTER. Natalie F. Barley, Alison Howard, Stephen Legon, Julian Rf Walters, Imperial Coil Sch of Medicine, London, United Kingdom.
THE COLORECTAL MUCOSA OF OLD RATS IS MORE SUSCEPTffiLE TO FOLATE DEPLETION THAN YOUNG RATS: IMPLICATIONS FOR FOLATE-RELATED COLORECTAL CARCINO· GENESIS. Sang-Woon Choi, Pamela 1. Bagley, Annie Gee, Kim Tsui, Joel B. Mason, USDA Human Nutrition Research Ctr at Tufts Univ, Boston, MA. Since inadequate folate status is associated with colorectal carcinogenesis and since age is a major determinant of colorectal cancer risk, we investigated the effect of aging on the colonic folate status. Thirty nine weanling male Sprague-Dawley rats and an equal number of 1 yr old animals were divided into three groups and fed amino-acid defined diets containing Omg folatelkg (deplete), 2mglkg (replete), and 8mglkg (supplemented). Five rats from each group were killed at 8 wks after initiation of diets and 8 rats from each group were killed at 20 wks. The folate concentrations were measured using the L. casei microbial assay. Total plasma homocysteine, S-adenosylmethionine (SAM) concentration, and the distribution of the different co-enzymatic forms of folate in colonic tissue were each measured by established HPLC methods. Old rats fed the folate-deplete diet showed a significantly lower weight compared to folate-replete and supplemented rats begining at 10 wks(p<0.05), while young rats which were folatedeplete did not show any impairment in weight gain. The colonic folate concentrations of old, folate-deplete rats were significantly lower at 20 wks compared to the young rats(p<0.05). Similarly, old folate-replete rats had significantly lower colonic folate concentrations than young replete rats(p<0.05). However, there was no significant difference in colonic folate concentrations between the young and old, folate-supplemented groups. Colonic concentrations of SAM, which are determined in part by tissue folate levels, were marginally lower in the old, deplete animals compared to the young, deplete animals(p=0.051). By 20 wks the folatedeplete old rats showed significant decreases in the proportion of total colonic folate in the form of methyltetrahydrofolate compared to the folate-replete and supplemented groups. However, young deplete rats did not show any significant difference in folate distribution. Although folate depletion produced significantly higher elevations in homocysteine levels in old rats compared to young, supplementation led to normal levels in both groups. Conclusions: The colon of old rats is more susceptible to folate depletion than juvenile animals. However folate supplementation, at four times the basal requirement, is just as effective at eliminating evidence of inadequate folate status in old animals as it is in the young.
Absorption of dietary calcium by the intestine is necessary for bone mineralization and the prevention of osteoporosis. Fractional calcium absorption shows considerable individual variability, and the well-studied association with the active metabolite of vitamin D, 1,25(OHhD3, accounts for only a proportion of this. Calcium absorption by the epithelial cells involves transport through the apical membrane, the cytoplasm (binding to the vitamin D dependent protein calbindin-D9k), and the basolateral membrane (by the calcium pumping ATPase PMCAI). Novel apical membrane calcium influx channels have been described in rabbit kidney and rat duodenum, and named ECaC (for epithelial calcium channel) or CaTI (for calcium transport protein). Using sequence data from those species to construct mixed primers, we amplified by RT-PCR a partial sequence from human duodenal RNA, and used this eDNA probe to study expression on northern blots. In this 446-base region, human CaTl has 97% and 86% amino-acid similarity with the rat and rabbit channels. A transcript approximately 3 kb in size was detectable in duodenum, but not ileum or colon. A similar sized transcript was also found in human placenta and pancreas, where there was also evidence of a larger transcript. A faint transcript of different size could also be detected in brain. No expression of any transcript was detectable in kidney. In duodenal biopsies from 20 previously characterised normal volunteers, CaTl RNA levels were measured and corrected for differences in GAPDH expression. There was considerable (lO-fold) variability in the levels of CaTl signal between subjects. CaTl signal correlated significantly with calbindin-D9k transcripts (r=0.48, p<0.05) but not with 1,25(OHhD, 25-0HD or with age. There was an unexpected strong correlation (r=0.83, p
549 THE EFFECTS OF LONG-TERM FEEDING WITH FISH OIL ON LIPID ABSORPTION IN THE RAT IN VIVO. Lone F. Larsen, Axel K. Hansen, Peter Marckmann, Klaus Bukhave, Tech Univ of Denmark, Copenhagen, Denmark; The Royal Veterinary and Agricul Univ, Copenhagen, Denmark. Background: It is well established that dietary fish oil counteracts the accumulation of postprandial plasma triglyceride (TG). Aims: To explore whether this effect originates in a decreased lipid absorption rate and/or an increased accumulation of lipid within the enterocytes. Methods: Twelve male Wistar rats (weight 197 g (192-201). median (Q50 range» were fed a basic chow (80% w/w) enriched with an oil-mixture (20% w/w), a-tocopherol (0.01% w/w). In the control chow all polyunsaturated fatty acids (PUFA) were n-6, while approximately 20% n-6 PUFA in fish oil chow were replaced by n-3 PUFA. The oil-mixtures were balanced as regards saturated (24%), monounsaturated (44%), and PUFA (32%). After 3 weeks the rats received an intragastric instillation of 0.8 g olive oil and the postprandial plasma TG was determined following 3 hours. One week later, 2 mL RPMJTM containing 61 mg 3H-triolein (33 nCi/mg) and 2 p,ffiol bile salts were instilled into a 15 em intestinal tied-off loop starting 2 em orally to the bile duct. At 15, 30, 45, and 60 min, a 2 em segment of the intestinal loop was removed from the aboral end and the incubation was continued for another 30 min. Enterocytes were isolated by calcium chelation and the total amount of absorped lipid determined by liquid scintillation counting. The distribution of radioactivity into different lipid classes was assessed by TLC and the results were normalized to enterocyte-DNA. Results: Plasma TG following a fat load was significantly (p
sst THE ROLE OF CAVEOLIN IN CLASS B SCAVENGER RECEPTOR-BI MEDIATED CHOLESTEROL UPTAKE IN THE INTESTINE. Willem 1. de Villiers, Gary W. Varilek, Lei Cai, Deneys R. van der Westhuyzen, Univ of Kentucky, Lexington, KY. Scavenger receptor-BI (SR-BI) is of central importance in cholesterol metabolism and homeostasis. It plays a key role in the selective lipid uptake of free and esterified cholesterol from high-density lipoprotein (HDL) to cells as well as in the reverse process; namely efflux of cholesterol from cells to HDL. SR-BI was recently detected in the small intestinal brush border membrane and may be the first identified receptor mediating dietary cholesterol absorption in the intestine. SR-BI colocalizes on the plasma membrane with caveolin-l, an integral protein component of caveolae. Caveolae are plasma membrane micro-domains receiving increasing attention as important in cellular cholesterol trafficking. In the present study we tested the hypothesis that caveolin-l expression promotes selective cholesterol ester uptake. We characterized the role of SR-BI and caveolin-l in selective lipid uptake and cholesterol efflux in two human colon cancer epithelial cell lines. Both CaCo-2 and HT-29 cells express SR-BI protein endogenously, but only HT-29 cells express caveolin-I protein. We overexpressed SR-BI and caveolin-I expression in these two cell lines by adenoviral-mediated gene transfer. The presence of caveolin-l did not alter the expression level of SR-BI. Expression of caveolin-I did not affect the amount of J25I_HDL associated with the cells, but decreased selective 3H-cholesterol ether uptake by the epithelial cells by 50%. Dosedependent adenoviral-mediated caveolin overexpression further reduced 3H-cholesterol ether uptake by the cell lines. The efflux of 3H-cholesterol did not change in each cell type with respect to caveolin-l expression. We conclude that caveolin-l negatively regulates SR-BI dependent selective cholesterol ester uptake without affecting free cholesterol efflux in colonic epithelial cells.
552 IDENTIFICATION OF A NOVEL NA-DEPENDENT TRANSPORT MECHANISM, HIGHLY SPECIFIC FOR GLUTAMINE, IN ISOLATED INTESTINAL VILLUS AND CRYPT CELLS. Jesus R. del Castillo, Maria C. Sulbaran, Luis O. Burguillos, Ivic, Caracas, Venezuela. Glutamine is the most abundant amino acid in the bloodstream and it constitutes the main metabolic substrate for intestinal epithelial cells. It has been postulated that the enterocyte transport glutamine by the Na-dependent System B. In this report, we evaluated glutamine transport mechanisms by measuring [3H]-glutamine in isolated villus and crypt cells from guinea pig small intestine. Both, villus and crypt cells transport glutamine by Na-dependent and Na-independent mechanisms. The Na-dependent
GASTROENTEROLOGY Vol. 118, No.4
548
550
IDENTIFICATION AND EXPRESSION OF THE HUMAN INTESTINAL EPITHELIAL APICAL MEMBRANE CALCIUM TRANSPORTER. Natalie F. Barley, Alison Howard, Stephen Legon, Julian Rf Walters, Imperial Coil Sch of Medicine, London, United Kingdom.
THE COLORECTAL MUCOSA OF OLD RATS IS MORE SUSCEPTffiLE TO FOLATE DEPLETION THAN YOUNG RATS: IMPLICATIONS FOR FOLATE-RELATED COLORECTAL CARCINO· GENESIS. Sang-Woon Choi, Pamela 1. Bagley, Annie Gee, Kim Tsui, Joel B. Mason, USDA Human Nutrition Research Ctr at Tufts Univ, Boston, MA. Since inadequate folate status is associated with colorectal carcinogenesis and since age is a major determinant of colorectal cancer risk, we investigated the effect of aging on the colonic folate status. Thirty nine weanling male Sprague-Dawley rats and an equal number of 1 yr old animals were divided into three groups and fed amino-acid defined diets containing Omg folatelkg (deplete), 2mglkg (replete), and 8mglkg (supplemented). Five rats from each group were killed at 8 wks after initiation of diets and 8 rats from each group were killed at 20 wks. The folate concentrations were measured using the L. casei microbial assay. Total plasma homocysteine, S-adenosylmethionine (SAM) concentration, and the distribution of the different co-enzymatic forms of folate in colonic tissue were each measured by established HPLC methods. Old rats fed the folate-deplete diet showed a significantly lower weight compared to folate-replete and supplemented rats begining at 10 wks(p<0.05), while young rats which were folatedeplete did not show any impairment in weight gain. The colonic folate concentrations of old, folate-deplete rats were significantly lower at 20 wks compared to the young rats(p<0.05). Similarly, old folate-replete rats had significantly lower colonic folate concentrations than young replete rats(p<0.05). However, there was no significant difference in colonic folate concentrations between the young and old, folate-supplemented groups. Colonic concentrations of SAM, which are determined in part by tissue folate levels, were marginally lower in the old, deplete animals compared to the young, deplete animals(p=0.051). By 20 wks the folatedeplete old rats showed significant decreases in the proportion of total colonic folate in the form of methyltetrahydrofolate compared to the folate-replete and supplemented groups. However, young deplete rats did not show any significant difference in folate distribution. Although folate depletion produced significantly higher elevations in homocysteine levels in old rats compared to young, supplementation led to normal levels in both groups. Conclusions: The colon of old rats is more susceptible to folate depletion than juvenile animals. However folate supplementation, at four times the basal requirement, is just as effective at eliminating evidence of inadequate folate status in old animals as it is in the young.
Absorption of dietary calcium by the intestine is necessary for bone mineralization and the prevention of osteoporosis. Fractional calcium absorption shows considerable individual variability, and the well-studied association with the active metabolite of vitamin D, 1,25(OHhD3, accounts for only a proportion of this. Calcium absorption by the epithelial cells involves transport through the apical membrane, the cytoplasm (binding to the vitamin D dependent protein calbindin-D9k), and the basolateral membrane (by the calcium pumping ATPase PMCAI). Novel apical membrane calcium influx channels have been described in rabbit kidney and rat duodenum, and named ECaC (for epithelial calcium channel) or CaTI (for calcium transport protein). Using sequence data from those species to construct mixed primers, we amplified by RT-PCR a partial sequence from human duodenal RNA, and used this eDNA probe to study expression on northern blots. In this 446-base region, human CaTl has 97% and 86% amino-acid similarity with the rat and rabbit channels. A transcript approximately 3 kb in size was detectable in duodenum, but not ileum or colon. A similar sized transcript was also found in human placenta and pancreas, where there was also evidence of a larger transcript. A faint transcript of different size could also be detected in brain. No expression of any transcript was detectable in kidney. In duodenal biopsies from 20 previously characterised normal volunteers, CaTl RNA levels were measured and corrected for differences in GAPDH expression. There was considerable (lO-fold) variability in the levels of CaTl signal between subjects. CaTl signal correlated significantly with calbindin-D9k transcripts (r=0.48, p<0.05) but not with 1,25(OHhD, 25-0HD or with age. There was an unexpected strong correlation (r=0.83, p
549 THE EFFECTS OF LONG-TERM FEEDING WITH FISH OIL ON LIPID ABSORPTION IN THE RAT IN VIVO. Lone F. Larsen, Axel K. Hansen, Peter Marckmann, Klaus Bukhave, Tech Univ of Denmark, Copenhagen, Denmark; The Royal Veterinary and Agricul Univ, Copenhagen, Denmark. Background: It is well established that dietary fish oil counteracts the accumulation of postprandial plasma triglyceride (TG). Aims: To explore whether this effect originates in a decreased lipid absorption rate and/or an increased accumulation of lipid within the enterocytes. Methods: Twelve male Wistar rats (weight 197 g (192-201). median (Q50 range» were fed a basic chow (80% w/w) enriched with an oil-mixture (20% w/w), a-tocopherol (0.01% w/w). In the control chow all polyunsaturated fatty acids (PUFA) were n-6, while approximately 20% n-6 PUFA in fish oil chow were replaced by n-3 PUFA. The oil-mixtures were balanced as regards saturated (24%), monounsaturated (44%), and PUFA (32%). After 3 weeks the rats received an intragastric instillation of 0.8 g olive oil and the postprandial plasma TG was determined following 3 hours. One week later, 2 mL RPMJTM containing 61 mg 3H-triolein (33 nCi/mg) and 2 p,ffiol bile salts were instilled into a 15 em intestinal tied-off loop starting 2 em orally to the bile duct. At 15, 30, 45, and 60 min, a 2 em segment of the intestinal loop was removed from the aboral end and the incubation was continued for another 30 min. Enterocytes were isolated by calcium chelation and the total amount of absorped lipid determined by liquid scintillation counting. The distribution of radioactivity into different lipid classes was assessed by TLC and the results were normalized to enterocyte-DNA. Results: Plasma TG following a fat load was significantly (p
sst THE ROLE OF CAVEOLIN IN CLASS B SCAVENGER RECEPTOR-BI MEDIATED CHOLESTEROL UPTAKE IN THE INTESTINE. Willem 1. de Villiers, Gary W. Varilek, Lei Cai, Deneys R. van der Westhuyzen, Univ of Kentucky, Lexington, KY. Scavenger receptor-BI (SR-BI) is of central importance in cholesterol metabolism and homeostasis. It plays a key role in the selective lipid uptake of free and esterified cholesterol from high-density lipoprotein (HDL) to cells as well as in the reverse process; namely efflux of cholesterol from cells to HDL. SR-BI was recently detected in the small intestinal brush border membrane and may be the first identified receptor mediating dietary cholesterol absorption in the intestine. SR-BI colocalizes on the plasma membrane with caveolin-l, an integral protein component of caveolae. Caveolae are plasma membrane micro-domains receiving increasing attention as important in cellular cholesterol trafficking. In the present study we tested the hypothesis that caveolin-l expression promotes selective cholesterol ester uptake. We characterized the role of SR-BI and caveolin-l in selective lipid uptake and cholesterol efflux in two human colon cancer epithelial cell lines. Both CaCo-2 and HT-29 cells express SR-BI protein endogenously, but only HT-29 cells express caveolin-I protein. We overexpressed SR-BI and caveolin-I expression in these two cell lines by adenoviral-mediated gene transfer. The presence of caveolin-l did not alter the expression level of SR-BI. Expression of caveolin-I did not affect the amount of J25I_HDL associated with the cells, but decreased selective 3H-cholesterol ether uptake by the epithelial cells by 50%. Dosedependent adenoviral-mediated caveolin overexpression further reduced 3H-cholesterol ether uptake by the cell lines. The efflux of 3H-cholesterol did not change in each cell type with respect to caveolin-l expression. We conclude that caveolin-l negatively regulates SR-BI dependent selective cholesterol ester uptake without affecting free cholesterol efflux in colonic epithelial cells.
552 IDENTIFICATION OF A NOVEL NA-DEPENDENT TRANSPORT MECHANISM, HIGHLY SPECIFIC FOR GLUTAMINE, IN ISOLATED INTESTINAL VILLUS AND CRYPT CELLS. Jesus R. del Castillo, Maria C. Sulbaran, Luis O. Burguillos, Ivic, Caracas, Venezuela. Glutamine is the most abundant amino acid in the bloodstream and it constitutes the main metabolic substrate for intestinal epithelial cells. It has been postulated that the enterocyte transport glutamine by the Na-dependent System B. In this report, we evaluated glutamine transport mechanisms by measuring [3H]-glutamine in isolated villus and crypt cells from guinea pig small intestine. Both, villus and crypt cells transport glutamine by Na-dependent and Na-independent mechanisms. The Na-dependent