1
April 1995 Intestinal Disorders A317 DUAL ROLE FOR ALUMINUM FLUORIDE SENSITIVE G-PROTEINS IN THE FUNCTION OF Tu EPITHELIAL CELLS: TRANSPORT AND BARRIER EFFECTS J. Ries. J. Stein, A. E. Traynor-Kaplan and K. E. Barrett, Dept. Medicine, Univ. of California,San Diego, Sch. of Medicine, San Diego,CA 92103. G-proteins play important roles in several signal traneduction pathways. The ability of aluminum fluoride (AIF4) to activate G-proteins by mimicking the y- phosphate group of GTP was exploited to examine roles for G-proteins in epithelial function. Studies were conductedusing the human colonic epithelial cell line, Tu. For most experiments, cells were grown on permeable supports and mounted in Ussing chambers. Ceils were preincnhated with pertnssis toxin (14 ng/ml) for 12 h, or the intracellular Ca2. chelator BAPTA (0.25 raM) for 45 rain, or 200 ~M deferoxamine, a heavy metal chelator, for 10 min prior to adding 5 mM NaF + 10 pM AICI~to generate AIF 4 in situ. CI" secretion was assessed as changes in short circuit current (I~). cAMP levels were measured in cell extracts via a commercial ELISA. All values are means _+ SEMfor n experiments, measured 20 rain at~erthe addition of NaF. Basolateral, but not apical, addition of AIF4 increased I~ and decreased resistance (table). Both effects were reversed by pertnssis toxin and deferoxamine. The effect on I~, but not resistance, was reversed by BAPTA and Cl'-free medium. Neither action of AIF4 was altered by staurosporine (0.1 #M, n=6), brefeldin A (10/tM, n=6), charybdotoxin (50nM, n=4), or a phospholipase C inhibitor, U-73122 (10/tM, n=4). AIF 4 also had no effect on cAMP levels relative to vasoactive intestinal polypeptide (VIP) as a positive control (negative control 14.5+1.1; A1F4 16.1+1.3; VIP 793+1.1 pmol/mg protein; n=4). n Relative resistance I~ (baseline= 1) (~A/em 2) NaF +AIC13 apical (10) 1.03.+0.03" 2.6+0.5" NaF +AIC13 basolateral (21) 0,70+0.03 13.5+2.4 + Pertussis toxin (4) 0.96+0.01" 3.8+ 1.3" + Bapta (6) 0.75+0.08 0.9+0.2" + Deferoxamine (5) 0.97+0.02* 3.1+0.7" + Cl-free media (6) 0.72+0.08 0.8+0.3" • p<0.05 or better vs. NaF+AICI3 basolateral We conclude that AlF4-sensitive G-proteins, likely localized to the basolateral membrane, regulate transport and barrier function of intestinal epithelial cells. The effect on CI" secretion, but not that on resistance, involves Ca2+ but not, phospholipase C activity, and thus occurs by an unknown mechanism. The effect on resistance is independent of Ca2+ and other known second messengers. Incidence and severity of osteopenic bone disease in adult celiac disease. AJ.M. Rijnders 1, C.J.J. Mulder 1, C.M. Spronk 1, J.C. Netelenbos2. Hospital Rijnstate Arnhem, Free University Hospital Amsterdam. Celiac disease may present with calcium malabsorption and predispose to the development of osteoporosis. The aim of this study was to determine the incidence and the severity of bone loss in a population of Dutch celiacs. Using a Norland XR-26 x-ray densitometer, bone mineral density (BMD) measurements of the lumbar spine (L2-4), the femoral neck (FN) and the greater trochanter (Tro) of the left femur, were obtained in 104 female and 16 male, adult, celiac patients, regard- less of duration of disease and lenght and quality of dietary measu- rements. The mean age was 49.7 years, ranging from 22 to 78 years. Measurements were expressed as the difference in Standard Deviations (SD) from the age/sex/ weight matched mean (Z-score, database of normals Norland). Degree of bone loss was subdivided in Z values below -1 SD (significant) and below -2 SD (severe) respectively. The BMD was moderately lower then the mean at the Spine and Tro (Spine -0.2, Tro -0.66) measurement sites whereas the BMD at the FN measurement site did not differ from the mean (+0.01). Z-values in men were lower then in women at all three sites (spine -0.41 vs -0.19, FN -0.41 vs +0.07, Tro -0.77 vs - 0.64). Z-values below -1 SD at one or more sites were found in 52 patients (43%) and did not differ between men and women (44% and 43% respectively). Z-values below -2 SD were measured in 18 patients (15%). Spine and FN measurements showed less frequently (24% and 16% resp) significant bone loss, compared to the Tro (37%). Conclusions: I) The incidence of significant and severe osteope- nic bone disease in this random population of Dutch celiacs is high. 2) Male and female celiac patients seem equally affected. 3) Routi- ne measurement of BMD in adult celiacs patients is indicated. STIMULATION OF GLUCAGON-LIKE PEPTIDE-1 RELEASE IN RESPONSE TO CARBOHYDRATES IN THE PERFUSED RAT ILEUM. U. Ritzel, U. Leonhardt, A. Fromme, F. Sfiickmann, G. Ramadori. Department of Medicine, Division of Gastroenterology and Endocrinology, University of G~ttingen, Gtttingen, Germany Glucagon-like peptide (7-36) amide (GLP-1) is the strongest peptidergic stimulator of insulin release. It is released from intestinal L-cells after fooduptake. The mechanisms of GLP-1 secretion are unclear. The effect of carbohydrates on GLP-1 release was studied in male Wistar- rats (200-230 g) by experimental perfusion of the distal ileum in vivo. GLP-1 levels were quantitated in venous effluent of a superior mesenteric vein by a specific radio- immuno-assay. Plasma GLP-1 levels were significantly increased by luminal administration of 5% glucose or 5% fructose in isotonic NaCI or 5% fructose in destilled water. 5% Galactose, 3-O.methylglucose, galactose and alpha- methylglucoside were dissolved in isotonic NaCI and induced a significantly increased GLP-1 secretion. Isotonic NaCI, N- acetylglucosamine, 2-desoxyglucose and the alpha- glucosidase inhibitor acarbose were given in the same concentration and did not affect GLP-1 release. It is concluded that carbohydrates that represent a substrate for the sodium-dependent glucose cotransporter (galactose, a- methylglucoside, and 3-O-methylglucose) are equally effective in raising plasma GLP-1 levels. The results further suggest that intracellular metabolism and removal from the cells are not necessary for stimulation of GLP-1 release, since 3-O-methyglucose is not metabolized within intestinal cells and a-methylglucoside is not a substrate for the glucose transporter at the basolateral membrane, The ability of fructose to induce sodium-independent GLP-1 release suggests that the sodium-dependent glucose cotrausporter is not the only trigger for carbohydrate-induced GLP-1 secretion. NPY INHIBITION OF VIP-INDUCED JEJUNAL HYPERSECRETION IN RATS; INVOLVEMENT OF ENDOGENOUS SOMATOSTATIN AND NERVOUS PATHWAYS. P.J.M. Rivi~re, M. Chovet, H. Fargeau, X. Pascaud, J.L. Junien. Institut de Recherche Jouveinal, Fresnes, France. The potent intestinal antisecretory action of neuropoptide Y (NPY) depends on a direct action on the enterocyte ~ as well as an indirect action through nervous pathways 2. In this last case, the final neuromediator is not identified. The present study investigates the possibility of the involvement of endogenous somatostatin in the NPY inhibition of VIP-induced jejunal hypersecretion in rats. Methods: In anesthetized (pentobarbital, 60 mg/kg i.p.) fasted Sprague-Dawley rats (160-180 g), a jejunal loop was isolated by two ligations at 5 and 25 cm distal to the ligament of Treitz. The loop was filled with saline (2 ml, 37°C). Jejunal secretion was stimulated by a 30 rain intraarterial infusion of VIP. At the end of the VIP infusion, the loop was collected, measured and weighed before and after fluid removal to determine water net flux (mg/cm). Intravenous (i,v.) bolus injections of NPY or octreotide were performed 15 rain before starting VIP infusion. Tetrodotoxin (TTX) or cyclosomatostatin were given by i.v. route 10 rain before NPY or octreotide. Results: In the basal state the net water flux was positive (+ 3.14 -+ 3.0 mg/cm). VIP (0.03-0.33 lag.rain -~) induced a dose- related inversion of net flux. The submaximal effect (- 28.5 + 6.5 mg/cm) was obtained at the dose of 0.1 ~g.min -~. VIP (0.1 gg.min"[) induced jejunal hypersecretion was inhibited in a dose-related manner by NPY and octreotide (EDs0:0.008 and 0.132 gg/kg i,v., respectively). NPY (0.03 ~g/kg) and octreotide (1 ~tg/kg) responses were inhibited (-85% and -100%, respectively) by cyelosomatostatin (1 pg/kg). TTX (5 ~g/kg) inhibited by 88% and 45% NPY and octreotide responses, respectively. Neither TTX nor eyclosomatostatin altered per se the VIP response. Conclusion: VIP induced a jejunal hypersecretion by a direct action on the enterocyte (TTX-resistant). VIP response was blocked by NPY in a TTX and cyclosomatostatin sensitive manner, suggesting an indirect action on the enterocyte through both nerve and somatostatin pathways. Octreotide response was partly TTX- sensitive, suggesting that in addition to a direct effect on the enterocyte, nerves are also involved. (1) Cox et al. J Physiol 1988; 398: 65. (2) Rivi~re et al. J Pharm Exp Ther 1993; 264:1268. This study was funded by Institut de Recherche Jouveinal, Fresnes, France.

Incidence and severity of osteopenic bone disease in adult celiac disease

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April 1995 Intestinal Disorders A317

• DUAL ROLE FOR ALUMINUM FLUORIDE SENSITIVE G-PROTEINS IN THE FUNCTION OF Tu EPITHELIAL CELLS: TRANSPORT AND BARRIER EFFECTS J. Ries. J. Stein, A. E. Traynor-Kaplan and K. E. Barrett, Dept. Medicine, Univ. of California,San Diego, Sch. of Medicine, San Diego,CA 92103.

G-proteins play important roles in several signal traneduction pathways. The ability of aluminum fluoride (AIF4) to activate G-proteins by mimicking the y- phosphate group of GTP was exploited to examine roles for G-proteins in epithelial function. Studies were conducted using the human colonic epithelial cell line, Tu. For most experiments, cells were grown on permeable supports and mounted in Ussing chambers. Ceils were preincnhated with pertnssis toxin (14 ng/ml) for 12 h, or the intracellular Ca 2. chelator BAPTA (0.25 raM) for 45 rain, or 200 ~M deferoxamine, a heavy metal chelator, for 10 min prior to adding 5 mM NaF + 10 pM AICI~ to generate AIF 4 in situ. CI" secretion was assessed as changes in short circuit current (I~). cAMP levels were measured in cell extracts via a commercial ELISA. All values are means _+ SEMfor n experiments, measured 20 rain at~er the addition of NaF. Basolateral, but not apical, addition of AIF 4 increased I~ and decreased resistance (table). Both effects were reversed by pertnssis toxin and deferoxamine. The effect on I~, but not resistance, was reversed by BAPTA and Cl'-free medium. Neither action of AIF4 was altered by staurosporine (0.1 #M, n=6), brefeldin A (10/tM, n=6), charybdotoxin (50nM, n=4), or a phospholipase C inhibitor, U-73122 (10/tM, n=4). AIF 4 also had no effect on cAMP levels relative to vasoactive intestinal polypeptide (VIP) as a positive control (negative control 14.5+1.1; A1F4 16.1+1.3; VIP 793+1.1 pmol/mg protein; n=4).

n Relative resistance I~ (baseline= 1) (~A/em 2)

NaF +AIC13 apical (10) 1.03.+0.03" 2.6+0.5" NaF +AIC13 basolateral (21) 0,70+0.03 13.5+2.4

+ Pertussis toxin (4) 0.96+0.01" 3.8+ 1.3" + Bapta (6) 0.75+0.08 0.9+0.2" + Deferoxamine (5) 0.97+0.02* 3.1+0.7" + Cl-free media (6) 0.72+0.08 0.8+0.3"

• p<0.05 or better vs. NaF+AICI3 basolateral We conclude that AlF4-sensitive G-proteins, likely localized to the basolateral membrane, regulate transport and barrier function of intestinal epithelial cells. The effect on CI" secretion, but not that on resistance, involves Ca 2+ but not, phospholipase C activity, and thus occurs by an unknown mechanism. The effect on resistance is independent of Ca 2+ and other known second messengers.

Incidence and severity of osteopenic bone disease in adult celiac disease. AJ .M. Rijnders 1, C.J.J. Mulder 1, C.M. Spronk 1, J.C. Netelenbos 2. Hospital Rijnstate Arnhem, Free University Hospital Amsterdam.

Celiac disease may present with calcium malabsorption and predispose to the development of osteoporosis. The aim of this study was to determine the incidence and the severity of bone loss in a population of Dutch celiacs.

Using a Norland XR-26 x-ray densitometer, bone mineral density (BMD) measurements of the lumbar spine (L2-4), the femoral neck (FN) and the greater trochanter (Tro) of the left femur, were obtained in 104 female and 16 male, adult, celiac patients, regard- less of duration of disease and lenght and quality of dietary measu- rements. The mean age was 49.7 years, ranging from 22 to 78 years. Measurements were expressed as the difference in Standard Deviations (SD) from the age/sex/ weight matched mean (Z-score, database of normals Norland). Degree of bone loss was subdivided in Z values below -1 SD (significant) and below -2 SD (severe) respectively.

The BMD was moderately lower then the mean at the Spine and Tro (Spine -0.2, Tro -0.66) measurement sites whereas the BMD at the FN measurement site did not differ from the mean (+0.01). Z-values in men were lower then in women at all three sites (spine -0.41 vs -0.19, FN -0.41 vs +0.07, Tro -0.77 vs - 0.64). Z-values below -1 SD at one or more sites were found in 52 patients (43%) and did not differ between men and women (44% and 43% respectively). Z-values below -2 SD were measured in 18 patients (15%). Spine and FN measurements showed less frequently (24% and 16% resp) significant bone loss, compared to the Tro (37%).

Conclusions: I) The incidence of significant and severe osteope- nic bone disease in this random population of Dutch celiacs is high. 2) Male and female celiac patients seem equally affected. 3) Routi- ne measurement of BMD in adult celiacs patients is indicated.

• STIMULATION OF GLUCAGON-LIKE PEPTIDE-1 RELEASE IN RESPONSE TO CARBOHYDRATES IN THE PERFUSED RAT ILEUM. U. Ritzel, U. Leonhardt, A. Fromme, F. Sfiickmann, G. Ramadori. Department of Medicine, Division of Gastroenterology and Endocrinology, University of G~ttingen, Gtttingen, Germany

Glucagon-like peptide (7-36) amide (GLP-1) is the strongest peptidergic stimulator of insulin release. It is released from intestinal L-cells after fooduptake. The mechanisms of GLP-1 secretion are unclear. The effect of carbohydrates on GLP-1 release was studied in male Wistar- rats (200-230 g) by experimental perfusion of the distal ileum in vivo. GLP-1 levels were quantitated in venous effluent of a superior mesenteric vein by a specific radio- immuno-assay. Plasma GLP-1 levels were significantly increased by luminal administration of 5% glucose or 5% fructose in isotonic NaCI or 5% fructose in destilled water. 5% Galactose, 3-O.methylglucose, galactose and alpha- methylglucoside were dissolved in isotonic NaCI and induced a significantly increased GLP-1 secretion. Isotonic NaCI, N- acetylglucosamine, 2-desoxyglucose and the alpha- glucosidase inhibitor acarbose were given in the same concentration and did not affect GLP-1 release. It is concluded that carbohydrates that represent a substrate for the sodium-dependent glucose cotransporter (galactose, a- methylglucoside, and 3-O-methylglucose) are equally effective in raising plasma GLP-1 levels. The results further suggest that intracellular metabolism and removal from the cells are not necessary for stimulation of GLP-1 release, since 3-O-methyglucose is not metabolized within intestinal cells and a-methylglucoside is not a substrate for the glucose transporter at the basolateral membrane, The ability of fructose to induce sodium-independent GLP-1 release suggests that the sodium-dependent glucose cotrausporter is not the only trigger for carbohydrate-induced GLP-1 secretion.

NPY INHIBITION OF VIP-INDUCED JEJUNAL HYPERSECRETION IN RATS; INVOLVEMENT OF ENDOGENOUS SOMATOSTATIN AND NERVOUS PATHWAYS. P.J.M. Rivi~re, M. Chovet, H. Fargeau, X. Pascaud, J.L. Junien. Institut de Recherche Jouveinal, Fresnes, France.

The potent intestinal antisecretory action of neuropoptide Y (NPY) depends on a direct action on the enterocyte ~ as well as an indirect action through nervous pathways 2. In this last case, the final neuromediator is not identified. The present study investigates the possibility of the involvement of endogenous somatostatin in the NPY inhibition of VIP-induced jejunal hypersecretion in rats. Methods: In anesthetized (pentobarbital, 60 mg/kg i.p.) fasted Sprague-Dawley rats (160-180 g), a jejunal loop was isolated by two ligations at 5 and 25 cm distal to the ligament of Treitz. The loop was filled with saline (2 ml, 37°C). Jejunal secretion was stimulated by a 30 rain intraarterial infusion of VIP. At the end of the VIP infusion, the loop was collected, measured and weighed before and after fluid removal to determine water net flux (mg/cm). Intravenous (i,v.) bolus injections of NPY or octreotide were performed 15 rain before starting VIP infusion. Tetrodotoxin (TTX) or cyclosomatostatin were given by i.v. route 10 rain before NPY or octreotide. Results: In the basal state the net water flux was positive (+ 3.14 -+ 3.0 mg/cm). VIP (0.03-0.33 lag.rain -~) induced a dose- related inversion of net flux. The submaximal effect (- 28.5 + 6.5 mg/cm) was obtained at the dose of 0.1 ~g.min -~. VIP (0.1 gg.min "[) induced jejunal hypersecretion was inhibited in a dose-related manner by NPY and octreotide (EDs0:0.008 and 0.132 gg/kg i,v., respectively). NPY (0.03 ~g/kg) and octreotide (1 ~tg/kg) responses were inhibited (-85% and -100%, respectively) by cyelosomatostatin (1 pg/kg). TTX (5 ~g/kg) inhibited by 88% and 45% NPY and octreotide responses, respectively. Neither TTX nor eyclosomatostatin altered p e r se the VIP response. Conclusion: VIP induced a jejunal hypersecretion by a direct action on the enterocyte (TTX-resistant). VIP response was blocked by NPY in a TTX and cyclosomatostatin sensitive manner, suggesting an indirect action on the enterocyte through both nerve and somatostatin pathways. Octreotide response was partly TTX- sensitive, suggesting that in addition to a direct effect on the enterocyte, nerves are also involved. (1) Cox et al. J Physiol 1988; 398: 65. (2) Rivi~re et al. J Pharm Exp Ther 1993; 264:1268. This study was funded by Institut de Recherche Jouveinal, Fresnes, France.