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The role of actin in EGF-induced alterations in microvillus membrane function and surface area
A338
AGA ABSTRACTS
PARACELLULAR TRANSPORT PLAYS A MINOR ROLE IN D-GLUCOSE ABSORPTION IN CONSCIOUS DOGS. E.E. Whane. J.C. Duun, H. Mahanty, T. Newton, M.J. Ziuner, D.W. McFadden, 3. Diamond, S.W. Ashley. Depts. of Surgery and Physiology, UCLA School of Medicine, Los Angeles, CA. Studies in anesthetized rodents have been interpreted to suggest that carrier-mediated D-glucose absorption enhances intestinal paracellular permeability, with the result that the majority of luminal D-glucose is absorbed through passive means. We tested this hypothesis in awake, unanesthetized dogs in viv0. METHODS: Transport studies were conducted using 25 cm jejunal Thiry-Vella loops previously created in 5 dogs. Isotonic electrolyte solution, containing 14C-PEG as the volume marker and 3H-L-ghicose (50 laCi/L) as the probe for glucose paracellular permeability, was infused into the loops at 4.7 ml/min. Infusate solutions of two different D-glucose concentrations spanning the physiological range, one at 1 mM and the other at 50 raM, were used. Loop effluents Were collected and assayed for 14C and 3H activities and D-glucose concentration. Mean D-glucose loads were calculated assuming a linear decline in glucose concentration as infus~tte traverses the loops. The fractional absorption of 3H-L-glucose was used to determine the portion Of D-glucose absorption that occul"s through passive permeation. RESULTS: The fractional absorption 3H-L-glucose was less than 10% ifi all cases. Paracellular permeability clearly did not vary with luminal Dglucose concentration. At the physiological concentrations used in this study, only a small minority o f D-glucose absorption occurs through passive permeation in the jejunum of unanesthetized dogs. Studies examining paracellular permeability in vitro or in anesthetized animals may bear little phYsiological relevance.
• COMBINATION OF ENTEROSCOPY WITH ENTEROCLYSIS FOR EVALUATION OF SMALL BOWEL LESIONS. PRELIMINARY EXPERIENCE. Jeff R. Willis, Gary R. Zuckerman, Hitesh R. Chokshi, Giuseppe Aliperti. Washington University School of Medicine, St. Louis, Missouri. Diagnostic and therapeutic capabilities in the proximal small bowel (SB) have improved with video enteroscopes. The distal small bowel is still inaccessible to routine endoscopy, therefore enteroclysis remains the diagnostic technique of choice for distal SB lesions. We herein report our experience with a previously described combination of the two techniques~ METHODS: 45 patients (mean age 59+2, 26 women) were referred for further evaluation of gastrointestinal bleeding atter negative endoscopy of the upper and lower tract (group A, 33 patients), or equivocal radiologie studies (group B, 12 patients). Push enteroscopy with the Olympus SIF-100 enteroscope was performed in all patients with the aid of an overtube during conscious sedation. SB enteroclysis was performed after enteroscopy in 26 patients with findings insufficient to explain the clinical picture. Technique: after enteroscopy, a naso-enteral tube was passed over a guidewire left in place upon withdrawing the enteroscope, and after transferring the guidewire from the oropharynx to the nose via a soft catheter. Routine enteroclysis was then performed after recovery from sedation, except in patients with preceding endoscopic biopsy or therapy. RESULTS: The overtube was passed in the duodenum in all patients. Enteroseopy alone identified a bleeding site in 16/33 (48%) group A patients (9 angiodysplasia, 6 ulcers, 1 vessel), and in 8/12 (67%) group B patients (5 ulcers, 2 strictures, 1 mass). Enteroclysis after enteroscopy identified a lesion in 1/16 (6%) group A patients (1 mass), and in 3/10 (30%) group B patients (1 mass, 1 stricture, 1 large diverticulum). No complications occurred. Passage of the enteroclysis catheter was successful in all cases. Patient satisfaction, assessed by telephone follow-up was c6mparable to other endoscopic procedures in all patients (45/45). CONCLUSIONS: (1)Combination of small bowel enteroscopy with enteroclysis in patients with suspected lesions of the small bowel is safe. (2) This combination is convenient to the patient, as it allows successful, comfortable passage of the nasoenteric catheter under sedation, and a single trip to the hospital. (3) Though only 6% of patients with gastrointestinal bleeding had a finding identified by enteroclysis, the radiographic study excluded lesions of the distal SB other than angiodysplasia, (4) This combination also improved the diagnostic yield in patients with equivocal radiographic findings.
GASTROENTEROLOGY, Vol. 108, No. 4
• GUT-I: A GLYCOSYLATION-DEPENDENT REGTJLATOR OF EPITHELIAL CELL PROLIFERATION B.M. WICE. J.I. GORDON. Department of Molecular Biology & Pharmacology, Washington University School of Medicine. St Louis, MO HT29 cells represent a useful model s y s t e m for studying the molecular m e c h a n i s m s that regulate intestinal epithelial cell proliferation and differentiation programs. In the absence of g l u c o s e lHT29/G-), t h e s e ceils u n d e r g o growth a r r e s t at confluence a n d differentiate to express goblet and enterocytic markers. In the presence of glucose (HT29/G÷), confluent cells continue to proliferate a n d fail to terminally differentiate, We used subtraction hybridization to isolate cDNAs encoding proteins expressed in HT29/G-, b u t not h u m a n cervical carcinoma (HeLa) cells. One s u c h protein (found only in intestine and liver), GUT1. represents a new m e m b e r of the Tetraspan Membrane Family (TMF) of proteins implicated in regulating cell growth, adhesion and signaling. Immunohistochemical analysis of h u m a n t i s s u e s revealed that GUT- 1 is confined to the apical m e m b r a n e s of nondividing villus-associated epithelial ceils and to the canalicular m e m b r a n e s of hepatocytes located in zone l of the liver acinus. Expression of GUT-1 in HT29 cells increases dramatically j u s t prior to confluence. It is extensively glycosylated only in HT'29/Geelis. T h e pattern of glycosylation is similar to what is Observed in the h u m a n small intestine i n v i v o , Neither HeLa nor SW480 colon adenocarcinoma cells actively transcribe the endogenous GUT-I gene. Therefore. t h e s e epithelial cells were used for a series of transfection studies designed to a s s e s s the effects of expressing GUT-l on growth regulation. During log phase. GUT1 h a s no effect on the growth rate of HeLa or SW480 cells. At confluence. HeLa cells e x p r e s s i n g GUT-I s h o w a d r a m a t i c reduction in cell density and focus formation. The pattern of glycosylation of GUT-1 in these HeLa cells resembles the pattern in HT29/G- cells (which. like GUT-l-positive HeLa cells, undergo growth arrest at confluence). GUT-I expression does not effect the growth properties of confluent SW480 cells. G1ycosylation of GUT-I in t h e s e cells resembles the pattern in HT29 G+ cells. which also fail to growth arrest at confluence. These findings suggest that GUT-1 may act as a cell contact-dependent, negative regulator of epithelial cell proliferation. This function may, in turn, may be dependent upon its pattern of glycosylation. Funded in part by grants from the NIH'(DK30292) and Amgen.
• THE ROLE OF ACTIN IN EGF-INDUCED ALTERATIONS IN MICROVILLUS MEMBRANE FUNCTION AND SURFACE AREA. J.K. Wong, J.A. Hardin, D.G. Gall. DePt. of PediatriCs, University of Calgary, Calgary, Alberta, Canada. We previously demonstrated that luminal EGF acutely upregulates brush border surface area and glucose transport in rabbit jejunum. Aim: The aim of this study was to examine the role of mierovillus eytoskeletal aetin in EGF-indueed alterations in glucose transport and absorptive surface area. Methods: New Zealand White rabbits (800-1000 g) were fasted, and two 15 era blind loops, were tied off distal to the ligament of Treitz. In separate experiments, EGF or EGF + eytoehalasin D was incubated in one loop and compared to the control loop. After a 1 hour exposure, loops were removed, and, in separate animals, either mucosa scraped, brush border membrane vesicles (BBMV) prepared and glucose transport determined by a rapid filtration technique, or total absorptive surface area of loops determined b y partitioning of the fluophore 1-[4-(trimethylamino) phenyl]-6-phenylhexatriene (TMA-DPH). Results: In EGF treated loops, Vmax for g!ueose transport (9.3 --- 0.6) was signifieantly (p<0.001) increased compared to control 'loops (7.7 ± 0.6 nmol/min/mg protein; n=6). Administration of cytoehalasin D abolished the EGF effeet on Vmax (EGE,+eytoehalasin D 6.3 ± 0.5; n=4). Km was not significantly altered in either treatment group compared to control. Total absorptive surface area was significantly (p < 0.05) increased in the EGF treated loops compared to control (EGF 7,3 x 109 --_3.6 x 109 vs. control 2.2 x 109 • 9.2 x l0 s p.m2; n=4). The increase in total absorptive surface area indueed by EGF was als0 completely inhibited by eytoehalasin D (EGF + eytoehalasin D 1.2 x 109 -+ 3.2 x i0 s, n=4) Conclusion.' Cytoehalasin inhibits the EGF induced inerease in glucose transport and surface area indicating that aetin plays a central role in the mechanism whereby EGF alters jejunal structure and funCtiori.
AGA ABSTRACTS
PARACELLULAR TRANSPORT PLAYS A MINOR ROLE IN D-GLUCOSE ABSORPTION IN CONSCIOUS DOGS. E.E. Whane. J.C. Duun, H. Mahanty, T. Newton, M.J. Ziuner, D.W. McFadden, 3. Diamond, S.W. Ashley. Depts. of Surgery and Physiology, UCLA School of Medicine, Los Angeles, CA. Studies in anesthetized rodents have been interpreted to suggest that carrier-mediated D-glucose absorption enhances intestinal paracellular permeability, with the result that the majority of luminal D-glucose is absorbed through passive means. We tested this hypothesis in awake, unanesthetized dogs in viv0. METHODS: Transport studies were conducted using 25 cm jejunal Thiry-Vella loops previously created in 5 dogs. Isotonic electrolyte solution, containing 14C-PEG as the volume marker and 3H-L-ghicose (50 laCi/L) as the probe for glucose paracellular permeability, was infused into the loops at 4.7 ml/min. Infusate solutions of two different D-glucose concentrations spanning the physiological range, one at 1 mM and the other at 50 raM, were used. Loop effluents Were collected and assayed for 14C and 3H activities and D-glucose concentration. Mean D-glucose loads were calculated assuming a linear decline in glucose concentration as infus~tte traverses the loops. The fractional absorption of 3H-L-glucose was used to determine the portion Of D-glucose absorption that occul"s through passive permeation. RESULTS: The fractional absorption 3H-L-glucose was less than 10% ifi all cases. Paracellular permeability clearly did not vary with luminal Dglucose concentration. At the physiological concentrations used in this study, only a small minority o f D-glucose absorption occurs through passive permeation in the jejunum of unanesthetized dogs. Studies examining paracellular permeability in vitro or in anesthetized animals may bear little phYsiological relevance.
• COMBINATION OF ENTEROSCOPY WITH ENTEROCLYSIS FOR EVALUATION OF SMALL BOWEL LESIONS. PRELIMINARY EXPERIENCE. Jeff R. Willis, Gary R. Zuckerman, Hitesh R. Chokshi, Giuseppe Aliperti. Washington University School of Medicine, St. Louis, Missouri. Diagnostic and therapeutic capabilities in the proximal small bowel (SB) have improved with video enteroscopes. The distal small bowel is still inaccessible to routine endoscopy, therefore enteroclysis remains the diagnostic technique of choice for distal SB lesions. We herein report our experience with a previously described combination of the two techniques~ METHODS: 45 patients (mean age 59+2, 26 women) were referred for further evaluation of gastrointestinal bleeding atter negative endoscopy of the upper and lower tract (group A, 33 patients), or equivocal radiologie studies (group B, 12 patients). Push enteroscopy with the Olympus SIF-100 enteroscope was performed in all patients with the aid of an overtube during conscious sedation. SB enteroclysis was performed after enteroscopy in 26 patients with findings insufficient to explain the clinical picture. Technique: after enteroscopy, a naso-enteral tube was passed over a guidewire left in place upon withdrawing the enteroscope, and after transferring the guidewire from the oropharynx to the nose via a soft catheter. Routine enteroclysis was then performed after recovery from sedation, except in patients with preceding endoscopic biopsy or therapy. RESULTS: The overtube was passed in the duodenum in all patients. Enteroseopy alone identified a bleeding site in 16/33 (48%) group A patients (9 angiodysplasia, 6 ulcers, 1 vessel), and in 8/12 (67%) group B patients (5 ulcers, 2 strictures, 1 mass). Enteroclysis after enteroscopy identified a lesion in 1/16 (6%) group A patients (1 mass), and in 3/10 (30%) group B patients (1 mass, 1 stricture, 1 large diverticulum). No complications occurred. Passage of the enteroclysis catheter was successful in all cases. Patient satisfaction, assessed by telephone follow-up was c6mparable to other endoscopic procedures in all patients (45/45). CONCLUSIONS: (1)Combination of small bowel enteroscopy with enteroclysis in patients with suspected lesions of the small bowel is safe. (2) This combination is convenient to the patient, as it allows successful, comfortable passage of the nasoenteric catheter under sedation, and a single trip to the hospital. (3) Though only 6% of patients with gastrointestinal bleeding had a finding identified by enteroclysis, the radiographic study excluded lesions of the distal SB other than angiodysplasia, (4) This combination also improved the diagnostic yield in patients with equivocal radiographic findings.
GASTROENTEROLOGY, Vol. 108, No. 4
• GUT-I: A GLYCOSYLATION-DEPENDENT REGTJLATOR OF EPITHELIAL CELL PROLIFERATION B.M. WICE. J.I. GORDON. Department of Molecular Biology & Pharmacology, Washington University School of Medicine. St Louis, MO HT29 cells represent a useful model s y s t e m for studying the molecular m e c h a n i s m s that regulate intestinal epithelial cell proliferation and differentiation programs. In the absence of g l u c o s e lHT29/G-), t h e s e ceils u n d e r g o growth a r r e s t at confluence a n d differentiate to express goblet and enterocytic markers. In the presence of glucose (HT29/G÷), confluent cells continue to proliferate a n d fail to terminally differentiate, We used subtraction hybridization to isolate cDNAs encoding proteins expressed in HT29/G-, b u t not h u m a n cervical carcinoma (HeLa) cells. One s u c h protein (found only in intestine and liver), GUT1. represents a new m e m b e r of the Tetraspan Membrane Family (TMF) of proteins implicated in regulating cell growth, adhesion and signaling. Immunohistochemical analysis of h u m a n t i s s u e s revealed that GUT- 1 is confined to the apical m e m b r a n e s of nondividing villus-associated epithelial ceils and to the canalicular m e m b r a n e s of hepatocytes located in zone l of the liver acinus. Expression of GUT-1 in HT29 cells increases dramatically j u s t prior to confluence. It is extensively glycosylated only in HT'29/Geelis. T h e pattern of glycosylation is similar to what is Observed in the h u m a n small intestine i n v i v o , Neither HeLa nor SW480 colon adenocarcinoma cells actively transcribe the endogenous GUT-I gene. Therefore. t h e s e epithelial cells were used for a series of transfection studies designed to a s s e s s the effects of expressing GUT-l on growth regulation. During log phase. GUT1 h a s no effect on the growth rate of HeLa or SW480 cells. At confluence. HeLa cells e x p r e s s i n g GUT-I s h o w a d r a m a t i c reduction in cell density and focus formation. The pattern of glycosylation of GUT-1 in these HeLa cells resembles the pattern in HT29/G- cells (which. like GUT-l-positive HeLa cells, undergo growth arrest at confluence). GUT-I expression does not effect the growth properties of confluent SW480 cells. G1ycosylation of GUT-I in t h e s e cells resembles the pattern in HT29 G+ cells. which also fail to growth arrest at confluence. These findings suggest that GUT-1 may act as a cell contact-dependent, negative regulator of epithelial cell proliferation. This function may, in turn, may be dependent upon its pattern of glycosylation. Funded in part by grants from the NIH'(DK30292) and Amgen.
• THE ROLE OF ACTIN IN EGF-INDUCED ALTERATIONS IN MICROVILLUS MEMBRANE FUNCTION AND SURFACE AREA. J.K. Wong, J.A. Hardin, D.G. Gall. DePt. of PediatriCs, University of Calgary, Calgary, Alberta, Canada. We previously demonstrated that luminal EGF acutely upregulates brush border surface area and glucose transport in rabbit jejunum. Aim: The aim of this study was to examine the role of mierovillus eytoskeletal aetin in EGF-indueed alterations in glucose transport and absorptive surface area. Methods: New Zealand White rabbits (800-1000 g) were fasted, and two 15 era blind loops, were tied off distal to the ligament of Treitz. In separate experiments, EGF or EGF + eytoehalasin D was incubated in one loop and compared to the control loop. After a 1 hour exposure, loops were removed, and, in separate animals, either mucosa scraped, brush border membrane vesicles (BBMV) prepared and glucose transport determined by a rapid filtration technique, or total absorptive surface area of loops determined b y partitioning of the fluophore 1-[4-(trimethylamino) phenyl]-6-phenylhexatriene (TMA-DPH). Results: In EGF treated loops, Vmax for g!ueose transport (9.3 --- 0.6) was signifieantly (p<0.001) increased compared to control 'loops (7.7 ± 0.6 nmol/min/mg protein; n=6). Administration of cytoehalasin D abolished the EGF effeet on Vmax (EGE,+eytoehalasin D 6.3 ± 0.5; n=4). Km was not significantly altered in either treatment group compared to control. Total absorptive surface area was significantly (p < 0.05) increased in the EGF treated loops compared to control (EGF 7,3 x 109 --_3.6 x 109 vs. control 2.2 x 109 • 9.2 x l0 s p.m2; n=4). The increase in total absorptive surface area indueed by EGF was als0 completely inhibited by eytoehalasin D (EGF + eytoehalasin D 1.2 x 109 -+ 3.2 x i0 s, n=4) Conclusion.' Cytoehalasin inhibits the EGF induced inerease in glucose transport and surface area indicating that aetin plays a central role in the mechanism whereby EGF alters jejunal structure and funCtiori.