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The down regulated in adenoma gene (DRA) encodes an intestine-specific membrane sulfate transport protein
April 1 9 9 5
• THE DOWN REGULATED IN ADENOMA GENE (DRA) ENCODES AN INTESTINE-SPECIFIC MEMBRANE SULFATE TRANSPORT PROTEIN D.G. Silberg*, W Wang #, R. Moseley#, and P.G. Traber*. Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA*; Division of Gastroenterology, VAMC and University of Michigan, Ann Arbor, MI#. The gene, Down Regulated in Adenoma (DRA), is expressed i n normal colon and small intestine, but is absent in the majority of colon adenomas and adenocarcinomas. A computer analysis of the amino acid sequences of DRA revealed varying homology with a soybean nodulin clone (GMAKI70), Neospora crassa sulphate permease II (Cys-14), and sulfate anion transporter-1 (sat-1) of rat hepatocytes. Both Cys-14 and sat-1 are known transporters of sulfate ions, therefore DRA was analyzed to determine if it functions as a sulfate transporter. The full coding sequence of the human DRA cDNA was cloned and studied in a Xenopus laevis oocyte expression system. RNA derived from in vitro transcription of DRA was injected into Xenopus oocytes and sulfate transport activity was assayed. Na +- independent [35S] sulfate uptake into RNA injected oocytes was significantly greater than uptake in waterinjected oocytes. Sulfate uptake was sensitive to the anion exchange inhibitor 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) and oxalate, exhibiting a cis-inhihition pattern similar to sat-1. Oxalate appears to be a substrate for hepatic, intestinal and renal sulfate transporters; therefore, oxalate transport was also examined. Na÷-independent [14C] oxalate uptake into RNA-injected oocytes was significantly greater than uptake in water-injected oocytes. These functional studies demonstrated that DRA does encode for an intestine-specific sodium independent sulfate transporter with similarities to the sat-1 liver transporter. DRA was originally analyzed because of its loss of expression in colonic neoplasms. Knowing the function of DRA, it remains unknown how the loss of a sulfate transporter protein would be important in the development of malignant ceils. It is possible that abnormal synthesis of sulfated proteoglycans or mucins may affect the growth of intestinal cells. Further investigation is necessary to determine if the loss of DRA is involved in the development of the malignant phenotype or simply a lost function that is inconsequential to the tumor cell.
MONOCLONAL AND POLYCLONAL ANTIBODIES TO RECOMBINANT HUMAN XANTHINE OXIDASE" DEVELOPMENT OF AN ELISA ASSAY. K.A. Skinner =, H.B. Skinner 3, and D.A. Parks l'z. Depts. of Anesthesiology 1 and Physiology 2, University of Alabama at Birmingham, Birmingham, AL and Dept. of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX Introduction: Reactive oxygen species generated from the enzyme xanthine oxidase (XO) may contribute to such diverse pathologies as ischemia-reperfusion injury, sepsis, thermal injury, acute viral infection and hemorrhagic shock. Traditional assays for determination of XO require that ×O be enzymatically active; however, the reactive oxygen products can inactivate the enzyme and lead to an underestimate of total XO. Also the demonstration that antibodies raised against purified XO may cross react with IgG necessitates reexamination of previous data by using antibodies directed against a recombinant XO. The purpose of these studies was to: 1) develop monoclonal and polyclonal antibodies against a recombinant human XO; and 2) determine the specificity of the XO antibody; and 3) develop an ELISA assay for determination of XO in biologic fluids;, Methods: A region of the human XO coding sequence was amplified by polymerase chain reaction (PCR) and the product cloned into an expression vector. The XO fusion protein was expressed, purified to near homogeneity and injected into mice and rabbits for production of monoclonal and polyclonal antibodies. Results/Conclusions: The antibodies recognized XO in plasma and milk of normal healthy volunteers. In addition, XO was recognized in the preservation solution obtained during a human liver just prior to transplant. Neither human nor bovine IgG was recognized by either the monoclonal or the polyclonal antibodies. This novel ELISA assay system specifically detects XO and should eliminate many of the problems associated with traditional assays for quantification of XO in the plasma.
Intestinal Disorders
A325
AN APICAL P E R M E A B I L I T Y B A R R I E R T O NH3 IN ISOLATED, PERFUSED COLONIC CRYPTS. SK Sinah. JP Geibel, HJ Binder, WF Boron. Depts. of Medicine and C&M Physiology, Yale School of Medicine, New Haven, CT 06510 Ammonia is a weak base that exists at high concentration (15-20 mM) in the colon as a result of bacterial metabolic activity. It is well-accepted that NH3 crosses membranes by nonionic diffusion, typically causing rapid intracellular alkalinization. To determine whether colonocytes are protected from chronic NH3 loading, we studied isolated crypts from rabbit proximal colon. Adapting methods from renal tubule microperfusion, we measured changes in intraceliular pH (pH i) to assess the NH3 permeability of the apical (lumen) and basolateral (bath) membrane surfaces of crypts. Crypt cells were loaded with the pH-sensitive dye, BCECF. pH i was recorded in single ceils by digital processing of fluorescent images. Ultrastmcturally, the crypt preparation is a n intact epithelium composed of two polarized cell-types, goblet and vacuolated-nongoblet cells. Dye uptake was heterogeneous, with fluorescence signal predominating in vacuolated cells. At the conclusion of each experiment, perfusion was confirmed with luminal trypan blue. Crypts with <95% exclusion of trypan blue from the bath and lumen were disregarded. All experiments were performed at 37"C, pH 7.4 unless otherwise indicated. All solutions were normotonic, nominally HCO3-free, and buffered with 32 mM HEPES. Perfusing the lumen with 20 mM NH4C1 for 15 mins resulted in no change from resting pHi: 7.07 :i:0.02 to 7.06 :k-0.02, mean -+.SEM; A p H i = -0.0l 5-'0.01; N=66 cells, 7 crypts. In contrast, after luminal washout, the basolateral surface of the same crypt was exposed to 20 mM NH4CI, resulting in an immediate alkalinization: 7.07 -L-0.02to 7.30 _+0.02; ApH~ +0.21 !O.01. Additionally, perfusing the lumen with 20 mM NH,C1 at pH 8.0, which has an [NH3] 4x higher than at pH 7.4, also resulted in no change from baseline pH i, To test if mucus is a component of the barrier to NH3, we cleared the lumen of visible mucus by perfusing with 2 mM DTI'; subsequently, 20 mM NH4CI did not produce alkalinization from the lumen. Finally, luminal perfusion with 100 mM NH4CI at pH 8.0 did not increase resting PHi: April= -0.0l _40.01, N= 54 cells, 4 crypts. Subsequent exposure of the same crypts to 4 mM NH4C1 at pH 7.4 from the bath consistently produced a rapid alkalinization: ApH F +0.08 -'20.01. Given that [NH3] was 100-fold greater at the luminal vs. the basolateral surface of the crypt, we conclude that the permeability-areaproduct for NH3 at the basolateral membrane is substantially greater than that for the apical barrier. As rapid diffusion of NH3 across apical membranes would be expected to increase pH i , possibly provoking cellular injury, we speculate that the apical barrier to the diffusion of NH3 in crypts reflects a cellular adaptation to the unique symbiotic environment of the colon.
PROSTAGLANDIN E= (PG) MITIGATES MICROTUBULE DAMAGE IN CULTURED RAT ENTEROCYTES (IEC). G.S. Smith*t, W.B. VanWinkle*, M.B. Snuggs*, R.J. Bick*, L.M. Buja*, and T.A. MillerJ'. Departments of Surgeryt and Pathology*, University of Texas Medical School, Houston, Texas. Prostaglandin E= (PG) pretreatment has been shown to confer significant protection to rat gastrointestinal epithelia exposed to high concentrations of ethanol (EtOH) in vivo. The ability of PG to provide this protection under in vitro conditions remains controversial and was the basis for this study. Rat IEC were incubated in Dulbecco's Modified Eagle Media (DMEM) _+ PG (2.6 ~M) for 15 minutes. 5% EtOH was added to fresh media and the cells were incubated for an additional 5 minutes, and fixed for 5 minutes in 80 mM PIPES, 5 mM EGTA, 1 mM MgCI=, 1% Triton X-100, 0.25% glutaraldehyde, and 3.7% pareformaldehyde (pH 6.8). Nonspecific blocking was attained by incubating the cells for 1 hour with 10% goat serum at 37°C. Subsequently, IEC were incubated with a primary antibody against stable microtubules for 30 minutes at 37°CI Microtubules were then examined for cell damage by confocal laser microscopy. 100 cells were counted for each group (n=3) and microtubule integrity was assessed and expressed as a percentage of cells appearing normal or damaged, regarding microtubule disruption. Ethanol treated control cells showed that 21.0 + 7% remained normal (80 _+8% damaged) while PG treated cells showed 63.0 _+_3% normal, 37 + 3% damaged. The majority of control cells exposed to EtOH demonstrated obvious microtubule disruption, visible as extensive kinking and dissociation from anchoring proteins. In contrast, only a minority of PG treated cells exposed to EtOH exhibited a similar pattern of injury. This study demonstrates therefore that PG significantly protects IEC from damage by EtOH and suggests that a major site of this protection is at the level of cytoplasmic microtubule. (Supported by N/H Grant DK25838)
• THE DOWN REGULATED IN ADENOMA GENE (DRA) ENCODES AN INTESTINE-SPECIFIC MEMBRANE SULFATE TRANSPORT PROTEIN D.G. Silberg*, W Wang #, R. Moseley#, and P.G. Traber*. Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA*; Division of Gastroenterology, VAMC and University of Michigan, Ann Arbor, MI#. The gene, Down Regulated in Adenoma (DRA), is expressed i n normal colon and small intestine, but is absent in the majority of colon adenomas and adenocarcinomas. A computer analysis of the amino acid sequences of DRA revealed varying homology with a soybean nodulin clone (GMAKI70), Neospora crassa sulphate permease II (Cys-14), and sulfate anion transporter-1 (sat-1) of rat hepatocytes. Both Cys-14 and sat-1 are known transporters of sulfate ions, therefore DRA was analyzed to determine if it functions as a sulfate transporter. The full coding sequence of the human DRA cDNA was cloned and studied in a Xenopus laevis oocyte expression system. RNA derived from in vitro transcription of DRA was injected into Xenopus oocytes and sulfate transport activity was assayed. Na +- independent [35S] sulfate uptake into RNA injected oocytes was significantly greater than uptake in waterinjected oocytes. Sulfate uptake was sensitive to the anion exchange inhibitor 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) and oxalate, exhibiting a cis-inhihition pattern similar to sat-1. Oxalate appears to be a substrate for hepatic, intestinal and renal sulfate transporters; therefore, oxalate transport was also examined. Na÷-independent [14C] oxalate uptake into RNA-injected oocytes was significantly greater than uptake in water-injected oocytes. These functional studies demonstrated that DRA does encode for an intestine-specific sodium independent sulfate transporter with similarities to the sat-1 liver transporter. DRA was originally analyzed because of its loss of expression in colonic neoplasms. Knowing the function of DRA, it remains unknown how the loss of a sulfate transporter protein would be important in the development of malignant ceils. It is possible that abnormal synthesis of sulfated proteoglycans or mucins may affect the growth of intestinal cells. Further investigation is necessary to determine if the loss of DRA is involved in the development of the malignant phenotype or simply a lost function that is inconsequential to the tumor cell.
MONOCLONAL AND POLYCLONAL ANTIBODIES TO RECOMBINANT HUMAN XANTHINE OXIDASE" DEVELOPMENT OF AN ELISA ASSAY. K.A. Skinner =, H.B. Skinner 3, and D.A. Parks l'z. Depts. of Anesthesiology 1 and Physiology 2, University of Alabama at Birmingham, Birmingham, AL and Dept. of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX Introduction: Reactive oxygen species generated from the enzyme xanthine oxidase (XO) may contribute to such diverse pathologies as ischemia-reperfusion injury, sepsis, thermal injury, acute viral infection and hemorrhagic shock. Traditional assays for determination of XO require that ×O be enzymatically active; however, the reactive oxygen products can inactivate the enzyme and lead to an underestimate of total XO. Also the demonstration that antibodies raised against purified XO may cross react with IgG necessitates reexamination of previous data by using antibodies directed against a recombinant XO. The purpose of these studies was to: 1) develop monoclonal and polyclonal antibodies against a recombinant human XO; and 2) determine the specificity of the XO antibody; and 3) develop an ELISA assay for determination of XO in biologic fluids;, Methods: A region of the human XO coding sequence was amplified by polymerase chain reaction (PCR) and the product cloned into an expression vector. The XO fusion protein was expressed, purified to near homogeneity and injected into mice and rabbits for production of monoclonal and polyclonal antibodies. Results/Conclusions: The antibodies recognized XO in plasma and milk of normal healthy volunteers. In addition, XO was recognized in the preservation solution obtained during a human liver just prior to transplant. Neither human nor bovine IgG was recognized by either the monoclonal or the polyclonal antibodies. This novel ELISA assay system specifically detects XO and should eliminate many of the problems associated with traditional assays for quantification of XO in the plasma.
Intestinal Disorders
A325
AN APICAL P E R M E A B I L I T Y B A R R I E R T O NH3 IN ISOLATED, PERFUSED COLONIC CRYPTS. SK Sinah. JP Geibel, HJ Binder, WF Boron. Depts. of Medicine and C&M Physiology, Yale School of Medicine, New Haven, CT 06510 Ammonia is a weak base that exists at high concentration (15-20 mM) in the colon as a result of bacterial metabolic activity. It is well-accepted that NH3 crosses membranes by nonionic diffusion, typically causing rapid intracellular alkalinization. To determine whether colonocytes are protected from chronic NH3 loading, we studied isolated crypts from rabbit proximal colon. Adapting methods from renal tubule microperfusion, we measured changes in intraceliular pH (pH i) to assess the NH3 permeability of the apical (lumen) and basolateral (bath) membrane surfaces of crypts. Crypt cells were loaded with the pH-sensitive dye, BCECF. pH i was recorded in single ceils by digital processing of fluorescent images. Ultrastmcturally, the crypt preparation is a n intact epithelium composed of two polarized cell-types, goblet and vacuolated-nongoblet cells. Dye uptake was heterogeneous, with fluorescence signal predominating in vacuolated cells. At the conclusion of each experiment, perfusion was confirmed with luminal trypan blue. Crypts with <95% exclusion of trypan blue from the bath and lumen were disregarded. All experiments were performed at 37"C, pH 7.4 unless otherwise indicated. All solutions were normotonic, nominally HCO3-free, and buffered with 32 mM HEPES. Perfusing the lumen with 20 mM NH4C1 for 15 mins resulted in no change from resting pHi: 7.07 :i:0.02 to 7.06 :k-0.02, mean -+.SEM; A p H i = -0.0l 5-'0.01; N=66 cells, 7 crypts. In contrast, after luminal washout, the basolateral surface of the same crypt was exposed to 20 mM NH4CI, resulting in an immediate alkalinization: 7.07 -L-0.02to 7.30 _+0.02; ApH~ +0.21 !O.01. Additionally, perfusing the lumen with 20 mM NH,C1 at pH 8.0, which has an [NH3] 4x higher than at pH 7.4, also resulted in no change from baseline pH i, To test if mucus is a component of the barrier to NH3, we cleared the lumen of visible mucus by perfusing with 2 mM DTI'; subsequently, 20 mM NH4CI did not produce alkalinization from the lumen. Finally, luminal perfusion with 100 mM NH4CI at pH 8.0 did not increase resting PHi: April= -0.0l _40.01, N= 54 cells, 4 crypts. Subsequent exposure of the same crypts to 4 mM NH4C1 at pH 7.4 from the bath consistently produced a rapid alkalinization: ApH F +0.08 -'20.01. Given that [NH3] was 100-fold greater at the luminal vs. the basolateral surface of the crypt, we conclude that the permeability-areaproduct for NH3 at the basolateral membrane is substantially greater than that for the apical barrier. As rapid diffusion of NH3 across apical membranes would be expected to increase pH i , possibly provoking cellular injury, we speculate that the apical barrier to the diffusion of NH3 in crypts reflects a cellular adaptation to the unique symbiotic environment of the colon.
PROSTAGLANDIN E= (PG) MITIGATES MICROTUBULE DAMAGE IN CULTURED RAT ENTEROCYTES (IEC). G.S. Smith*t, W.B. VanWinkle*, M.B. Snuggs*, R.J. Bick*, L.M. Buja*, and T.A. MillerJ'. Departments of Surgeryt and Pathology*, University of Texas Medical School, Houston, Texas. Prostaglandin E= (PG) pretreatment has been shown to confer significant protection to rat gastrointestinal epithelia exposed to high concentrations of ethanol (EtOH) in vivo. The ability of PG to provide this protection under in vitro conditions remains controversial and was the basis for this study. Rat IEC were incubated in Dulbecco's Modified Eagle Media (DMEM) _+ PG (2.6 ~M) for 15 minutes. 5% EtOH was added to fresh media and the cells were incubated for an additional 5 minutes, and fixed for 5 minutes in 80 mM PIPES, 5 mM EGTA, 1 mM MgCI=, 1% Triton X-100, 0.25% glutaraldehyde, and 3.7% pareformaldehyde (pH 6.8). Nonspecific blocking was attained by incubating the cells for 1 hour with 10% goat serum at 37°C. Subsequently, IEC were incubated with a primary antibody against stable microtubules for 30 minutes at 37°CI Microtubules were then examined for cell damage by confocal laser microscopy. 100 cells were counted for each group (n=3) and microtubule integrity was assessed and expressed as a percentage of cells appearing normal or damaged, regarding microtubule disruption. Ethanol treated control cells showed that 21.0 + 7% remained normal (80 _+8% damaged) while PG treated cells showed 63.0 _+_3% normal, 37 + 3% damaged. The majority of control cells exposed to EtOH demonstrated obvious microtubule disruption, visible as extensive kinking and dissociation from anchoring proteins. In contrast, only a minority of PG treated cells exposed to EtOH exhibited a similar pattern of injury. This study demonstrates therefore that PG significantly protects IEC from damage by EtOH and suggests that a major site of this protection is at the level of cytoplasmic microtubule. (Supported by N/H Grant DK25838)