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Enterocyte integrin expression varies with growth
A1016
AGA ABSTRACTS
GASTROENTEROLOGY, Vol. 108, No. 4
• THE LIVER IN TRANSFORMING GROWTH FACTOR-J~I (TGF-131) NULl MICE. A.Olufemi Williams, Alan D. Knapton, *A. G.. Geiser, *J. J Letterio, *Anita B. Roberts. Lab of Experimental Pathology and *Lab ol Chemoprevention, Division of Cancer Etiology, National Cancer Institute, Bethesda Maryland, 20892. Deletion of TGF-131 gene in mice, provides a model for the study of this polypeptide known for its control of cell growth and differentiation, The localization of TGF-131 in the mitochondria of rat liver has been reported (Heine et a1.1991) but the significance of this observation is not known. This report describes the fine structure of liver and cultures of hepatocytes derived from livers of homozygote TGF-p 1 gene knockout (null) mice (young < 21days; old > 21days) and control mice. Light, transmission electronmicroscopic(TEM) and immunohistochemical techniques were used for the studies, in old knockout mice (>21days), hepatocytes had non-communicating intracytoplasmic lumina, on TEM, adjacent to centrioles; these were not seen in control mice. Hypertrophy of Golgi complex was seen more frequently in hepatocytes of older knockout mice (50%) than in the hepatocytes of younger knockout mice (40%) and normal mice (10%). The number of mitocbondria in each bepatocyte was increased in the older knockout mice compared with those in young knockout and normal mice (p > 0.001 }. TGF-131 protein was immunolocalized in the livers of young knockout mice{< 21 days), consistent with maternal transfer of TGF-I~I protein(Letterio et a1.1994}. Hepatocyte cultures, derived from livers of old and young TGF-I~I knockout mice, also revealed intracytoplasmic lumina, dilated Golgi vesicles with inspissated, osmiophilic material but no increase in number of mitochondria. Following the addition of exogenous TGF-131 (12.5ng/ml), the number of intracytoplasmic vacuoles was reduced, from an average of 52 to 2 vacuoles in 4 hours, Secretory products, not: seen before TGF-131 treatment, became visible. Antibody to TGF-131 blocked the TGF-131induced reduction of vacuoles. This suggests that exogenous TGF-~I may be responsible for the release of constitutively secreted products. Non-communicating intrecytoplasmic lumina have been described in benign and malignant lesions, including the liver, but this is the first description in cells lacking TGF-I31. We suggest that this molecule plays a significant role in the pathways that regulate the number of hepatic mitochondria and also in the secretory function of the Golgi apparatus. The functional significance of these subcellular changes is not understood.
• IMMUNOHISTOCHEMICAL LOCALIZATION OF SOMATOSTATIN RECEPTORS 2, 3, AND 5 IN THE RAT GUT. HC Won~, D Su, C Stemini, J Reeves, JH Walsh. CURE/Gastroenteric Biology Center, Department of Medicine, University of California, Los Angeles. Several somatostatin receptor subtypes have recently been cloned. Immnnospeeifie polyelonal antibodies to these receptors ha~,e been made using synthetic peptides of selected regions as the antigen. Each peptide was conjugatad to keyholelimpet haemocyaninnsing bisdiazotized benzidine reagent, which couples through tyrosine and histidine residues. Rabbits were immunized and boosted at least two times before use. Antibody activitieswere first screened by ELISA using its corresponding peptide fragment. The antibodies were affinity purified using peptide fragments conjugated to EAH sepharose. Affinity purified IgG was dialyzed and used for immunocytochemical studies in rat tissues. Antibodies used in this study were: Snecificitv SSRec2(361-369) SSRec2(331-340) SSRee3(413-42g) SSRec5(I-11)
Amino Acid Seeuences (d)YLNGDLQTSI (d)YKVSGAEDGER (d)YPQEATAGDKASTLSHL MEPLSLASTPSY
Antibodv# #9431 #9452 #9472 #9462
Results:
Both anti-SSRec2 antibodies(9431 & 9452) stained enteric neurons and processes of the stomach and intestine. In addition to the enteric plexuses, immunoreactive fibers were distributed to the circular muscle, vasculature and mucosa. Antibody #9452 also stained mueosal epithelial cells in the stomach. A weak staining was observed in the enteric plexuses with both anti-SSRec3(#9472) and antiSSRec5(#9462) antibodies. These studies reveal that the stomach is innervated by nerves that express somatostatin receptorsubtypes 2, 3, and 5. One SSTR2 receptor antibody also stained mucosal epithelial ceils in the stomach, but precise identificationof these cells has not been made. The specific localization of somatostatin subtypes in the gut presumably determines biological responses to subtype specific agonists.
ENTEROCYTE INTEGRIN E X P R E S S I O N VARIES WITH GROWTH. S. Wolpert, M. Wong, and B.L. Bass. Dept of Surgery, Baltimore VAMC and U n i v e r s i t y of Maryland, Baltimore, Maryland. Integrins are h e t e r o d i m e r i c transmembrane proteins that serve both as anchors to the e x t r a c e l l u l a r m a t r i x and as receptors. Enterocyte integrin e x p r e s s i o n varies along the villus axis, a process p o t e n t i a l l y m e d i a t e d by components of the basement membrane. If integrin e x p r e s s i o n is dependent on epithelial cell-matrix interactions, then enterocytes grown on a single matrix should m a i n t a i n stable integrin sub-populations. To test this hypothesis, ~-i, ~-2, and ~-i integrins were evaluated using i m m u n o h i s t o c h e m i s t r y at different points r e l a t i v e to confluence, a transition point between p r o l i f e r a t i o n and quiescence. Caco-2 cells, a human colon cancer cell line that spontaneously differentiates in culture; and IEC-6 cells, a rat enterocyte that remains undifferentiated, were grown on plastic chamberslides, fixed in-sftu for immunohistochemistry and stained for in~egrin at several points s u r r o u n d i n g confluence. In Cacc-2 cells, ~-i and ~-2 integrins were present at confluence (seen as a thick fluorescent rim staining the cell membrane), but absent at days 7, and out to 21 postconfluence. ~-I integrin was present a t p r e - c o n f l u e n c e in the Caco-2 cells and remained present to 21 days post-confluence. In IEC-6 cells, ~-I and ~-2 integrins were present at p r e - c o n f l u e n c e and out to 21 days post confluence. This data 'suggests that v a r i a t i o n in integrin e x p r e s s i o n is i) not e n t i r e l y dependent on specific e p i t h e l i a l - m a t r i x i n t e r a c t i o n and 2) undergoes significant change coincident With a change in d i f f e r e n t i a t i o n rather than p r o l i f e r a t i v e state. The v a r i a b i l i t y of ~ integrin e x p r e s s i o n suggests unique roles for these species at specific times during cell proliferation and/or differentiation, whereas ~-i appears to play a more constitutive role. This data suggests that w i t h i n these integrin sub-populations, e x p r e s s i o n is at least p a r t l y independent of basement membrane components and related to either intrinsic or paracrine factors.
• SPECIFIC HIGH- AND LOW-AFFINITY BINDING SITES FOR ENDOTHELIN-I IN GUINEA-PIG GALLBLADDER MEMBRANES. M. Woods, B. Battistini, T.D. Warner, L.J.D. O'Donnell,. M.J.G. Farthing and J.R. Vane. TheWilliam Harvey Research lnstimte and Dept. of Gastroenterology, St.BarthMomew's Hospital Medical College, CWarterhouseSquare, London, ECIM 6BQ, U.K. EllldMayo General Hospital, Department of Medici,e¢ Casllebar, Mayo, IREIAND.
Endothelin isopeptides (ET-1, -2, -3) and sarafotoxin 6e (SX6e) are potent contractors of the guinea-pig isolated gallbladder. Based on the relative, potencies of ET agonists and the effects of several E l ' receptor antagonists, we have reported that two receptors, an ETB and an additional uneharacterised receptor, mediate these contractions. Here, we have characterized the binding of ET to guinea-pig gallbladder membranes. Guinea-pigs (250-350 g) were killed by cervical dislocation and the gallbladder rapidly excised, trimmed free of connective tissue and fat, minced to small pieces and homogenized for 10 rain in 10 volumes of icecold assay buffer. The homogenate was centrifuged for 15 rain at ,l 500 g (4°C), the supernatant collected and centrifuged for 60 min at 100 000g (4°C). The pellet formed was resuspended in 3 ml of ice-cold assay buffer (as above) and its protein content determined. The membranes (20 ug of protein) were incubated in binding buffer with 125I-ET-] (2000 Ci/mmol) in the presence of 10-15 M to 10-6 M ET-I, ET-3 or SX6e. After 240 min at room temperature, the binding was stopped by filtering the incubate through a Whatman glass filter followed by washing with 3 x 4 ml of ice-cold binding buffer. The amount of radioactivity present on the filter was measured in a gamma counter for 60 sec. In competition binding studies using membranes prepared from the guinea-pig gallbladder, I0 -11 M and 10 6 M ET-I inhibited by 76.9_+3.1 and 95.7_+1.1%, respectively, the binding of [12511-ET=I (n=3). The displacement of 1251-ET-1 by ET-I was biphasie. A very high-affinity site (1C50:35 fM) and a high affinity site (IC50:0.18 nM) were observed, They represented 75% and 25%, respectively, of the total population of ET receptors. Using ET-3 as the cold Iigand, a biphasic displacement was also observed (IC5o: 0.10 nM and 70 nM, each representing about 50% of total binding). Using SX6c, a monophasic curve was observed with one site of low affinity (IC5o: >70 riM). This study shows that ET agonists in the guinea-pig gallbladder act through at least two high affinity sites and one lower affinity site. This supports our previous reports showing the existence of at least two receptors in this tissue. Further Studies will be conducted to determine the exact nature of these receptors. This work was supported in part by the Parke-Davis Pharmaceutical,a Division of Warner-Lambert Co (Ann Arbor, MI, USA). B.B. is a Fellowof the MedicalResearchCouncilof Canada.
AGA ABSTRACTS
GASTROENTEROLOGY, Vol. 108, No. 4
• THE LIVER IN TRANSFORMING GROWTH FACTOR-J~I (TGF-131) NULl MICE. A.Olufemi Williams, Alan D. Knapton, *A. G.. Geiser, *J. J Letterio, *Anita B. Roberts. Lab of Experimental Pathology and *Lab ol Chemoprevention, Division of Cancer Etiology, National Cancer Institute, Bethesda Maryland, 20892. Deletion of TGF-131 gene in mice, provides a model for the study of this polypeptide known for its control of cell growth and differentiation, The localization of TGF-131 in the mitochondria of rat liver has been reported (Heine et a1.1991) but the significance of this observation is not known. This report describes the fine structure of liver and cultures of hepatocytes derived from livers of homozygote TGF-p 1 gene knockout (null) mice (young < 21days; old > 21days) and control mice. Light, transmission electronmicroscopic(TEM) and immunohistochemical techniques were used for the studies, in old knockout mice (>21days), hepatocytes had non-communicating intracytoplasmic lumina, on TEM, adjacent to centrioles; these were not seen in control mice. Hypertrophy of Golgi complex was seen more frequently in hepatocytes of older knockout mice (50%) than in the hepatocytes of younger knockout mice (40%) and normal mice (10%). The number of mitocbondria in each bepatocyte was increased in the older knockout mice compared with those in young knockout and normal mice (p > 0.001 }. TGF-131 protein was immunolocalized in the livers of young knockout mice{< 21 days), consistent with maternal transfer of TGF-I~I protein(Letterio et a1.1994}. Hepatocyte cultures, derived from livers of old and young TGF-I~I knockout mice, also revealed intracytoplasmic lumina, dilated Golgi vesicles with inspissated, osmiophilic material but no increase in number of mitochondria. Following the addition of exogenous TGF-131 (12.5ng/ml), the number of intracytoplasmic vacuoles was reduced, from an average of 52 to 2 vacuoles in 4 hours, Secretory products, not: seen before TGF-131 treatment, became visible. Antibody to TGF-131 blocked the TGF-131induced reduction of vacuoles. This suggests that exogenous TGF-~I may be responsible for the release of constitutively secreted products. Non-communicating intrecytoplasmic lumina have been described in benign and malignant lesions, including the liver, but this is the first description in cells lacking TGF-I31. We suggest that this molecule plays a significant role in the pathways that regulate the number of hepatic mitochondria and also in the secretory function of the Golgi apparatus. The functional significance of these subcellular changes is not understood.
• IMMUNOHISTOCHEMICAL LOCALIZATION OF SOMATOSTATIN RECEPTORS 2, 3, AND 5 IN THE RAT GUT. HC Won~, D Su, C Stemini, J Reeves, JH Walsh. CURE/Gastroenteric Biology Center, Department of Medicine, University of California, Los Angeles. Several somatostatin receptor subtypes have recently been cloned. Immnnospeeifie polyelonal antibodies to these receptors ha~,e been made using synthetic peptides of selected regions as the antigen. Each peptide was conjugatad to keyholelimpet haemocyaninnsing bisdiazotized benzidine reagent, which couples through tyrosine and histidine residues. Rabbits were immunized and boosted at least two times before use. Antibody activitieswere first screened by ELISA using its corresponding peptide fragment. The antibodies were affinity purified using peptide fragments conjugated to EAH sepharose. Affinity purified IgG was dialyzed and used for immunocytochemical studies in rat tissues. Antibodies used in this study were: Snecificitv SSRec2(361-369) SSRec2(331-340) SSRee3(413-42g) SSRec5(I-11)
Amino Acid Seeuences (d)YLNGDLQTSI (d)YKVSGAEDGER (d)YPQEATAGDKASTLSHL MEPLSLASTPSY
Antibodv# #9431 #9452 #9472 #9462
Results:
Both anti-SSRec2 antibodies(9431 & 9452) stained enteric neurons and processes of the stomach and intestine. In addition to the enteric plexuses, immunoreactive fibers were distributed to the circular muscle, vasculature and mucosa. Antibody #9452 also stained mueosal epithelial cells in the stomach. A weak staining was observed in the enteric plexuses with both anti-SSRec3(#9472) and antiSSRec5(#9462) antibodies. These studies reveal that the stomach is innervated by nerves that express somatostatin receptorsubtypes 2, 3, and 5. One SSTR2 receptor antibody also stained mucosal epithelial ceils in the stomach, but precise identificationof these cells has not been made. The specific localization of somatostatin subtypes in the gut presumably determines biological responses to subtype specific agonists.
ENTEROCYTE INTEGRIN E X P R E S S I O N VARIES WITH GROWTH. S. Wolpert, M. Wong, and B.L. Bass. Dept of Surgery, Baltimore VAMC and U n i v e r s i t y of Maryland, Baltimore, Maryland. Integrins are h e t e r o d i m e r i c transmembrane proteins that serve both as anchors to the e x t r a c e l l u l a r m a t r i x and as receptors. Enterocyte integrin e x p r e s s i o n varies along the villus axis, a process p o t e n t i a l l y m e d i a t e d by components of the basement membrane. If integrin e x p r e s s i o n is dependent on epithelial cell-matrix interactions, then enterocytes grown on a single matrix should m a i n t a i n stable integrin sub-populations. To test this hypothesis, ~-i, ~-2, and ~-i integrins were evaluated using i m m u n o h i s t o c h e m i s t r y at different points r e l a t i v e to confluence, a transition point between p r o l i f e r a t i o n and quiescence. Caco-2 cells, a human colon cancer cell line that spontaneously differentiates in culture; and IEC-6 cells, a rat enterocyte that remains undifferentiated, were grown on plastic chamberslides, fixed in-sftu for immunohistochemistry and stained for in~egrin at several points s u r r o u n d i n g confluence. In Cacc-2 cells, ~-i and ~-2 integrins were present at confluence (seen as a thick fluorescent rim staining the cell membrane), but absent at days 7, and out to 21 postconfluence. ~-I integrin was present a t p r e - c o n f l u e n c e in the Caco-2 cells and remained present to 21 days post-confluence. In IEC-6 cells, ~-I and ~-2 integrins were present at p r e - c o n f l u e n c e and out to 21 days post confluence. This data 'suggests that v a r i a t i o n in integrin e x p r e s s i o n is i) not e n t i r e l y dependent on specific e p i t h e l i a l - m a t r i x i n t e r a c t i o n and 2) undergoes significant change coincident With a change in d i f f e r e n t i a t i o n rather than p r o l i f e r a t i v e state. The v a r i a b i l i t y of ~ integrin e x p r e s s i o n suggests unique roles for these species at specific times during cell proliferation and/or differentiation, whereas ~-i appears to play a more constitutive role. This data suggests that w i t h i n these integrin sub-populations, e x p r e s s i o n is at least p a r t l y independent of basement membrane components and related to either intrinsic or paracrine factors.
• SPECIFIC HIGH- AND LOW-AFFINITY BINDING SITES FOR ENDOTHELIN-I IN GUINEA-PIG GALLBLADDER MEMBRANES. M. Woods, B. Battistini, T.D. Warner, L.J.D. O'Donnell,. M.J.G. Farthing and J.R. Vane. TheWilliam Harvey Research lnstimte and Dept. of Gastroenterology, St.BarthMomew's Hospital Medical College, CWarterhouseSquare, London, ECIM 6BQ, U.K. EllldMayo General Hospital, Department of Medici,e¢ Casllebar, Mayo, IREIAND.
Endothelin isopeptides (ET-1, -2, -3) and sarafotoxin 6e (SX6e) are potent contractors of the guinea-pig isolated gallbladder. Based on the relative, potencies of ET agonists and the effects of several E l ' receptor antagonists, we have reported that two receptors, an ETB and an additional uneharacterised receptor, mediate these contractions. Here, we have characterized the binding of ET to guinea-pig gallbladder membranes. Guinea-pigs (250-350 g) were killed by cervical dislocation and the gallbladder rapidly excised, trimmed free of connective tissue and fat, minced to small pieces and homogenized for 10 rain in 10 volumes of icecold assay buffer. The homogenate was centrifuged for 15 rain at ,l 500 g (4°C), the supernatant collected and centrifuged for 60 min at 100 000g (4°C). The pellet formed was resuspended in 3 ml of ice-cold assay buffer (as above) and its protein content determined. The membranes (20 ug of protein) were incubated in binding buffer with 125I-ET-] (2000 Ci/mmol) in the presence of 10-15 M to 10-6 M ET-I, ET-3 or SX6e. After 240 min at room temperature, the binding was stopped by filtering the incubate through a Whatman glass filter followed by washing with 3 x 4 ml of ice-cold binding buffer. The amount of radioactivity present on the filter was measured in a gamma counter for 60 sec. In competition binding studies using membranes prepared from the guinea-pig gallbladder, I0 -11 M and 10 6 M ET-I inhibited by 76.9_+3.1 and 95.7_+1.1%, respectively, the binding of [12511-ET=I (n=3). The displacement of 1251-ET-1 by ET-I was biphasie. A very high-affinity site (1C50:35 fM) and a high affinity site (IC50:0.18 nM) were observed, They represented 75% and 25%, respectively, of the total population of ET receptors. Using ET-3 as the cold Iigand, a biphasic displacement was also observed (IC5o: 0.10 nM and 70 nM, each representing about 50% of total binding). Using SX6c, a monophasic curve was observed with one site of low affinity (IC5o: >70 riM). This study shows that ET agonists in the guinea-pig gallbladder act through at least two high affinity sites and one lower affinity site. This supports our previous reports showing the existence of at least two receptors in this tissue. Further Studies will be conducted to determine the exact nature of these receptors. This work was supported in part by the Parke-Davis Pharmaceutical,a Division of Warner-Lambert Co (Ann Arbor, MI, USA). B.B. is a Fellowof the MedicalResearchCouncilof Canada.