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Molecular and cellular approaches to epithelial differentiation in the intestinal mucosa
Cell Biology
CHOLINE TRANSPORT BY PLASMA MEMBRANE VESICLES FROM A549 CELLS Aron B Fisher, Chandra Dodia, Avinash Chander, Arnost Kleinzeller, Institute For Environmental of Physiology, University of Medicine & Dept. Pennsylvania School of Medicine, Philadelphia, PA 19104 USA. were Plasma membrane vesicles prepared by sucrose density gradient centrifugation from A549 cells, a human lung alveolar epithelial cell line. We have previously shown that the choline transport system of these cells is similar to that of granular pneumocytes, the secretory cell for lung surfactant (Am. J. Resp. Cell. Mol. Biol. 1:455, 1989). Vesicles showed ten-fold enrichment activity over the cell in 5'-nucleotidase and minimal contamination with NADPHhomogenate cyto c red or SDH. Uptake of 3%choline (5 uM) by vesicles suspended in 0.32 M sucrose-O.01 M HEPES0.01 M Tris (pH 7.4) was 504 + 10 pmol/mg prot in 90 set (mean + SE; n=5). Binding to disrupted vesicles represented 5 15% of total uptake. Uptake was time-dependent and at 30 min of incubation (5 UM choline) was 1600 pmol/mg protein, representing an internal concentration of a 62-fold accumulation ratio (in/out). 308 uM, The estimated kinetic constants for uptake were Km 26 uM and V,,, 1.89 nmol/mg prot per min. Choline uptake was inhibited approx. 50% in the presence of 10 uM hemicholinium-3 or dimethylethanolamine and 90% by 1 uM decylcholine. These results indicate the successful isolation of plasma membrane vesicles from lung epithelial cells that will permit study of the low-affinity choline transport system.
P665
P667
MOLECULAR AND CELLULAR APPROACHES TO EPITRELIAL DIFFRRENTIATION IN THE INTRSTINAL MUCOSA Ghuiiaa Pemzai, Olga Capasso. Da&la Bar& Giovanna Biaea, Fabio Nobili, Giulii Ranaldi, Yule Sambuy. Istituto Nazionale deBa Nu@i&me., Vii Ardeatim 546.00178 Roma,Italy. The functionally differentiated epithelium of the intestinal mucosa exerts a key role in the absorption of nuuients and in their vector&l transport to the circtdation. We have chosen the epithelium from rat intestineasamalelsystcmto~y~mechanismsimolvedinepltbelial dlff cnntiation.Our~includetheisolationandm~analysis of stage spccillc genes from intestinal cDNA libraries as well as the eseablishmuuofnormalin~epithelialcdlliacstobeusedeJanin vitro model of ditTerentiaIion. As a molecular approach we are using subtractive hybridization to isolategencstiarespe&callye~inthe@minallydiff=entiated state. We have mnsuucted a cDNA library in the bacmiopbage vector QtlO using poly(A)+ RNA extmcted from isolated villi of young m intestine. This library was smzened using a sub&acted poba that is highly enriched in sequences expessed in the differentiated epitheIium of the intestinal villi and not in the f-1 intesdne. ‘Ibe. clones lhat we have &olatal&omthisscreeningancmrentlyun&invesdgationtodetamine theiriisaespe&cityandUw.irn~aaquencc. As a cellular approach to the development of an in vitro model of epitheUeIdiffermtiadmwe.haveestablisbaddueeepimelial~IihesErom fetal rat intestines which exhibit morphological and functional characteristics suggestive of a priorly diffuendated, rapidly proBfa&g state. We have developed a battery of functional tests to assess tbe appearance of traits typical of the differentiated entezocyte. such as intestinal~~chydrolspeactivitie9,UIbecarricdoutonasmallnamber ofcells.usinglhisappror&wenteatblgondleducacelllinesvarious mnditicms known to affect epitbelid all cUtYe.
International
Reports,
P666
Vol. 14, Abstracts
LONG-TERM HJ%PATOCYTES
Supplement
1990
CULTIVATION OF HUMAN IN SERUM-FREE MEDIUM
E.Krasheninnikov, Research Center USSR Ministry
or or
The present study demonstrates an improved method for cultivation of fetal original and adult human liver cells in chemically defined argjnine-Free containing selective medium, lncry;y concentrations of some amino particular branched-chains ones, supple: mented with vitamins, insulin, epidermal hydrocortisone, rowth factor, glucagon, & ransferrin,thyrotro in-releasing factor, ethano Pamine,cholera toxin, somatotropin, microelements and linoleic acid. Fetal he atocytes were obtained from fetuses at 6 I: o 26 weeks df gestation and maintained in culture over a period of 3 months. The liver cells divided, formed a confluent monola er practically free from non-hepatic ce Y 1s and secreted alpha-fetoprotein The amount of and albumin.
hepatic resection, for 4 weeks, form;id a monolayer with morphologicalbiochemical of characteristics hepa:;Eytes and secreted albumin. Thus, culture suitable model for regulation 0r functions during effects, of various , carcinogenic and viral agents.
P666
EPITliELIALJ4ESENCHYMAL
INTERACITONS
AND THE GLUCOCORTWXD RESPONSE IN lNTESTINAL DEVELOPMENT. Aideen Moore, Emanuela Ferretti and Martin Post. Neonatal Research Division, M5G 1X8.
Hospital
for Sick Children,
Toronto,
Canada
Epithelial-mesenchymal interactions are important during development of the foregut derivatives and have also be-en shown to play a role in the glucocorticoid response which mediates final differentiation in many of these tlssuea. In the intact animal injection of hydrocortlsone to pre-weaning pups results in induction of the dlsac&a&ase enzymes (eg. sucrase and maltase) whicharenonnaUyfoundinmaturesmaUintestinalepitheiium. Sucrase and malw activity begins to appear 24hrs post injection and reaches maximum activity by 48hrs When this is ezamined at the RNA level we found that the mRNA for sucrase is maximally expressed 24hrs post injection. In organ culture experiments using 2&i fetal rat intestine sucrase activity is induced by dcxamethasone showing that the hormonal effect can be mediated locally. To examine the mntrlbution of epithelial and mescnchymal cells to the glucocortimid response we altured rat intestinal epithelial crypt cells (IEC 6 cells) on matrix deposited by fibroblasts or exposed to fibroblastconditioncd media (FCM). Sucrase and maltase activity was induced by 30% in the IEC 6 cells grown on matrix and there was a 2-3 fold induaion of sucfase and maltase activity in the cells exposed to 2Od fetal FCM. The mnditioaed media effect was seen with fibroblasts of intestinal or lung origin and was augmented by glummrdmids. l%is soggests that both soluble Eaaors and matrix mole&es produced by the mesenchyme can influence intestinal eplthallal dffferentiatlon. (Supported by MRC Qmada ahd Physicialls Servicea Incorporated.)
CHOLINE TRANSPORT BY PLASMA MEMBRANE VESICLES FROM A549 CELLS Aron B Fisher, Chandra Dodia, Avinash Chander, Arnost Kleinzeller, Institute For Environmental of Physiology, University of Medicine & Dept. Pennsylvania School of Medicine, Philadelphia, PA 19104 USA. were Plasma membrane vesicles prepared by sucrose density gradient centrifugation from A549 cells, a human lung alveolar epithelial cell line. We have previously shown that the choline transport system of these cells is similar to that of granular pneumocytes, the secretory cell for lung surfactant (Am. J. Resp. Cell. Mol. Biol. 1:455, 1989). Vesicles showed ten-fold enrichment activity over the cell in 5'-nucleotidase and minimal contamination with NADPHhomogenate cyto c red or SDH. Uptake of 3%choline (5 uM) by vesicles suspended in 0.32 M sucrose-O.01 M HEPES0.01 M Tris (pH 7.4) was 504 + 10 pmol/mg prot in 90 set (mean + SE; n=5). Binding to disrupted vesicles represented 5 15% of total uptake. Uptake was time-dependent and at 30 min of incubation (5 UM choline) was 1600 pmol/mg protein, representing an internal concentration of a 62-fold accumulation ratio (in/out). 308 uM, The estimated kinetic constants for uptake were Km 26 uM and V,,, 1.89 nmol/mg prot per min. Choline uptake was inhibited approx. 50% in the presence of 10 uM hemicholinium-3 or dimethylethanolamine and 90% by 1 uM decylcholine. These results indicate the successful isolation of plasma membrane vesicles from lung epithelial cells that will permit study of the low-affinity choline transport system.
P665
P667
MOLECULAR AND CELLULAR APPROACHES TO EPITRELIAL DIFFRRENTIATION IN THE INTRSTINAL MUCOSA Ghuiiaa Pemzai, Olga Capasso. Da&la Bar& Giovanna Biaea, Fabio Nobili, Giulii Ranaldi, Yule Sambuy. Istituto Nazionale deBa Nu@i&me., Vii Ardeatim 546.00178 Roma,Italy. The functionally differentiated epithelium of the intestinal mucosa exerts a key role in the absorption of nuuients and in their vector&l transport to the circtdation. We have chosen the epithelium from rat intestineasamalelsystcmto~y~mechanismsimolvedinepltbelial dlff cnntiation.Our~includetheisolationandm~analysis of stage spccillc genes from intestinal cDNA libraries as well as the eseablishmuuofnormalin~epithelialcdlliacstobeusedeJanin vitro model of ditTerentiaIion. As a molecular approach we are using subtractive hybridization to isolategencstiarespe&callye~inthe@minallydiff=entiated state. We have mnsuucted a cDNA library in the bacmiopbage vector QtlO using poly(A)+ RNA extmcted from isolated villi of young m intestine. This library was smzened using a sub&acted poba that is highly enriched in sequences expessed in the differentiated epitheIium of the intestinal villi and not in the f-1 intesdne. ‘Ibe. clones lhat we have &olatal&omthisscreeningancmrentlyun&invesdgationtodetamine theiriisaespe&cityandUw.irn~aaquencc. As a cellular approach to the development of an in vitro model of epitheUeIdiffermtiadmwe.haveestablisbaddueeepimelial~IihesErom fetal rat intestines which exhibit morphological and functional characteristics suggestive of a priorly diffuendated, rapidly proBfa&g state. We have developed a battery of functional tests to assess tbe appearance of traits typical of the differentiated entezocyte. such as intestinal~~chydrolspeactivitie9,UIbecarricdoutonasmallnamber ofcells.usinglhisappror&wenteatblgondleducacelllinesvarious mnditicms known to affect epitbelid all cUtYe.
International
Reports,
P666
Vol. 14, Abstracts
LONG-TERM HJ%PATOCYTES
Supplement
1990
CULTIVATION OF HUMAN IN SERUM-FREE MEDIUM
E.Krasheninnikov, Research Center USSR Ministry
or or
The present study demonstrates an improved method for cultivation of fetal original and adult human liver cells in chemically defined argjnine-Free containing selective medium, lncry;y concentrations of some amino particular branched-chains ones, supple: mented with vitamins, insulin, epidermal hydrocortisone, rowth factor, glucagon, & ransferrin,thyrotro in-releasing factor, ethano Pamine,cholera toxin, somatotropin, microelements and linoleic acid. Fetal he atocytes were obtained from fetuses at 6 I: o 26 weeks df gestation and maintained in culture over a period of 3 months. The liver cells divided, formed a confluent monola er practically free from non-hepatic ce Y 1s and secreted alpha-fetoprotein The amount of and albumin.
hepatic resection, for 4 weeks, form;id a monolayer with morphologicalbiochemical of characteristics hepa:;Eytes and secreted albumin. Thus, culture suitable model for regulation 0r functions during effects, of various , carcinogenic and viral agents.
P666
EPITliELIALJ4ESENCHYMAL
INTERACITONS
AND THE GLUCOCORTWXD RESPONSE IN lNTESTINAL DEVELOPMENT. Aideen Moore, Emanuela Ferretti and Martin Post. Neonatal Research Division, M5G 1X8.
Hospital
for Sick Children,
Toronto,
Canada
Epithelial-mesenchymal interactions are important during development of the foregut derivatives and have also be-en shown to play a role in the glucocorticoid response which mediates final differentiation in many of these tlssuea. In the intact animal injection of hydrocortlsone to pre-weaning pups results in induction of the dlsac&a&ase enzymes (eg. sucrase and maltase) whicharenonnaUyfoundinmaturesmaUintestinalepitheiium. Sucrase and malw activity begins to appear 24hrs post injection and reaches maximum activity by 48hrs When this is ezamined at the RNA level we found that the mRNA for sucrase is maximally expressed 24hrs post injection. In organ culture experiments using 2&i fetal rat intestine sucrase activity is induced by dcxamethasone showing that the hormonal effect can be mediated locally. To examine the mntrlbution of epithelial and mescnchymal cells to the glucocortimid response we altured rat intestinal epithelial crypt cells (IEC 6 cells) on matrix deposited by fibroblasts or exposed to fibroblastconditioncd media (FCM). Sucrase and maltase activity was induced by 30% in the IEC 6 cells grown on matrix and there was a 2-3 fold induaion of sucfase and maltase activity in the cells exposed to 2Od fetal FCM. The mnditioaed media effect was seen with fibroblasts of intestinal or lung origin and was augmented by glummrdmids. l%is soggests that both soluble Eaaors and matrix mole&es produced by the mesenchyme can influence intestinal eplthallal dffferentiatlon. (Supported by MRC Qmada ahd Physicialls Servicea Incorporated.)