title: Development of stably transfected monolayer overexpressing the human apical sodium-dependent bile acid transporter (hASBT).
abstract: OBJECTIVE
  The human apical sodium-dependent bile acid transporter (hASBT) represents a potential target for prodrug design to increase oral drug absorption. Unfortunately, available monolayer cell culture models do not reliably express hASBT, and nonpolarized cells only allow for uptake assessment, which limits prodrug development efforts. The objective of this study was to develop and characterize a stably transfected hASBT-MDCK cell line.
  
  
  METHODS
  cDNA encoding hASBT was cloned into pcDNA3.1-V5-polyHis-B to generate an expression plasmid that was then transfected into MDCK-II cells. Clonal populations were chosen based on high hASBT activity and monolayer integrity. Western blot confirmed the expression of the recombinant hASBT; functionality was characterized using taurocholic acid.
  
  
  RESULTS
  In the selected clone, hASBT-mediated taurocholate permeability across hASBT-MDCK monolayers was almost 25-fold higher with sodium, than without sodium where hASBT is not functional. In the presence of sodium, taurocholate and mannitol permeabilities were 23.0x10(-6) cm/sec and 2.60x10(-6) cm/s, respectively, indicating high hASBT functionality and monolayer integrity. hASBT-MDCK monolayer properties were stable over 6 months and demonstrated low within-day variability. Taurocholate uptake and inhibition kinetic parameters from hASBT-MDCK were similar to those obtained from hASBT-COS7 model, confirming hASBT functionality in hASBT-MDCK.
  
  
  CONCLUSIONS
  Results indicate that the developed hASBT-MDCK system is a competent, high-expression, stable assay for hASBT transport and inhibition studies.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland 21201, USA.
authors.forename: Anand
authors.initials: A
authors.lastname: Balakrishnan
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Sussman
authors.forename: James E
authors.initials: JE
authors.lastname: Polli
mesh_terms: D000818:Animals; D001693:Biological Transport, Active; D015153:Blotting, Western; D002478:Cells, Cultured; D002635:Chenodeoxycholic Acid / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008353:Mannitol / Q000494:pharmacology; D029363:Organic Anion Transporters, Sodium-Dependent / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics*; D012964:Sodium / Q000378:metabolism; D027981:Symporters / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics*; D013656:Taurocholic Acid / Q000378:metabolism; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D029363:Organic Anion Transporters, Sodium-Dependent; D027981:Symporters; D002635:Chenodeoxycholic Acid; C071908:sodium-bile acid cotransporter; D008353:Mannitol; D013656:Taurocholic Acid; D012964:Sodium