title: Mechanistic roles of neutral surfactants on concurrent polarized and passive membrane transport of a model peptide in Caco-2 cells.
abstract: The transport of the model peptide Acf(NMef)2NH2 across Caco-2 cell monolayers was studied in the apical (AP) to basolateral (BL) and the BL to AP direction in the presence of Polysorbate 80 or Cremophore EL in the AP compartment. Increasing surfactant concentrations resulted in increasing AP-->BL peptide permeability and decreasing BL-->AP permeability. In either direction, limiting permeabilities were achieved at concentrations less than the critical micellar concentrations (cmc's) of the surfactants, and remained constant at much higher concentrations. These plateau permeabilities were not equivalent in the two directions. This residual assymetry was abolished by increasing the peptide concentration. Altogether, the observations support the presence of at least two pumps in Caco-2 cells for this peptide, polarized in the BL-->AP direction. These experimental results were analyzed within the context of a quantitative biophysical model incorporating concurrent passive diffusion across the AP and BL membranes accompanied by surfactant-inhibitable active polarized efflux across the AP membrane. The model was also used to locate the additional transport activity at the BL membrane as an uptake pump. Under conditions of complete inhibition, the intrinsic passive diffusional permeability of Acf(NMef)2NH2 was found to be 13 x 10(-6) cm/s, essentially identical with results reported earlier with this peptide utilizing verapamil as an inhibitor. With respect to the mechanism of surfactant inhibition of the apical efflux transport, the monomeric species was found to be responsible with no contribution from micelles. Modeling the mode of inhibition as a noncompetitive Michaelis-Menten process gave identical Kis of 0.5 microM for the two surfactants. Finally, increase of either surfactant beyond 750 microM resulted in a decrease of peptide permeability in the AP-->BL direction. This was attributed to weak association of the peptide with micelles in the AP compartment, which effectively decreased the thermodynamic activity of the peptide at surfactant concentrations greater than 20 times their cmc. Both the experimental approach and accompanying theoretical model demonstrated in this work will allow for further characterization of the inhibitory potencies of surfactants for the nonpassive efflux pathway in vitro and in vivo.
authors.affiliation: Department of Pharmaceutical Chemistry, University of Kansas, Lawrence 66047, USA.
authors.forename: M M
authors.initials: MM
authors.lastname: Nerurkar
authors.forename: N F
authors.initials: NF
authors.lastname: Ho
authors.forename: P S
authors.initials: PS
authors.lastname: Burton
authors.forename: T J
authors.initials: TJ
authors.lastname: Vidmar
authors.forename: R T
authors.initials: RT
authors.lastname: Borchardt
mesh_terms: D001692:Biological Transport / Q000187:drug effects; D001693:Biological Transport, Active / Q000187:drug effects; D018938:Caco-2 Cells; D004058:Diffusion; D005990:Glycerol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D008823:Micelles; D010455:Peptides / Q000378:metabolism*; D010539:Permeability; D011136:Polysorbates / Q000737:chemistry / Q000494:pharmacology*; D013500:Surface Tension; D013501:Surface-Active Agents / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D008823:Micelles; D010455:Peptides; D011136:Polysorbates; D013501:Surface-Active Agents; C000515:cremophor EL; D005990:Glycerol