MECHANISTIC ROLES OF NEUTRAL SURFACTANTS ON CONCURRENT POLARIZED AND PASSIVE MEMBRANE-TRANSPORT OF A MODEL PEPTIDE IN CACO-2 CELLS

The transport of the model peptide Acf(NMef)(2)NH2 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 i n increasing AP-->BL peptide permeability and decreasing BL-->AP perme ability. 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 concentratio ns. These plateau permeabilities were not equivalent in the two direct ions. 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 c ontext of a quantitative biophysical model incorporating concurrent pa ssive diffusion across the AP and BL membranes accompanied by surfacta nt-inhibitable active polarized efflux across the AP membrane. The mod el 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)(2)NH2 was found to be 13 x 10(-6) cm/s, essentially identical with results repor ted earlier with this peptide utilizing verapamil as an inhibitor. Wit h respect to the mechanism of surfactant inhibition of the apical effl ux tranport, the monomeric species was found to be responsible with no contribution from micelles. Modeling the mode of inhibition as a nonc ompetitive Michaelis-Menten process gave identical K-i's of 0.5 mu M f or the two surfactants. Finally, increase of either surfactant beyond 750 mu M resulted in a decrease of peptide permeability in the AP-->BL direction. This was attributed to weak association of the peptide wit h micelles in the AP compartment, which effectively decreased the ther modynamic activity of the peptide at surfactant concentrations greater than 20 times their cmc. Both the experimental approach and accompany ing theoretical model demonstrated in this work will allow for further characterization of the inhibitory potencies of surfactants for the n onpassive efflux pathway in vitro and in vivo.