Analysis of absorption-enhancing mechanisms for combinatorial use of spermine with sodium taurocholate in Caco-2 cells

Previously, we reported that the combined use of spermine (SPM) and sodium taurocholate (STC) (SPM–STC) significantly improves the oral absorption of rebamipide (BCS class IV) and pulmonary absorption of interferon-α without any harmful histopathological changes in the gastrointestinal tract and lungs, respectively. In the present study, we examined the effect of SPM–STC on the transport of fluorescein isothiocyanate-labeled dextrans (FDs) across Caco-2 cell monolayers and attempted to clarify the mechanisms underlying the transport enhancement caused by SPM–STC. SPM–STC were found to significantly enhance the transport of FDs, while the treatment with SPM–STC was not harmful, and the decrease in transepithelial electrical resistance was transient and reversible. The voltage-clamp study clearly indicated that the opening of the paracellular route could be mainly responsible for the enhanced transport of FD-4. As for the mechanisms, it was found that SPM–STC caused a significant increase in membrane fluidity, which would lead to the enhanced transport of small-molecule drugs such as rebamipide. Since SPM–STC increased intracellular Ca²⁺ via Ca²⁺ uptake through Ca²⁺ channels and Ca²⁺ release from the endoplasmic reticulum stimulated by the IP₃ pathway, the subsequent possible activation of the MLCK signaling pathway would have led to the contraction of the actin–myosin ring. The rearrangement of tight junction-constituting proteins induced through the MAPK pathway has also been suggested as a possible mechanism for opening tight junctions. Claudin-4, a key protein constituting the tight junction, merged with F-actin along with the plasma membrane, was significantly decreased, which would be at least partial structural evidence for the tight-junction opening.