Localization and Differential Activity of P-glycoprotein in the Bovine Olfactory and Nasal Respiratory Mucosae

Abstract

The purpose of this study was to demonstrate that P-glycoprotein (P-gp) is localized in the olfactory mucosa and is capable of limiting the nose-to-brain transport of substrates. Bovine olfactory and nasal respiratory mucosae were compared to both localize P-gp and to measure its activity within the epithelia. Immunolocalization was performed on the bovine olfactory and nasal respiratory mucosa using the C219 monoclonal antibody. Flux of etoposide, a substrate reported to be primarily effluxed by P-gp, across bovine olfactory and nasal respiratory mucosae was measured using Sweetana-Grass (Navicyte) vertical diffusion cells. Experiments were performed to evaluate the effect of directionality, donor concentration, and the presence of inhibitors. Dense staining was observed on the apical surface of the ciliated epithelial cells and within the submucosal lymphatics/vasculature and mucosal glands of the bovine olfactory and nasal respiratory mucosae. Staining in the nasal respiratory epithelium was weak and patchy when compared to that observed in the olfactory mucosa. The secretory transport (Js-m) kinetics of etoposide in the olfactory (Km = 260.5 microM, Vmax = 0.179 microM/cm(2) min) and nasal respiratory (Km = 46.9 microM, Vmax = 0.034 microM/cm(2) min) mucosae were observed to be saturable and concentration-dependent. The flux of etoposide in the submucosal-mucosal (Js-m) direction was significantly greater than the flux in the mucosal-submucosal (Jm-s) direction in both the olfactory and nasal respiratory mucosa. The efflux ratios (Js-m/Jm-s) of etoposide across the olfactory and the nasal respiratory mucosae were 2.02 and 2.10, respectively. In the presence of inhibitors such as 2,4-dinitrophenol (1 mM) and quinidine (1 mM), etoposide showed an increase in Jm-s and a decrease in Js-m. The etoposide efflux was unaffected in the presence of a specific multiresistance associated protein 1 (MRP1) inhibitor (MK571) and methotrexate, a substrate for BCRP and MRP1-4. P-gp was localized in the epithelial cells, nasal glands, and the vascular endothelium of both the bovine olfactory and nasal respiratory mucosae, and the expressed P-gp was capable of effluxing a substrate such as etoposide. The Km and Vmax of etoposide efflux were higher in the olfactory mucosa compared to the nasal respiratory mucosa, and the expression of P-gp seems to be greater in the olfactory epithelium compared to the nasal respiratory epithelium based on the staining density observed using immunohistochemistry.