| Permeability-glycoprotein (Pgp) actively exports numerous potentially toxic compounds once they diffuse into the cell membrane of intestinal epithilia cells. We adapted the everted sleeve technique that uses small intestinal sections (1 cm) and relatively short incubation times (8--12 min) to make the first measures of intestinal Pgp function in an avian (chicken) and wild mammalian species. Tissues maintained both structural and functional integrity. We defined Pgp activity as the difference in accumulation of a specific Pgp substrate, [3H]-digoxin, in sleeves incubated in ringer solution with and without a maximally inhibitory concentration of a known Pgp inhibitor, cyclosporin A. We demonstrated significant variation in Pgp activity along the intestine, between chickens fed different diets, and found that chicken and woodrats have higher Pgp activity than Sprague-Dawley rats. In chicken, we also found that genistein (200 muM) and sterigmatocystin (5 muM), but not quercetin (10--330 muM), isoquercetrin (100 muM), or aflatoxin B1 (5 muM) inhibited Pgp.;We hypothesized that Pgp activity would be higher in Neotoma stephensi, a specialist on Juniperus monosperma known to be high in plant toxins, than the sympatric generalist, N. albigula, which consumes juniper in the field, but is unable to tolerate a high juniper diet. With these first measures of Pgp in wild mammals, we found that Pgp activity per mg tissue averaged over the entire intestine in N. stephensi was 2.4 fold higher than N. albigula . This result suggests that Pgp may play a role in the ability of N. stephensi to tolerate juniper.;We also tested whether Pgp recognizes alpha-pinene (1--100 muM), the dominant monoterpene in J. monosperma. We found no significant increase in digoxin accumulation using racemic alpha-pinene as a competitive inhibitor in Caco-2 cells which over-express Pgp, or everted sleeves from N. stephensi, N. albigula, N. cinerea, and Sprague-Dawley rats. We also found no increase in phosphate production from Pgp ATP-ase in transfected insect membrane vesicles exposed to alpha-pinene. Additionally, we acclimated 5 N. stephensi to a juniper diet for 5 days, but found this diet treatment did not induce intestinal Pgp activity. Our data suggest alpha-pinene is not a Pgp substrate. |
