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Most amino acids are cotransported with sodium. Deoxynivalenol (DON) decreases glucose absorption in the chicken small intestine in vivo and in vitro, and this effect is apparently mediated by the inhibition of the sodium D-glucose cotransporter. DON could selectively modulate the activities of other intestinal transporters. In order to assess this hypothesis, a study was conducted to characterize the in vitro effects of DON in the presence of mucosal amino acids, using L-proline as a model, on the electrophysiological parameters in the jejunums of laying hens. L-Proline (mucosal concentration of 1 mmol/L) was added to a stripped proximal part of jejunum sheets mounted in Ussing chambers in Ringer buffer, and the electrical properties were measured. The transmural potential difference (PD) was nearly constant between the treatments. The tissue resistance (Rt) was higher (P < 0.05) in the tissues exposed to DON compared with basal values and the values after addition of L-proline. Addition of L-proline on the luminal side of the isolated mucosa increased (P < 0.05) the short circuit-current (Isc), and it decreased (P < 0.05) after addition of DON, indicating that the proline-induced Isc was altered by DON. The addition of proline after incubation of the tissues with DON had no effect (P > 0.05) on PD or Rt. Proline did not increase the Isc under these conditions. DON decreased (P < 0.1) the Isc after addition of proline, indicating that DON inhibited the Na+-amino acid co-transport. We concluded from the present study that the amino acid cotransporter activity appears to be highly sensitive to DON suppression.