Volume-sensitive Chloride Channel Activity Does Not Depend on Endogenous P-glycoprotein

To determine whether endogenous P-glycoprotein, the MDR1 gene product that functions as a drug transport pump, is a volume-sensitive Cl- channel molecule or a protein kinase C-mediated regulator of the Cl- channel, whole-cell patch-clamp and molecular biological experiments were carried out in a human small intestinal epithelial cell line. Endogenous expression of P-glycoprotein was confirmed by Northern blot analysis, reverse transcription-polymerase chain reaction, Western blot analysis, and immunostaining. The P-glycoprotein expression was abolished by the antisense (but not sense) oligonucleotide for the MDR1 gene, whereas the magnitude of the Cl- current activated by osmotic swelling was not distinguishable between both antisense- and sense-treated cells. The volume-sensitive Cl- currents were not specifically affected by the anti-P-glycoprotein monoclonal antibodies, MRK16, C219, and UIC2. An inhibitor of P-glycoprotein-mediated pump activity, verapamil, was found to never affect the Cl- current. A substrate for the P-glycoprotein-mediated drug pump, vincristine or daunomycin, did not prevent swelling-induced activation of the Cl- current. Furthermore, the Cl- current was not affected by an activator of protein kinase C (12-O-tetradecanoylphorbol-13-acetate or 1-oleoyl-2-acetyl-sn-glycerol). Thus, it is concluded that the endogenous P-glycoprotein molecule is not itself a volume-sensitive Cl- channel nor a protein kinase C-mediated regulator of the channel in the human epithelial cells. To determine whether endogenous P-glycoprotein, the MDR1 gene product that functions as a drug transport pump, is a volume-sensitive Cl- channel molecule or a protein kinase C-mediated regulator of the Cl- channel, whole-cell patch-clamp and molecular biological experiments were carried out in a human small intestinal epithelial cell line. Endogenous expression of P-glycoprotein was confirmed by Northern blot analysis, reverse transcription-polymerase chain reaction, Western blot analysis, and immunostaining. The P-glycoprotein expression was abolished by the antisense (but not sense) oligonucleotide for the MDR1 gene, whereas the magnitude of the Cl- current activated by osmotic swelling was not distinguishable between both antisense- and sense-treated cells. The volume-sensitive Cl- currents were not specifically affected by the anti-P-glycoprotein monoclonal antibodies, MRK16, C219, and UIC2. An inhibitor of P-glycoprotein-mediated pump activity, verapamil, was found to never affect the Cl- current. A substrate for the P-glycoprotein-mediated drug pump, vincristine or daunomycin, did not prevent swelling-induced activation of the Cl- current. Furthermore, the Cl- current was not affected by an activator of protein kinase C (12-O-tetradecanoylphorbol-13-acetate or 1-oleoyl-2-acetyl-sn-glycerol). Thus, it is concluded that the endogenous P-glycoprotein molecule is not itself a volume-sensitive Cl- channel nor a protein kinase C-mediated regulator of the channel in the human epithelial cells.

Hali tarjima qilinmagan