Inhibition of P-glycoprotein-mediated transport by a hydrophobic contaminant in commercial gluconate salts

The substitution of gluconate for Cl- is commonly used to characterize Cl- transport or Cl--dependent transport mechanisms. We evaluated the effects o f substituting gluconate for Cl- on the transport of the P-glycoprotein sub strate rhodamine 123 (R123). The replacement of Ringer solution containing Cl- (Cl--Ringer) with gluconate-Ringer inhibited R123 efflux, whereas the r eplacement of Cl- by other anions (sulfate or cyclamate) had no effect. The inhibition of R123 efflux by gluconate-Ringer was absent after chloroform extraction of the sodium gluconate salt. The readdition of the sodium gluco nate-chloroform extract to the extracted gluconate-Ringer or to cyclamate-R inger inhibited R123 efflux, whereas its addition to Cl--Ringer had no effe ct. These observations indicate that the inhibition of P-glycoprotein-media ted R123 transport by gluconate is due to one or more chloroform-soluble co ntaminants and that the inhibition is absent in the presence of Cl-. The re sults are consistent with the fact that P-glycoprotein substrates are hydro phobic. Care should be taken when replacing ions to evaluate membrane trans port mechanisms because highly pure commercial preparations may still conta in potent contaminants that affect transport.