FUNCTIONAL-STUDIES OF P-GLYCOPROTEIN IN INSIDE-OUT PLASMA-MEMBRANE VESICLES DERIVED FROM MURINE ERYTHROLEUKEMIA-CELLS OVEREXPRESSING MDR-3 - PROPERTIES AND KINETICS OF THE INTERACTION OF VINBLASTINE WITH P-GLYCOPROTEIN AND EVIDENCE FOR ITS ACTIVE MEDIATED TRANSPORT

Active [H-3]vinblastine (VBL) transport (efflux) was documented for in side-out plasma membrane vesicles from murine erythroleukemia cells (M EL/VCR-6) resistant to vinca alkaloids and overexpressing MDR 3 P-glyc oprotein (P-gp) 80-fold. Uptake of [H-3]VBL at 37 degrees C by these i nside-out vesicles, but not rightside-out vesicles or inside-out vesic les from wild-type cells, was obtained in the form of a rapid, initial phase (0-1 min) and a slower later phase (>1 min). The rapidity of ea ch phase correlated with relative P-gp content among different MEL/VCR cell lines. The initial MDR-specific phase was temperature- and pH-de pendent (optimum at pH 7), osmotically insensitive, and did not requir e ATP. The second MDR-specific phase was temperature-dependent, osmoti cally sensitive, and strictly dependent upon the presence of ATP (K-m = 0.37 +/- 0.04 mM). Although other triphosphate nucleotides were part ially effective in replacing ATP, the nonhydrolyzable analogue ATP gam ma S (adenosine 5'-O-(thiotriphosphate)) was ineffective. This time co urse appears to represent tandem binding of [H-3]VBL by P-gp and its m ediated transport, with the latter process representing the rate-limit ing step. In support of this conclusion, both binding and transport we re inhibited by verapamil, quinidine, and reserpine, all known to be i nhibitors of photoaffinity labeling of P-gp, but only transport was in hibited by C219 anti-P-gp antibody or orthovanadate. Although the rate of transport of [H-3]VBL was 7-7.5-fold lower than the rate of bindin g (V-max = 104 +/- 15 pmol/min/mg protein, K-m = 1.5 - 2 x 10(5) mol(- 1) s(-1)) to P-gp, each phase exhibited saturation kinetics and values for apparent K-m and K-D for each process were approximately the same (215 +/- 35 and 195 +/- 30 nM). Intravesicular accumulation of [H-3]V BL was almost completely eliminated by high concentrations of nonradio active VBL, suggesting that simple diffusion does not contribute appre ciably to total accumulation of [H-3]VBL in this vesicle system. This could be at least partially explained by the fact that these inside-ou t vesicles under the conditions employed did not maintain a P-gp media ted pH gradient. However, ATP-dependent, intravesicular accumulation o f osmotically sensitive [H-3]VBL occurred against a substantial permea nt concentration gradient in both a time- and concentration-dependent manner consistent with an active, saturable process.