| Abstract: |
Active [3H]vinblastine (VBL) transport (efflux) was documented for inside-out plasma membrane vesicles from murine erythroleukemia cells (MEL/VCR-6) resistant to vinca alkaloids and overexpressing MDR 3 P- glycoprotein (P-gp) 80-fold. Uptake of [3H]VBL at 37°C by these inside-out vesicles, but not rightside-out vesicles or inside-out vesicles 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 each phase correlated with relative P-gp content among different MEL/VCR cell lines. The initial MDR- specific phase was temperature- and pH-dependent (optimum at pH 7), osmotically insensitive, and did not require ATP. The second MDR-specific phase was temperature-dependent, osmotically sensitive, and strictly dependent upon the presence of ATP (K(m) = 0.37 ± 0.04 mM). Although other triphosphate nucleotides were partially effective in replacing ATP, the nonhydrolyzable analogue ATPγS (adenosine 5'-O-(thiotriphosphate)) was ineffective. This time course appears to represent tandem binding of [3H]VBL by P-gp and its mediated transport, with the latter process representing the rate-limiting step. In support of this conclusion, both binding and transport were inhibited by verapamil, quinidine, and reserpine, all known to be inhibitors of photoaffinity labeling of P-gp, but only transport was inhibited by C219 anti-P-gp antibody or orthovanadate. Although the rate of transport of [3H]VBL was 7-7.5-fold lower than the rate of binding (V(max) = 104 ± 15 pmol/min/mg protein, K(on) = 1.5 - 2 x 105 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 [3H]VBL was almost completely eliminated by high concentrations of nonradioactive VBL, suggesting that simple diffusion does not contribute appreciably to total accumulation of [3H]VBL in this vesicle system. This could be at least partially explained by the fact that these inside-out vesicles under the conditions employed did not maintain a P-gp mediated pH gradient. However, ATP-dependent, intravesicular accumulation of osmotically sensitive [3H]VBL occurred against a substantial permeant concentration gradient in both a time- and concentration-dependent manner consistent with an active, saturable process. |
| Keywords: |
human cell; animal; mice; gene expression; protein binding; ph; tumor cells, cultured; vinblastine; kinetics; cell membrane; murinae; protons; adenosine triphosphate; verapamil; membrane transport; biological transport; concentration response; multidrug resistance; hydrogen-ion concentration; p-glycoprotein; drug resistance, multiple; membrane vesicle; glycoprotein p; dose time effect relation; leukemia, erythroblastic, acute; temperature dependence; quinidine; mice, inbred dba; reserpine; human; priority journal; article; osmosis; support, non-u.s. gov't; support, u.s. gov't, p.h.s.; erythroleukemia cell; vinca
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