The Issues of Transport Multiplicity and Energetics Pertaining to Methotrexate Efflux in LI210 Cells Addressed by an Analysis of Cis and Trans Effects of Inhibitors'

Studies arc described addressing the controversial issue of the multi plicity of efflux routes for |'H|M TX in 1.1210 cells. \\e examined efflux multiplicity under conditions that do and do not maintain cellular ATP at the physiological level. In ATP-replete cells, the results delineate a probenccid-, bromosulfophthalcin-, and vcrapamil-inhibitable route that accounts for nearly 90% of |TI|MTX efflux. Efflux of |'H|MTX by this route is inhibited by bromosulfophthalein in the trans orientation only, inhibited by probenecid only «henpresent simultaneously in the els and trans orientation and inhibited by verapamil only in the cis orientation. The remaining efflux of this folate analogue in ATP-replete cells appears to be mediated by the one-carbon, reduced folate system (MTX influx route), in that it is not inhibited by bromosulfophtalein or verapamil but is inhibited by the A'-hydroxysuccinimide ester of MTX, a specific inhibitor of MTX influx, and a 10-fold higher concentration of probenecid than that required to inhibit ATP-dependent efflux. I nder these condi tions, MTX did not /rani-stimulate |'II|MTX efflux. Also, no evidence was obtained for a putative bromosulfophthalein-insensitive, probcnecidinhibitable route for |'H|MTX efflux. In cells depleted to the extent of 90-95% of their ATP by 60-min incubation in medium in the absence of D-glucose and with 10 mM sodium azide, overall efflux of |'H|MTX was markedly reduced and appears to be mediated solely by the MTX influx route. Influx of |'II|MTX was both cis and trans inhibited by probenecid, and efflux under these conditions was markedly inhibited by the A'hydroxysuccinimide ester of MTX and fra/w-stimulated by MTX. Over all, the results of these studies are consistent with a model for methotrexate transport in L1210 cells derived (Dembo et al., J. Membrane Biol., 78: 9-17, 1984] in the authors' laboratory based solely upon a kinetic analysis of |'H|MTX influx and efflux in ATP-replete and de pleted L1210 cells. As such, these new results identify a single ATPdependent efflux route as the bromosulfophthalein-, probenecid-, and verapamil-inhibitable route in I.I 210 cells under conditions that maintain ATP levels at maximum.