The issues of transport multiplicity and energetics pertaining to methotrexate efflux in L1210 cells addressed by an analysis of cis and trans effects of inhibitors.

Studies are described addressing the controversial issue of the multiplicity of efflux routes for [3H]MTX in L1210 cells. We 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 probenecid-, bromosulfophthalein-, and verapamil-inhibitable route that accounts for nearly 90% of [3H]MTX efflux. Efflux of [3H]MTX by this route is inhibited by bromosulfophthalein in the trans orientation only, inhibited by probenecid only when present simultaneously in the cis 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 bromosulfophthalein or verapamil but is inhibited by the N-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. Under these conditions, MTX did not trans-stimulate [3H]MTX efflux. Also, no evidence was obtained for a putative bromosulfophthalein-insensitive, probenecid-inhibitable route for [3H]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 [3H]MTX was markedly reduced and appears to be mediated solely by the MTX influx route. Influx of [3H]MTX was both cis and trans inhibited by probenecid, and efflux under these conditions was markedly inhibited by the N-hydroxysuccinimide ester of MTX and trans-stimulated by MTX. Overall, 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 [3H]MTX influx and efflux in ATP-replete and depleted L1210 cells. As such, these new results identify a single ATP-dependent efflux route as the bromosulfophthalein-, probenecid-, and verapamil-inhibitable route in L1210 cells under conditions that maintain ATP levels at maximum.