Relationship between membrane transport of methotrexate and endogenous cyclic adenosine 3':5'-monophosphate in the...

Transport of methotrexate in mammalian cells is a complex process. Although the drug is pumped uphill into cells, metabolic poisons enhance influx and transmembrane gradients for this agent. Structurally unrelated inorganic and organic anions nonspecifically depress these parameters in a variety of tumor cells. It has been suggested that cell cyclic adenosine 3':5'-Monophosphate (cyclic AMP) plays a regulatory role in the transport of this agent. To evaluate this further, a number of different experimental approaches were used to analyze the relationship between methotrexate transport and cell cyclic AMP in the Ehrlich ascites tumor. The following results indicate that for the Ehrlich ascites tumor, at least, there is no evidence for a regulatory role for cyclic AMP in the transport of methotrexate. (a) A marked increase in cell cyclic AMP by cholera-toxin or reduction in cyclic AMP by ascorbate was unaccompanied by changes in methotrexate influx. (b) Instantaneous and constant inhibition of methotrexate influx by isobutylmethylxanthine was temporally dissociated from the slower rise in cell cyclic AMP that was induced by this agent. (c) Although isobutylmethylxanthine and dibutyryl cyclic AMP augment cell cyclic AMP and decrease methotrexate influx, they have different effects on efflux and net transport of methotrexate. Isobutylmethylxanthine stimulates net methotrexate uptake and slows methotrexate efflux but dibutyryl cyclic AMP depresses net methotrexate uptake without an effect on methotrexate efflux. (d) Azide markedly stimulates methotrexate influx and net transport without a change in cell cyclic AMP. (d) Dinitrophenol inhibits methotrexate influx and augments net methotrexate accumulation but does not alter cell cyclic AMP.