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Transport, Binding, and Polyglutamation of Methotrexate in Freshly Isolated Rat Hepatocytes¹

Departments of Medicine and Pharmacology, Medical College of Virginia, Richmond, Virginia 23298

Influx of [³H]methotrexate into freshly isolated hepatocytes in suspension is mediated by two routes, one with a high affinity (K_m = 5.9 µM) and another with a low affinity for methotrexate. Both transport routes are equally sensitive to the sulfhydryl group inhibitor, p-chloromercuriphenylsulfonic acid, alterations in temperature, substitution of extracellular Na⁺ with choline, and inhibition by ouabain or azide. The high-affinity pathway for methotrexate shows specificity for the 4-amino group of the pteridine moiety as methotrexate and aminopterin similarly inhibit influx of the labeled drug. On the other hand, 100 µM concentrations of the naturally occurring folates, folic acid, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate, are not inhibitory to influx of 1 µM methotrexate.

Once in the cell, methotrexate rapidly reaches molar equivalence with dihydrofolate reductase following which both exchangeable and nonexchangeable intracellular methotrexate accumulates. The exchangeable component reaches steady state within 0.5 hr while the nonexchangeable component increases for at least 1 hr. The nonexchangeable component represents both bound methotrexate and methotrexate polyglutamates. Polyglutamates of methotrexate are a trivial component of total ³H within the cell until about 15 min, but thereafter, their rate of accumulation is constant so that by 1 hr they represent 30% of total intracellular ³H. At steady state, there is a transmembrane chemical gradient for exchangeable methotrexate of 2.4:1; this is 24 times greater than the chemical gradient predicted for equilibrium when the transcellular membrane potential is considered.

These results indicate that there are multiple routes for methotrexate transport in the rat hepatocyte that appear to be, at least in part, distinct from the routes for folic acid and the tetrahydrofolate cofactors. The data suggest that transport is energy and Na⁺ dependent and that the transport carrier requires intact sulfhydryl groups. Net association of methotrexate with the cells is a complex process determined by transport and binding to multiple sites within the cell and metabolism to polyglutamate derivatives that are retained within the cell.

¹ Supported by Grants CA-16906 and AM-18796 from the NIH.

² Recipient of Training Grant HL-07110.

³ To whom requests for reprints should be addressed.

Received 7/30/79. Accepted 11/28/79.

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