TY - JOUR
T1 - K+-induced alterations of energetics and exchange diffusion in the carrier-mediated transport of the folic acid analog, methotrexate, in ehrlich ascites tumor cells
AU - Fry, David W.
AU - Cybulski, Raymond L.
AU - Goldman, I. David
N1 - Funding Information:
The excellent technical assistance of Linda Anderson is gratefully acknowledged. This research was supported by NIH grant No. CA-16906. D.W.F. was supported by NIH Training Grants AM-07150 and CA-09340. R.L.C. was supported by NIH Training Grants CA-09036 and CA-09340.
PY - 1980/12/2
Y1 - 1980/12/2
N2 - The bidirectional fluxes and energetics of methotrexate transport in Ehrlich ascites tumor cells were profoundly altered in a high [K+], low [Na+] buffer (K+ buffer). Incubation of cells for 30 min in K+ buffer reduced influx by 27% and the efflux rate constant by 53%. This asymmetrical inhibition of bidirectional fluxes increased the net exchangeable intracellular methotrexate level per cell, but the actual intracellular methotrexate concentration at the steady state was similar to that in Na+ buffer, since the high [K+] caused an increase in intracellular water. Because cells exposed to K+ buffer were depolarized, the apparent electrochemical potential difference for methotrexate was markedly reduced. However, the steady-state intracellular methotrexate level was still related to the extracellular concentration by an absorption isotherm, indicating asymmetry in the bidirectional fluxes similar to that observed in Na+ buffer and thus predicting that transmembrane gradients would be produced at very low extracellular methotrexate concentrations. Glucose, which had little effect on bidirectional fluxes and reduced the steady-state level of methotrexate in Na+ buffer, stimulated influx, inhibited efflux and rapidly increased the steady state in K+ buffer similar to the effects of glucose in the presence of iodoacetate in Na+ buffer. Finally, cells exposed to K+ buffer exhibited trans-stimulation of [3H]methotrexate influx when loaded with non-labeled methotrexate, a phenomenon not observed in Na+ buffer. The results indicate that although methotrexate transport is not affected by transient changes in the cationic composition of the extracellular compartment, prolonged exposure of cells to a high [K+], low [Na+] environment markedly alters the physical properties of the cells and the transport parameters for methotrexate and reveals characteristics of the methotrexate carrier system that are not evident in other buffer systems.
AB - The bidirectional fluxes and energetics of methotrexate transport in Ehrlich ascites tumor cells were profoundly altered in a high [K+], low [Na+] buffer (K+ buffer). Incubation of cells for 30 min in K+ buffer reduced influx by 27% and the efflux rate constant by 53%. This asymmetrical inhibition of bidirectional fluxes increased the net exchangeable intracellular methotrexate level per cell, but the actual intracellular methotrexate concentration at the steady state was similar to that in Na+ buffer, since the high [K+] caused an increase in intracellular water. Because cells exposed to K+ buffer were depolarized, the apparent electrochemical potential difference for methotrexate was markedly reduced. However, the steady-state intracellular methotrexate level was still related to the extracellular concentration by an absorption isotherm, indicating asymmetry in the bidirectional fluxes similar to that observed in Na+ buffer and thus predicting that transmembrane gradients would be produced at very low extracellular methotrexate concentrations. Glucose, which had little effect on bidirectional fluxes and reduced the steady-state level of methotrexate in Na+ buffer, stimulated influx, inhibited efflux and rapidly increased the steady state in K+ buffer similar to the effects of glucose in the presence of iodoacetate in Na+ buffer. Finally, cells exposed to K+ buffer exhibited trans-stimulation of [3H]methotrexate influx when loaded with non-labeled methotrexate, a phenomenon not observed in Na+ buffer. The results indicate that although methotrexate transport is not affected by transient changes in the cationic composition of the extracellular compartment, prolonged exposure of cells to a high [K+], low [Na+] environment markedly alters the physical properties of the cells and the transport parameters for methotrexate and reveals characteristics of the methotrexate carrier system that are not evident in other buffer systems.
KW - (Ehrlich ascites tumor cell)
KW - Electrochemical potential difference
KW - K
KW - Methotrexate transport
KW - Na
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U2 - 10.1016/0005-2736(80)90398-3
DO - 10.1016/0005-2736(80)90398-3
M3 - Article
C2 - 7192570
AN - SCOPUS:0019135877
SN - 0005-2736
VL - 603
SP - 157
EP - 170
JO - BBA - Biomembranes
JF - BBA - Biomembranes
IS - 1
ER -