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Transport of Amino Acid Amide Sarcosinamide and Sarcosinamide Chloroethylnitrosourea in Human Glioma SK-MG-1 Cells¹

Lady Davis Institute for Medical Research of the Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Quebec, Canada H3T 1E2 [V. S., L. C. P.], and the Montreal Neurological Institute, Montreal, Quebec, Canada, H3A2B4 [W. F.]

The transport of the amino acid amide N-[³H]sarcosinamide (methyl glycinamide) was investigated in human glioma SK-MG-1 cells. Sarcosinamide uptake was found to be temperature dependent, sodium independent, and linear up to 1 min at 22°C. Equilibrium was reached after 10 min at 22°C with accumulation slightly above unity. Sarcosinamide was not metabolized in the cells as shown by thin layer chromatography. The uptake of sarcosinamide was significantly decreased when the extracellular pH was lowered from 7.5 to 6.0 and significantly enhanced at pH values above 7.5. The latter effect may be due mainly to increased cell permeability at high pH. The uptake of the labeled sarcosinamide was trans-stimulated by excess cold sarcosinamide. Sarcosinamide up-take over a 200-fold range of concentrations followed Michaelis-Menten kinetics with a K_m of 0.284 ± 0.041 mM and a V_max of 0.154 ± 0.024 nmol/10⁶ cells/min. The uptake of sarcosinamide was significantly reduced by iodoacetate but not by the metabolic poisons NaF, ouabain, or dinitrophenyl, suggesting that the uptake is not dependent on energy, rather it proceeds by facilitated diffusion. Several naturally occurring substrates were unable to inhibit the uptake of sarcosinamide. Leucine significantly reduced the uptake of sarcosinamide, while sarcosinamide was a weak inhibitor of leucine transport. 2-Aminobicyclo[2,2,1]heptane-2-carboxylic acid, a specific substrate for the sodium-independent, 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid-sensitive amino acid system L failed to inhibit the uptake of sarcosinamide. Epinephrine reduced the uptake of sarcosinamide and sarcosinamide was equally potent as an inhibitor of epinephrine transport. Dixon plot analysis demonstrated that epinephrine (K_m = 0.270 mM) inhibits the uptake of sarcosinamide competitively (K_i = 0.260 mM). These results indicate that sarcosinamide is a substrate for the catecholamine transporter. The alkylating agent, sarcosinamide chloroethylnitrosourea, was tested for its ability to inhibit the uptake of sarcosinamide. The results of Dixon plot analysis were consistent with competitive inhibition of sarcosinamide uptake and the inhibition constant K_i for SarCNU was found to be 3.26 ± 0.57 mM. The steady-state intracellular concentration of SarCNU was found to be significantly higher (cell:medium ratio of 1.03 ± 0.01) than that of BCNU (cell:medium ratio of 0.52 ± 0.12). These findings indicate that SarCNU and sarcosinamide share the same carrier for uptake in SK-MG-1 cells. This transport mechanism may be responsible for the increased accumulation of SarCNU as compared to BCNU, a nitrosourea which enters cells by passive diffusion.

¹ This work was supported by National Institute of Neurological Disorders and Stroke Grant R01-NSC22230 and a grant from the Cancer Research Society (Montreal).

² Predoctoral fellow of the Cancer Research Society (Montreal).

³ To whom requests for reprints should be addressed, at the Lady Davis Institute for Medical Research of the Sir Mortimer B. Davis-Jewish General Hospital, 3755 Cote Ste. Catherine Road, Montreal, Quebec, Canada H3T 1E2.

Received 7/26/89. Revised 11/15/89.