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 degrees C. Equilibrium was reached after 10 min at 22 degrees 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 uptake over a 200-fold range of concentrations followed Michaelis-Menten kinetics with a Km of 0.284 +/- 0.041 mM and a Vmax of 0.154 +/- 0.024 nmol/10(6) 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 (Km = 0.270 mM) inhibits the uptake of
sarcosinamide competitively (Ki = 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 Ki 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.