Transport of (2-chloroethyl)-3-sarcosinamide-1-nitrosourea in the human glioma cell line SK-MG-1 is mediated by an epinephrine-sensitive carrier system

Abstract

The transport of (2-chloroethyl)-3-sarcosinamide-1-nitrosourea (SarCNU), an experimental anticancer compound, was investigated in the human glioma cell line SK-MG-1. The transport of [3H]SarCNU was examined in suspension. The uptake of [3H] SarCNU was found to be temperature dependent, with influx being linear to 4 sec at 37 degrees. Equilibrium was reached after 1 min at 22 degrees and 37 degrees, with accumulation slightly above unity. SarCNU was not significantly metabolized in the cells after a 60-min incubation at 37 degrees, as shown by thin layer chromatography. At 37 degrees, uptake of [3H]SarCNU was found to be saturable, sodium independent, and energy independent. Previous work demonstrated that SarCNU was able to inhibit the uptake of sarcosinamide, which is transported by the catecholamine uptake 2 system. This catecholamine system mediates the physiological transport of epinephrine. Epinephrine was able to significantly inhibit the uptake of [3H]SarCNU, at a concentration of 50 microM, by 40%. Additionally, several amino acids were unable to inhibit the uptake of SarCNU. The initial rate of SarCNU influx is mediated by both facilitated and nonfacilitated diffusion. The nonfacilitated diffusion rate could be estimated from the linear concentration dependence of the residual influx rate for SarCNU, which was not inhibited by the presence of excess co-permeant (epinephrine). Dixon plot analysis, corrected for nonfacilitated diffusion of SarCNU, revealed that epinephrine inhibited the uptake of SarCNU competitively, with a Ki of 163 +/- 15 microM, a value similar to the Km value for epinephrine influx in SK-MG-1 cells. Additionally, after appropriate corrections for nonfacilitated diffusion in the influx rates observed for SarCNU, it was revealed that SarCNU influx obeyed Michaelis-Menten kinetics over a 200-fold range of concentrations, with a Km of 2.39 +/- 0.37 mM and a Vmax of 236 +/- 53 pmol/microliters of intracellular water/sec. Metabolic poisons (2,4-dinitrophenol, iodoacetate, NaCN, NaF, or ouabain) were unable to inhibit the influx of SarCNU, suggesting that the carrier-mediated uptake of SarCNU is energy independent and mediated by facilitated diffusion. These findings indicate that SarCNU uptake in SK-MG-1 cells is mediated both by nonfacilitated diffusion and by facilitated diffusion via the catecholamine uptake 2 carrier system. SarCNU is the first chloroethylnitrosourea that has been demonstrated to have carrier-mediated uptake. Moreover, this carrier-mediated uptake may play a role in the increased cytotoxicity of SarCNU against gliomas, compared with that of 1,3-bis(2,-chloroethyl)-1-nitrosourea, which enters cells primarily by passive diffusion.