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

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 [H-3]SarCNU was examined in suspension. The uptake of [H-3] SarCNU was found to be temperature de pendent, with influx being linear to 4 sec at 37-degrees. Equilibrium was reached after 1 min at 22-degrees and 37-degrees, with accumulatio n slightly above unity. SarCNU was not significantly metabolized in th e cells after a 60-min incubation at 37-degrees, as shown by thin laye r chromatography. At 37-degrees, uptake of [H-3]SarCNU was found to be saturable, sodium independent, and energy independent. Previous work demonstrated that SarCNU was able to inhibit the uptake of sarcosinami de, which is transported by the catecholamine uptake 2 system. This ca techolamine system mediates the physiological transport of epinephrine . Epinephrine was able to significantly inhibit the uptake of [H-3]Sar CNU, at a concentration of 50 muM, by 40%. Additionally, several amino acids were unable to inhibit the uptake of SarCNU. The initial rate o f SarCNU influx is mediated by both facilitated and nonfacilitated dif fusion. 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 (epine phrine). Dixon plot analysis, corrected for nonfacilitated diffusion o f SarCNU, revealed that epinephrine inhibited the uptake of SarCNU com petitively, with a K(i) of 163 +/- 15 muM, a value similar to the K(m) value for epinephrine influx in SK-MG-1 cells. Additionally, after ap propriate corrections for nonfacilitated diffusion in the influx rates observed for SarCNU, it was revealed that SarCNU influx obeyed Michae lis-Menten kinetics over a 200-fold range of concentrations, with a K( m) of 2.39 +/- 0.37 mm and a V(max) of 236 +/- 53 pmol/mul of intracel lular water/sec. Metabolic poisons (2,4-dinitrophenol, iodoacetate, Na CN, NaF, or ouabain) were unable to inhibit the influx of SarCNU, sugg esting that the carrier-mediated uptake of SarCNU is energy independen t and mediated by facilitated diffusion. These findings indicate that SarCNU uptake in SK-MG-1 cells is mediated both by nonfacilitated diff usion and by facilitated diffusion via the catecholamine uptake 2 carr ier system. SarCNU is the first chloroethylnitrosourea that has been d emonstrated to have carrier-mediated uptake. Moreover, this carrier-me diated uptake may play a role in the increased cytotoxicity of SarCNU against gliomas, compared with that of 1,3-bis(2-chloroethyl)-1-nitros ourea, which enters cells primarily by passive diffusion.