GLUCOSE DEPRIVATION AND ACUTE CYCLOHEXIMIDE TREATMENT STIMULATE SYSTEM-L AMINO-ACID TRANSPORT IN CULTURED VASCULAR SMOOTH MUSCLE CELLS

The effect of glucose deprivation on the uptake of leucine has been ex amined in cultured vascular smooth muscle cells isolated from rat aort ae. Equimolar substitution of sucrose or fructose for glucose in the c ulture medium enhanced the uptake of leucine in a time- and concentrat ion-dependent manner. The effect was first detectable after 12 h and r eached the maximum, 2-fold, after 48 h with an apparent half-maximal e ffect at 1 mM glucose and could be reversed after 48 h of glucose refe eding. The enhanced leucine uptake was completely inhibited by 2-amino -2-norbornane-carboxylic acid, a specific substrate for System L, but not by alpha-(methylamino)isobutyric acid or lysine. Kinetic analyses indicated that this stimulation was mediated via a homogenous system w ith a 1.7-fold increase in the V-max without any change in the K-m (0. 15 mM). Prolonged treatments with cycloheximide (10 mu g/ml) or actino mycin D (10 mu g/ml) blocked this glucose deprivation effect and its r eversal. However, cycloheximide also very rapidly stimulated leucine u ptake, reaching the maximum, 2.5-fold over the basal at Ih. This effec t occurred at concentrations that matched its inhibition on protein sy nthesis (half-maximal at 0.1 mu g/ml) and could be reproduced with pur omycin as well as actinomycin D. The stimulatory effect of cycloheximi de was also accompanied by an increase in the V-max but not in the K-m , being sensitive to 2-amino-2-norbornane-carboxylic acid inhibition o nly, and appeared to occur in an additive manner to that of glucose de privation. Although the uptake of leucine was stimulated by glucose de privation and brief exposure to cycloheximide, these treatments had no effect on the efflux of the substrate. These results are all consiste nt with the System L amino acids transport activity in cultured rat va scular smooth muscle cells being under the control of at least two non -hormonal regulatory mechanisms, one that is likely to involve a labil e repressor molecule and the other involving de nova protein synthesis as a result of chronic glucose deprivation.