Low B C, Ross I K, Grigor M R
Department of Biochemistry, University of Otago, Dunedin, New Zealand.
J Biol Chem. 1994 Dec 23;269(51):32098-103.
The effect of glucose deprivation on the uptake of leucine has been examined in cultured vascular smooth muscle cells isolated from rat aortae. Equimolar substitution of sucrose or fructose for glucose in the culture medium enhanced the uptake of leucine in a time- and concentration-dependent manner. The effect was first detectable after 12 h and reached the maximum, 2-fold, after 48 h with an apparent half-maximal effect at 1 mM glucose and could be reversed after 48 h of glucose refeeding. 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 with a 1.7-fold increase in the Vmax without any change in the Km (0.15 mM). Prolonged treatments with cycloheximide (10 micrograms/ml) or actinomycin D (10 micrograms/ml) blocked this glucose deprivation effect and its reversal. However, cycloheximide also very rapidly stimulated leucine uptake, reaching the maximum, 2.5-fold over the basal at 1 h. This effect occurred at concentrations that matched its inhibition on protein synthesis (half-maximal at 0.1 micrograms/ml) and could be reproduced with puromycin as well as actinomycin D. The stimulatory effect of cycloheximide was also accompanied by an increase in the Vmax but not in the Km, being sensitive to 2-amino-2-norbornane-carboxylic acid inhibition only, and appeared to occur in an additive manner to that of glucose deprivation. Although the uptake of leucine was stimulated by glucose deprivation and brief exposure to cycloheximide, these treatments had no effect on the efflux of the substrate. These results are all consistent with the System L amino acids transport activity in cultured rat vascular smooth muscle cells being under the control of at least two non-hormonal regulatory mechanisms, one that is likely to involve a labile repressor molecule and the other involving de novo protein synthesis as a result of chronic glucose deprivation.
在从大鼠主动脉分离出的培养血管平滑肌细胞中，研究了葡萄糖剥夺对亮氨酸摄取的影响。在培养基中用等摩尔的蔗糖或果糖替代葡萄糖，以时间和浓度依赖的方式增强了亮氨酸的摄取。该效应在12小时后首次可检测到，48小时后达到最大值，为2倍，在1 mM葡萄糖时出现明显的半最大效应，并且在葡萄糖再喂养48小时后可以逆转。增强的亮氨酸摄取被2-氨基-2-降冰片烷羧酸（系统L的特异性底物）完全抑制，但不被α-（甲基氨基）异丁酸或赖氨酸抑制。动力学分析表明，这种刺激是通过一个同质系统介导的，Vmax增加了1.7倍，而Km（0.15 mM）没有任何变化。用环己酰亚胺（10微克/毫升）或放线菌素D（10微克/毫升）进行长时间处理可阻断这种葡萄糖剥夺效应及其逆转。然而，环己酰亚胺也非常迅速地刺激亮氨酸摄取，在1小时时达到最大值，比基础值高2.5倍。这种效应发生在与其对蛋白质合成的抑制相匹配的浓度下（在0.1微克/毫升时为半最大），并且嘌呤霉素和放线菌素D也可以重现。环己酰亚胺的刺激作用也伴随着Vmax的增加，但Km没有增加，仅对2-氨基-2-降冰片烷羧酸抑制敏感，并且似乎以与葡萄糖剥夺相加的方式发生。尽管亮氨酸的摄取受到葡萄糖剥夺和短暂暴露于环己酰亚胺的刺激，但这些处理对底物的流出没有影响。这些结果都与培养的大鼠血管平滑肌细胞中的系统L氨基酸转运活性受至少两种非激素调节机制控制一致，一种可能涉及不稳定的阻遏分子，另一种涉及慢性葡萄糖剥夺导致的从头蛋白质合成。
