Smith D W, Scriver C R, Tenenhouse H S, Simell O
Medical Research Council Genetics Group, McGill University-Montreal Children's Hospital Research Institute, Canada.
Proc Natl Acad Sci U S A. 1987 Nov;84(21):7711-5. doi: 10.1073/pnas.84.21.7711.
Lysinuric protein intolerance (LPI) is an autosomal recessive phenotype consistent with impaired transport of cationic amino acids at the basolateral membrane of intestinal and renal epithelia. On the assumption that the basolateral membrane of epithelial cells and plasma membrane of parenchymal cells are functional analogues, we studied transport of cationic amino acids by cultured skin fibroblasts from LPI and control subjects matched for age, sex, and site of biopsy. We measured Na+-independent transport of radiolabeled lysine, arginine, ornithine, and homoarginine on system y+, the carrier with preference for cationic amino acids, and leucine transport on system L (as the internal control). LPI cells had increased net uptake of cationic amino acids (nmol/mg of protein) relative to leucine. LPI cells also maintained increased steady-state intracellular pools of cationic amino acids. Neither increased metabolic utilization nor increased pool size were responsible for high uptake of cationic amino acids in LPI cells. We then measured trans-stimulated efflux of homoarginine as a specific test of system y+ activity. Homoarginine efflux was significantly impaired in LPI cells (P less than 0.05), whereas leucine efflux was similar in LPI and control cells. Percent trans-stimulation of homoarginine efflux was 1.0 +/- 0.5% in homozygous LPI cells, 10 +/- 0.5% in heterozygous cells, and 22 +/- 0.5% in control cells indicating a gene-dosage effect. The LPI mutation affects system y+ asymmetrically, selectively impairing efflux in fibroblast plasma membrane. To our knowledge, this appears to be the first demonstration that the skin fibroblast can be used to study a corresponding transport defect in intestinal and renal membranes.
赖氨酸尿性蛋白不耐受症（LPI）是一种常染色体隐性表型，与肠道和肾上皮细胞基底外侧膜上阳离子氨基酸转运受损一致。基于上皮细胞基底外侧膜与实质细胞的质膜具有功能相似性这一假设，我们研究了来自LPI患者及年龄、性别和活检部位相匹配的对照受试者的培养皮肤成纤维细胞对阳离子氨基酸的转运情况。我们测定了放射性标记的赖氨酸、精氨酸、鸟氨酸和高精氨酸在y+系统（优先转运阳离子氨基酸的载体）上的非钠依赖性转运，以及亮氨酸在L系统上的转运（作为内部对照）。相对于亮氨酸，LPI细胞对阳离子氨基酸的净摄取量（nmol/毫克蛋白）增加。LPI细胞还维持着阳离子氨基酸的稳态细胞内池增加。LPI细胞中阳离子氨基酸的高摄取量既不是代谢利用增加所致，也不是池大小增加所致。然后，我们测定了高精氨酸的转刺激外流，作为对y+系统活性的特异性检测。LPI细胞中的高精氨酸外流明显受损（P<0.05），而LPI细胞和对照细胞中的亮氨酸外流相似。纯合LPI细胞中高精氨酸外流的转刺激百分比为1.0±0.5%，杂合细胞中为10±0.5%，对照细胞中为22±0.5%，表明存在基因剂量效应。LPI突变对y+系统的影响不对称，选择性地损害成纤维细胞质膜中的外流。据我们所知，这似乎是首次证明皮肤成纤维细胞可用于研究肠道和肾膜中相应的转运缺陷。
