Taylor P M, Mackenzie B, Low S Y, Rennie M J
Department of Anatomy and Physiology, University of Dundee, Scotland, United Kingdom.
J Biol Chem. 1992 Feb 25;267(6):3873-7.
As a first step in attempting to isolate the Na(+)-dependent System N transporter from rat liver we have investigated the use of prophase-arrested oocytes from Xenopus laevis for the functional expression of rat liver glutamine transporters. Individual oocytes, defolliculated by collagenase treatment, were injected with 50 nl of a 1 mg.ml-1 solution of poly(A)+ RNA (mRNA) isolated from rat liver. 50 microM L-[3H]glutamine uptake was measured 1-5 days post-injection: after 48 h, poly(A)+ RNA-injected oocytes showed a 60 +/- 12% increase in Na(+)-dependent glutamine uptake compared to controls. This increased uptake showed characteristic features of hepatic System N: that is, it tolerated Li(+)-for-Na+ substitution and was inhibited by the System N substrate L-histidine (5 mM) in Li medium, unlike endogenous Na(+)-dependent glutamine transport. In subsequent experiments rat liver poly(A)+ RNA, size-fractionated by density gradient fractionation, was injected into oocytes. Injection of poly(A)+ RNA of 1.9-2.8 kilobases (kb) in size resulted in a significant stimulation of Na(+)-dependent glutamine transport to 0.362 +/- 0.080 pmol.min-1/oocyte from 0.178 +/- 0.060 pmol.min-1/oocyte in vehicle-injected oocytes (p less than 0.01). A lighter fraction, with poly(A)+ RNA of less than 1.9 kilobases size resulted in a similar increase in Na(+)-dependent glutamine uptake which was largely Li(+)-tolerant: Li(+)-stimulated glutamine uptake in oocytes injected with this fraction increased to 0.230 +/- 0.070 pmol.min-1/oocyte from 0.098 +/- 0.029 pmol.min-1/oocyte in controls (p less than 0.05). This enhanced rate of Li(+)-stimulated glutamine uptake was inhibited 28 and 70%, respectively, by 1 and 5 mM L-histidine. Na(+)-independent uptake of glutamine rose by 72 +/- 12% in oocytes injected with poly(A)+ RNA of 2.8-3.6 kb (p less than 0.001). These results demonstrate that glutamine transporters, with characteristics associated with hepatic Systems N, L, and A (or ASC), can be expressed in X. laevis oocytes injected with specific size fractions of rat liver mRNA.
作为从大鼠肝脏中分离出依赖钠离子的系统N转运体的第一步，我们研究了使用非洲爪蟾前期阻滞的卵母细胞来功能性表达大鼠肝脏谷氨酰胺转运体。通过胶原酶处理去除卵泡的单个卵母细胞，注射50 nl从大鼠肝脏分离的1 mg.ml-1的聚腺苷酸（poly(A)+）RNA（mRNA）溶液。在注射后1 - 5天测量50 microM L-[3H]谷氨酰胺的摄取：48小时后，与对照相比，注射poly(A)+ RNA的卵母细胞中依赖钠离子的谷氨酰胺摄取增加了60±12%。这种增加的摄取表现出肝脏系统N的特征：即它耐受锂离子替代钠离子，并且在锂培养基中被系统N底物L-组氨酸（5 mM）抑制，这与内源性依赖钠离子的谷氨酰胺转运不同。在随后的实验中，通过密度梯度分级分离大小的大鼠肝脏poly(A)+ RNA被注射到卵母细胞中。注射大小为1.9 - 2.8千碱基（kb）的poly(A)+ RNA导致依赖钠离子的谷氨酰胺转运显著刺激，从注射溶剂的卵母细胞中的0.178±0.060 pmol.min-1/卵母细胞增加到0.362±0.080 pmol.min-1/卵母细胞（p小于0.01）。一个较轻的组分，其poly(A)+ RNA大小小于1.9千碱基，导致依赖钠离子的谷氨酰胺摄取有类似增加，且在很大程度上耐受锂离子：注射该组分的卵母细胞中锂离子刺激的谷氨酰胺摄取从对照中的0.098±0.029 pmol.min-1/卵母细胞增加到0.230±0.070 pmol.min-1/卵母细胞（p小于0.05）。这种增强的锂离子刺激的谷氨酰胺摄取速率分别被1 mM和5 mM L-组氨酸抑制了28%和70%。在注射2.8 - 3.6 kb的poly(A)+ RNA的卵母细胞中，不依赖钠离子的谷氨酰胺摄取增加了72±12%（p小于0.001）。这些结果表明，具有与肝脏系统N、L和A（或ASC）相关特征的谷氨酰胺转运体可以在注射了特定大小的大鼠肝脏mRNA组分的非洲爪蟾卵母细胞中表达。
