Jeng Y J, Lolait S J, Strakova Z, Chen C, Copland J A, Mellman D, Hellmich M R, Soloff M S
Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston 77555, USA.
Neuropeptides. 1996 Dec;30(6):557-65. doi: 10.1016/s0143-4179(96)90039-6.
Oxytocin (OT) and vasopressin (AVP) stimulate insulin and glucagon release from the pancreas, and evoke insulin secretion from the rat insulinoma cell line, RINm5F. To determine which AVP/OT receptor subtype is expressed in RINm5F cells, we used PCR with degenerate primers to two transmembrane domains of the AVP (V1a, V1b (or V3), V2) and OT receptors (OTRs). The single PCR fragment identified was used to obtain a full length cDNA from a RINm5F cDNA library. Comparison of the deduced amino acid sequence of this clone with uterine OTR sequences from several species (human, sheep, bovine) and to the pig kidney epithelial cell (LLC-PK1) OTR reveals a very high degree of homology. After the RIN cell OTR cDNA was stably transfected into CHO cells (CHO-OTR), the cell membranes bound iodinated oxytocin antagonist with an apparent Kd comparable to that of RIN cell membranes and those from other OT target cells. Comparison of the ligand specificities of CHO-OTR and RIN cells membranes showed that the relative Ki values of a series of OT analogues were approximately equivalent in both preparations. The rank order of apparent Ki values also corresponded to published values for the rat myometrium, where OT elicits intracellular calcium transients, and increases inositol phosphate production. In uterin endometrium and amnion cells, OT stimulates prostaglandin release. Stimulation of CHO-OTR cells with OT caused an increase in cytosolic calcium concentration originating from both intracellular and extracellular sources, and a dose-dependent increase in inositol phosphate levels. Arachidonic acid release and PGE2 synthesis were also stimulated by OT. These findings (amino acid sequence homology, binding specificity, and signal transduction/second messenger production) suggest that OTRs from RINm5F cells are indistinguishable from OTRs that have been described in other tissues. The expression of OTR in pancreatic cells implies that OT plays a role in pancreatic function.
催产素（OT）和血管加压素（AVP）可刺激胰岛素和胰高血糖素从胰腺释放，并引发大鼠胰岛素瘤细胞系RINm5F分泌胰岛素。为确定RINm5F细胞中表达的是哪种AVP/OT受体亚型，我们使用简并引物对AVP（V1a、V1b（或V3）、V2）和OT受体（OTRs）的两个跨膜结构域进行了聚合酶链反应（PCR）。鉴定出的单一PCR片段用于从RINm5F cDNA文库中获取全长cDNA。将该克隆推导的氨基酸序列与几种物种（人类、绵羊、牛）子宫OTR序列以及猪肾上皮细胞（LLC-PK1）OTR进行比较，发现具有高度同源性。将RIN细胞OTR cDNA稳定转染到CHO细胞（CHO-OTR）后，细胞膜结合碘化催产素拮抗剂的表观解离常数（Kd）与RIN细胞膜及其他OT靶细胞膜的表观解离常数相当。CHO-OTR与RIN细胞膜配体特异性的比较表明，一系列OT类似物的相对抑制常数（Ki）值在两种制剂中大致相当。表观Ki值的排序也与大鼠子宫肌层的已发表值相对应，在大鼠子宫肌层中，OT可引发细胞内钙瞬变并增加肌醇磷酸生成。在子宫子宫内膜和羊膜细胞中，OT可刺激前列腺素释放。用OT刺激CHO-OTR细胞会导致细胞溶质钙浓度升高，其来源既有细胞内也有细胞外，同时肌醇磷酸水平呈剂量依赖性增加。花生四烯酸释放和前列腺素E2（PGE2）合成也受到OT的刺激。这些发现（氨基酸序列同源性、结合特异性以及信号转导/第二信使生成）表明，RINm5F细胞的OTRs与其他组织中描述的OTRs没有区别。OTR在胰腺细胞中的表达意味着OT在胰腺功能中发挥作用。
