Williams J A, McChesney D J, Calayag M C, Lingappa V R, Logsdon C D
Department of Physiology, University of California, San Francisco 94143.
Proc Natl Acad Sci U S A. 1988 Jul;85(13):4939-43. doi: 10.1073/pnas.85.13.4939.
The expression of receptors for cholecystokinin (CCK) and other similar acting Ca2+-mobilizing hormones was studied in Xenopus laevis oocytes. Poly(A)+ RNA was prepared from pancreatic AR42J cells, which normally express receptors for CCK and bombesin and the RNA injected into oocytes. The presence of these pancreatic receptors on the oocytes was then demonstrated by hormone-induced mobilization of 45Ca2+. CCK receptors were present 1 day (maximum, 2 days) after injection of RNA and were generally proportional to the amount of poly(A)+ RNA injected (1-50 ng). Oocyte CCK receptors retained selectivity for CCK analogs (CCK8 greater than unsulfated CCK8 greater than CCK4) and were blocked by the specific CCK receptor antagonist CR 1409. When poly(A)+ RNA was subjected to size fractionation on sucrose gradients, activity-inducing CCK receptors showed a single peak centered at 3 kilobases. The generality of this oocyte system for expressing Ca2+-mobilizing hormone receptors was further shown by expression of a response to bombesin after injection of AR42J cell RNA and a response to vasopressin and angiotensin II when poly(A)+ RNA from rat liver was injected. No response to CCK was demonstrable after injection of liver RNA, demonstrating the specificity of this assay.
在非洲爪蟾卵母细胞中研究了胆囊收缩素(CCK)及其他类似作用的钙动员激素受体的表达。从胰腺AR42J细胞制备多聚腺苷酸(poly(A)+)RNA,该细胞正常表达CCK和蛙皮素受体,并将RNA注入卵母细胞。然后通过激素诱导的45Ca2+动员证明卵母细胞上存在这些胰腺受体。注射RNA后1天(最多2天)出现CCK受体,且通常与注入的poly(A)+ RNA量(1 - 50 ng)成正比。卵母细胞CCK受体对CCK类似物保持选择性(CCK8大于未硫酸化的CCK8大于CCK4),并被特异性CCK受体拮抗剂CR 1409阻断。当poly(A)+ RNA在蔗糖梯度上进行大小分级分离时,诱导活性的CCK受体显示出一个以3千碱基为中心的单峰。注射AR42J细胞RNA后对蛙皮素产生反应,注射大鼠肝脏的poly(A)+ RNA后对血管加压素和血管紧张素II产生反应,进一步证明了该卵母细胞系统用于表达钙动员激素受体的通用性。注射肝脏RNA后未显示对CCK的反应,证明了该测定方法的特异性。