Smith P F, Neill J D
Proc Natl Acad Sci U S A. 1987 Aug;84(15):5501-5. doi: 10.1073/pnas.84.15.5501.
The quantitative relationship between receptor binding and hormone secretion at the single-cell level was investigated in the present study by combining a reverse hemolytic plaque assay for measurement of luteinizing hormone (LH) secretion from individual pituitary cells with an autoradiographic assay of 125I-labeled gonadotropin-releasing hormone (GnRH) agonist binding to the same cells. In the plaque assay, LH secretion induces complement-mediated lysis of the LH-antibody-coated erythrocytes around the gonadotropes, resulting in areas of lysis (plaques). LH release from individual gonadotropes was quantified by comparing radioimmunoassayable LH release to hemolytic area in similarly treated cohort groups of cells; plaque area was linearly related to the amount of LH secreted. Receptor autoradiography was performed using 125I-labeled GnRH-A (a superagonist analog of GnRH) both as the ligand and as the stimulant for LH release in the plaque assay; the developed silver grains appearing over cells in the center of plaques were measured microscopically. The grains appeared to represent specific and high-affinity receptors for GnRH because no pituitary cells other than gonadotropes bound the labeled ligand and grain development was progressively inhibited by coincubation with increasing doses of unlabeled GnRH-A. Despite high correlations between mean grain number and mean plaque area in dose-response curves, the correlation coefficients for these parameters were low (range 0.02-0.38) in the individual cells comprising these groups. We conclude that GnRH receptor number for any individual gonadotrope is a weak determinant of the amount of LH it can secrete; nevertheless, full occupancy of all its GnRH receptors is required for any gonadotrope to reach its full LH-secretory capacity. Apparently the levels of other factors comprising the steps along the secretory pathway determine the secretory capacity of an individual cell.
在本研究中，通过将用于测量单个垂体细胞促黄体生成素（LH）分泌的反向溶血空斑试验与125I标记的促性腺激素释放激素（GnRH）激动剂与相同细胞结合的放射自显影试验相结合，研究了单细胞水平上受体结合与激素分泌之间的定量关系。在空斑试验中，LH分泌诱导促性腺激素周围包被有LH抗体的红细胞发生补体介导的裂解，从而产生裂解区域（空斑）。通过比较放射免疫分析法可检测到的LH释放量与经类似处理的同组细胞的溶血面积，对单个促性腺激素细胞释放的LH进行定量；空斑面积与分泌的LH量呈线性相关。在空斑试验中，使用125I标记的GnRH-A（GnRH的一种超激动剂类似物）作为配体和LH释放的刺激剂进行受体放射自显影；在显微镜下测量出现在空斑中心细胞上的显影银颗粒。这些颗粒似乎代表了GnRH的特异性高亲和力受体，因为除促性腺激素细胞外，没有其他垂体细胞能结合标记的配体，并且随着未标记的GnRH-A剂量增加共同孵育，颗粒显影逐渐受到抑制。尽管在剂量反应曲线中平均颗粒数与平均空斑面积之间存在高度相关性，但在构成这些组的单个细胞中，这些参数的相关系数较低（范围为0.02 - 0.38）。我们得出结论，任何单个促性腺激素细胞的GnRH受体数量对其能够分泌的LH量的决定作用较弱；然而，任何促性腺激素细胞要达到其最大LH分泌能力，需要其所有GnRH受体完全被占据。显然，构成分泌途径各个步骤的其他因素的水平决定了单个细胞的分泌能力。
