Heiman M L, Nekola M V, Murphy W A, Lance V A, Coy D H
Endocrinology. 1985 Jan;116(1):410-5. doi: 10.1210/endo-116-1-410.
An improved rat anterior pituitary primary cell culture technique for studying GH-releasing activity of human pancreatic GH-releasing factor (hpGRF) and its analogs is described. Male pituitaries, dispersed by a combination of trypsin digestion and mechanical agitation, were plated at a density of 200,000 cells per well and cultured for 4 days. The attached cells were then stimulated with synthetic hpGRF which was comprised of the first 29 residues of the larger, originally isolated forms and which was amidated at the C-terminal (hpGRF-29). Analogs of hpGRF-29 which were modified in positions 1, 2, 3, or 7, and other secretagogues were similarly tested. Medium was collected after 3 h, and secreted hormone was measured by RIA. The cells were extremely sensitive to hpGRF-29 stimulation, and this effect was specific. The minimal effective dose of hpGRF-29 was an unprecedented 0.4 X 10(-15)M. No stimulation of LH, FSH, or PRL by hpGRF-29 was observed. Bombesin and vasoactive intestinal peptide were ineffective in stimulating GH release. [D-Trp6]LHRH (a potent LHRH agonist), also did not release GH but did stimulate secretion of LH and FSH at doses ranging from 0.4 X 10(-10)M to 1.0 X 10(-9)M. Responses of the cells to hpGRF-29 analogs were characterized by distinct heterologous dose-response curves. [D-Ala2]hpGRF-29 was 50 times more active than its parent 29-amino-acid peptide. [D-Thr7]hpGRF-29, another analog that differed from hpGRF-29 by the insertion of a D-isomer for the naturally occurring L-residue, was about 10,000 times less effective in stimulating GH secretion than was hpGRF-29 itself. Potencies of these and other analogs with respect to GH release in vitro were similar to those estimated in vivo. Thus, this primary cell culture provides an extremely sensitive, selective, and reproducible system for studying hpGRF structure-activity relationships. Further, such tremendous sensitivity to hpGRF can provide a system to study changes in pituitary sensitivity to hpGRF during different physiological states.
本文描述了一种改良的大鼠垂体前叶原代细胞培养技术，用于研究人胰腺生长激素释放因子（hpGRF）及其类似物的生长激素释放活性。雄性垂体通过胰蛋白酶消化和机械搅拌相结合的方法进行分散，以每孔200,000个细胞的密度接种并培养4天。然后用合成的hpGRF刺激贴壁细胞，该hpGRF由最初分离的较大形式的前29个残基组成，并在C末端酰胺化（hpGRF-29）。对在第1、2、3或7位进行修饰的hpGRF-29类似物以及其他促分泌素进行了类似的测试。3小时后收集培养基，并用放射免疫分析法测定分泌的激素。这些细胞对hpGRF-29刺激极为敏感，且这种作用具有特异性。hpGRF-29的最小有效剂量低至前所未有的0.4×10⁻¹⁵M。未观察到hpGRF-29对促黄体生成素（LH）、促卵泡生成素（FSH）或催乳素（PRL）的刺激作用。蛙皮素和血管活性肠肽在刺激生长激素释放方面无效。[D-色氨酸⁶]促性腺激素释放激素（LHRH，一种有效的LHRH激动剂）在0.4×10⁻¹⁰M至1.0×10⁻⁹M的剂量范围内也不释放生长激素，但能刺激LH和FSH的分泌。细胞对hpGRF-29类似物的反应具有明显不同的异源剂量反应曲线。[D-丙氨酸²]hpGRF-29的活性比其母体29个氨基酸的肽高50倍。[D-苏氨酸⁷]hpGRF-29是另一种类似物，它与hpGRF-29的不同之处在于用D-异构体取代了天然存在的L-残基，在刺激生长激素分泌方面比hpGRF-29本身的效力低约10,000倍。这些及其他类似物在体外对生长激素释放的效力与体内估计的效力相似。因此，这种原代细胞培养为研究hpGRF的结构-活性关系提供了一个极其敏感、选择性和可重复的系统。此外，对hpGRF的这种巨大敏感性可为研究不同生理状态下垂体对hpGRF敏感性的变化提供一个系统。
