Wormald P J, Abrahamson M J, Millar R P
Department of Chemical Pathology, University of Cape Town Medical School, Observatory, Republic of South Africa.
J Clin Endocrinol Metab. 1988 Jun;66(6):1272-7. doi: 10.1210/jcem-66-6-1272.
A method for culturing cells from human pituitaries obtained at autopsy for studies of the secretion of gonadotropins and other hormones is described. The EC50 of GnRH-stimulated LH secretion from human pituitary cells was 1.25 nmol/L, similar to that found with rat pituitary cells (2.45 nmol/L). The GnRH agonist (D-Ala6, N alpha MeLeu7, Pro9-NEt)GnRH was 10-fold more active in stimulating LH release from both human and rat pituitary cells (EC50, 0.21 and 0.35 nmol/L, respectively). The GnRH antagonists (Ac-D-Nal(2)1,D-alpha-Me-4-ClPhe2,D-3-Pal3,D-Arg6,D-Ala10 )GnRH and (D-pGlu1,D-Phe2,D-Trp3,6)GnRH both inhibited 3 nmol/L GnRH-stimulated LH release from human pituitary cells (IC50, 2.40 and 78.6 nmol/L, respectively) with potencies similar to those observed in the rat (IC50, 0.68 and 33.0 nmol/L, respectively). Stimulation of the human pituitary cells with 100 nmol/L GnRH for 44 min resulted in biphasic release of LH and FSH. The initial phase occurred during the first 4 min of stimulation, and the second protracted plateau phase continued for at least the ensuing 40 min. The kinetics of LH and FSH release were identical, with no evidence of differential release at any stage. GnRH-stimulated LH and FSH secretion was Ca2+ dependent. The presence of 3 mmol/L EGTA prevented the gonadotropin response to GnRH, and the Ca2+ ionophore A23187 stimulated release of both LH and FSH. The phorbol ester 12-O-tetradecanoyl phorbol-13-acetate also stimulated gonadotropin secretion, indicating that activation of protein kinase-C is able to elicit LH release. GnRH desensitized the cells to subsequent stimulation with GnRH. After initial 2-h incubation with 0.1-100 nmol/L GnRH, LH release during a second 2-h incubation with the same doses was reduced by 50-100%. The reduction in FSH ranged from 63-92%. Depletion of gonadotropin pools may contribute to desensitization, since total LH and FSH cell content decreased 48% and 49%, respectively, after 100 nmol/L GnRH stimulation for 4 h. We conclude that functionally active cells can be cultured from human pituitaries obtained postmortem and that the mechanism of GnRH action and the relative potencies of GnRH analogs closely parallel those in the rat.
本文描述了一种用于培养从尸检获得的人垂体细胞的方法，用于研究促性腺激素和其他激素的分泌。人垂体细胞中GnRH刺激的LH分泌的EC50为1.25 nmol/L，与大鼠垂体细胞（2.45 nmol/L）相似。GnRH激动剂（D-Ala6，NαMeLeu7，Pro9-NEt）GnRH在刺激人和大鼠垂体细胞释放LH方面的活性高10倍（EC50分别为0.21和0.35 nmol/L）。GnRH拮抗剂（Ac-D-Nal(2)1，D-α-Me-4-ClPhe2，D-3-Pal3，D-Arg6，D-Ala10）GnRH和（D-pGlu1，D-Phe2，D-Trp3,6）GnRH均抑制3 nmol/L GnRH刺激的人垂体细胞LH释放（IC50分别为2.40和78.6 nmol/L），其效力与在大鼠中观察到的相似（IC50分别为0.68和33.0 nmol/L）。用100 nmol/L GnRH刺激人垂体细胞44分钟导致LH和FSH的双相释放。初始阶段发生在刺激的前4分钟，第二个持续的平台期至少持续随后的40分钟。LH和FSH释放的动力学相同，在任何阶段都没有差异释放的证据。GnRH刺激的LH和FSH分泌是Ca2+依赖性的。3 mmol/L EGTA的存在阻止了促性腺激素对GnRH的反应，Ca2+离子载体A23187刺激了LH和FSH的释放。佛波酯12-O-十四烷酰佛波醇-13-乙酸酯也刺激促性腺激素分泌，表明蛋白激酶-C的激活能够引发LH释放。GnRH使细胞对随后的GnRH刺激脱敏。在用0.1 - 100 nmol/L GnRH初始孵育2小时后，在第二次用相同剂量孵育2小时期间LH释放减少了50 - 100%。FSH的减少范围为63 - 92%。促性腺激素池的耗尽可能导致脱敏，因为在100 nmol/L GnRH刺激4小时后，LH和FSH细胞总含量分别下降了48%和49%。我们得出结论，可从死后获得的人垂体中培养出功能活跃的细胞，并且GnRH作用机制和GnRH类似物的相对效力与大鼠中的情况密切相似。
