Iwasaki K, Fujii A, Merchenthaler I, Groot K, Arimura A
Department of Obstetrics and Gynecology, School of Medicine, Tokai University.
Tokai J Exp Clin Med. 1987 May;12(2):117-24.
Growth hormone releasing factor (GHRF) was examined to determine whether it affects somatostatin (SRIF) release from cultured rat hypothalamic cells and fragments in vitro. The hypothalami of rat fetuses were collected on the 17th day of pregnancy under a dissection microscope. Thirty hypothalami were placed in phosphate buffered saline, and the cells were dispersed with 0.1% collagenase. The dispersed cells were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% horse serum and 2.5% fetal calf serum at 37 degrees C under 5% CO2 in air. On the 12th day of culture, the cells were washed with Krebs Ringer bicarbonate buffer containing glucose (KRBG), and then incubated with KRBG for 1 hour. The medium was replaced with KRBG alone (control) or KRBG containing test substances, and incubated for another hour. SRIF released into the medium was quantitated by RIA. The mean basal release of SRIF was 14.7 +/- 0.9 pg/dish/hour. One-tenth, 1, and 10nM hpGRF44 stimulated SRIF release by 1.4, 1.5, and 1.8 fold respectively in a dose-related manner. Ten nM ovine corticotropin releasing factor (o-CRF) also stimulated SRIF release by 2.3 fold. One, 10, and 100 nM hpGRF44, 10nM o-CRF, 10nM thyrotropin releasing hormone (TRH) and 60 mM K+ also stimulated SRIF release from rat hypothalamic fragments. Removal of Ca++ from the medium resulted in a decrease of basal release of SRIF. In Ca++ free medium, 10nM hpGRF44 failed to release SRIF. One-tenth nM hpGRF44, 10nM GnRH, and 10nM VIP have no effect on SRIF release statistically. The results of this study demonstrate that a high concentration of GHRF stimulates SRIF release from the hypothalamus in vitro, suggesting a possibility that GHRF may increase the release of SRIF from the median eminence and the hypothalamus in vivo under certain conditions.
研究生长激素释放因子（GHRF），以确定其是否会影响体外培养的大鼠下丘脑细胞和组织块中生长抑素（SRIF）的释放。在解剖显微镜下于妊娠第17天收集大鼠胎儿的下丘脑。将30个下丘脑置于磷酸盐缓冲盐水中，并用0.1%的胶原酶分散细胞。分散的细胞在含有10%马血清和2.5%胎牛血清的杜尔贝科改良伊格尔培养基（DMEM）中，于37℃、5%二氧化碳的空气中培养。培养第12天，用含葡萄糖的克雷布斯-林格碳酸氢盐缓冲液（KRBG）洗涤细胞，然后用KRBG孵育1小时。将培养基换成单独的KRBG（对照）或含测试物质的KRBG，再孵育1小时。用放射免疫分析法（RIA）定量测定释放到培养基中的SRIF。SRIF的平均基础释放量为14.7±0.9皮克/培养皿/小时。十分之一、1和10纳摩尔的人胰高血糖素样肽-44（hpGRF44）分别以剂量相关的方式刺激SRIF释放1.4、1.5和1.8倍。10纳摩尔的羊促肾上腺皮质激素释放因子（o-CRF）也刺激SRIF释放2.3倍。1、10和100纳摩尔的hpGRF44、10纳摩尔的o-CRF、10纳摩尔的促甲状腺激素释放激素（TRH）和60毫摩尔的钾离子也刺激大鼠下丘脑组织块释放SRIF。从培养基中去除钙离子导致SRIF基础释放量减少。在无钙培养基中，10纳摩尔的hpGRF44未能释放SRIF。十分之一纳摩尔的hpGRF44、10纳摩尔的促性腺激素释放激素（GnRH）和10纳摩尔的血管活性肠肽（VIP）对SRIF释放无统计学影响。本研究结果表明，高浓度的GHRF在体外刺激下丘脑释放SRIF，提示在某些条件下GHRF可能在体内增加正中隆起和下丘脑SRIF的释放。
