Stachura M E, Tyler J M, Kent P G
Endocrinology. 1986 Sep;119(3):1245-53. doi: 10.1210/endo-119-3-1245.
We previously reported the differential stimulation of stored and newly synthesized rat (r)GH release by human GH-releasing factor-44 (hGRF-44). Those studies were performed over a 3-h period in a static in vitro incubation system. The present experiments focus on hGRF-44 effects upon release of new hormone (synthesized during tissue stimulation by the secretagogue) and were performed in in vitro perifusion to study the time course of the response. A double label ([14C], [3H]), immunoprecipitation protocol defined hormone release in relation to time of synthesis: intracellular stores of hormone were prelabeled with [14C]; subsequent exposure of prelabeled tissue to continuous concurrent [3H]leucine and 3 nM hGRF-44 defined newly synthesized hormone and associated it with the secretagogue. In a parallel set of experiments, 1 mM (Bu)2cAMP was substituted for hGRF-44. Prolonged exposure to hGRF-44 in perifusion stimulated an initial surge of stored [14C]rGH release which was followed by a rate of [14C]rGH release which declined rapidly but remained suprabasal. [14C]rGH release in response to (Bu)2cAMP was also biphasic, but the initial surge was delayed and the later stimulatory period was better sustained in comparison to responses to hGRF-44. Stimulation of stored [14C] rPRL release by hGRF-44 was observed in perifusion, confirming our observation in the static system. Release of newly synthesized, 3H-labeled rGH was immediately stimulated by either hGRF-44 or (Bu)2cAMP, and that stimulation was maintained throughout exposure to either secretagogue. In contrast, whereas newly synthesized [3H]rPRL release was stimulated by (Bu)2cAMP, its release in response to hGRF-44 resembled that in control experiments. No effect upon hormone synthesis was observed during the 3h of exposure to hGRF-44. In conclusion, these experiments confirm that hGRF-44 differentially stimulates release of both newly synthesized and stored rGH, and demonstrate differential dynamics in the response as well. Specifically, hGRF-44 stimulation of new rGH release is sustained while its effect on stored hormone simultaneously wanes. Further, when expressed as a percent of intracellular hormone available for release, new hormone is released more than 10 times faster than stored hormone. These observations argue for the existence of separate intracellular paths along which newly synthesized and stored hormone are released by the somatotroph. Finally, these data confirm that hGRF-44 stimulates release of stored rPRL without altering release of newly synthesized rPRL.
我们之前报道了人生长激素释放因子44（hGRF - 44）对储存的和新合成的大鼠（r）生长激素释放的不同刺激作用。那些研究是在静态体外孵育系统中进行3小时。本实验聚焦于hGRF - 44对新激素（在促分泌素刺激组织过程中合成）释放的影响，并在体外灌流实验中进行，以研究反应的时间进程。一种双标记（[14C]，[3H]）免疫沉淀方案确定了与合成时间相关的激素释放：激素的细胞内储存物预先用[14C]标记；随后将预先标记的组织暴露于连续同时存在的[3H]亮氨酸和3 nM hGRF - 44中，确定新合成的激素，并将其与促分泌素相关联。在一组平行实验中，用1 mM二丁酰环磷腺苷（（Bu）2cAMP）替代hGRF - 44。在灌流实验中长时间暴露于hGRF - 44刺激了储存的[14C]rGH释放的初始激增，随后是[14C]rGH释放速率迅速下降但仍高于基础水平。对（Bu）2cAMP反应的[14C]rGH释放也是双相的，但与对hGRF - 44的反应相比，初始激增延迟，后期刺激期维持得更好。在灌流实验中观察到hGRF - 44刺激储存的[14C]催乳素（rPRL）释放，证实了我们在静态系统中的观察结果。新合成的、3H标记的rGH释放立即受到hGRF - 44或（Bu）2cAMP的刺激，并且在整个暴露于任一促分泌素的过程中这种刺激都持续存在。相比之下，虽然新合成的[3H]rPRL释放受到（Bu）2cAMP的刺激，但其对hGRF - 44的反应释放与对照实验相似。在暴露于hGRF - 44的3小时内未观察到对激素合成的影响。总之，这些实验证实hGRF - 44对新合成的和储存的rGH释放有不同的刺激作用，并且在反应中也表现出不同的动力学。具体而言，hGRF - 44对新rGH释放的刺激持续存在，而其对储存激素的作用同时减弱。此外，当以可用于释放的细胞内激素百分比表示时，新激素的释放速度比储存激素快10倍以上。这些观察结果表明存在不同的细胞内途径，生长激素细胞通过这些途径释放新合成的和储存的激素。最后，这些数据证实hGRF - 44刺激储存的rPRL释放而不改变新合成的rPRL的释放。
