Simard M, Pekary A E, Smith V P, Hershman J M
Medical Services, Veterans Administration Wadsworth Medical Center, Los Angeles, California 90073.
Endocrinology. 1989 Jul;125(1):524-31. doi: 10.1210/endo-125-1-524.
In the present study we have examined the in vivo effects of thyroid hormone and TRH on secretory tissue concentrations of TRH and TRH-Gly (pGlu-His-Pro-Gly), a TRH precursor. Within secretory granules, TRH-Gly is converted to TRH through alpha-amidation of the C-terminal proline residue, using Gly as the NH2 donor. Using specific RIA, we measured the TRH-Gly immunoreactivity (TRH-Gly-IR) and TRH-IR concentrations in tissues from the reproductive and gastrointestinal systems, adrenals, and other internal organs in euthyroid, hypothyroid, and T4-treated 250-g Sprague-Dawley male rats. TRH-Gly-IR concentrations were more than 2-fold higher than TRH-IR concentrations within the adrenal, pancreas, bowel, and stomach at the time of death. Untreated hypothyroidism and exogenous TRH significantly increased adrenal TRH-Gly-IR levels. Pancreatic TRH-Gly levels increased about 2-fold in hypothyroid rats. Incubation at 60 C significantly increased TRH-Gly-IR levels in the pancreas, adrenal, bowel, stomach, and epididymis by 14-, 3-, 6-, 6-, and 6-fold, respectively. Also after 60 C incubation increases in the TRH-Gly-IR/TRH-IR ratio of 2.7-, 4-, and 1.7-fold were observed in the pancreas, epididymis, and bowel, respectively. Pooled tissue extracts were fractionated by cation exchange and reverse phase HPLC for characterization of TRH-Gly-IR. Both chromatographic methods revealed a major peak of TRH-Gly-IR coeluting with synthetic TRH-Gly. Incubation at 60 C caused 13.5-, 4.1-, 1.5-, and 5-fold increments in the TRH-Gly-IR for adrenal, pancreas, prostate, and thyroid, respectively, compared to the immediately extracted control aliquots. Cation exchange and reverse phase HPLC also revealed production of higher mol wt TRH precursor peptides after incubation at 60 C for 4 or 20 h. Only the TRH-Gly-IR peak coeluting with pGlu-His-Pro-Gly was converted into TRH by rat brain alpha-amidating enzyme. The data suggest that biosynthesis of TRH occurs in rat extrahypothalamic tissues and may be modulated by thyroid status, iv TRH, and selective thermal inactivation of enzymes that convert prepro-TRH to TRH.
在本研究中，我们检测了甲状腺激素和促甲状腺激素释放激素（TRH）对TRH及TRH前体TRH-甘氨酸（pGlu-His-Pro-Gly）分泌组织浓度的体内效应。在分泌颗粒内，TRH-甘氨酸通过以甘氨酸作为氨基供体，将C末端脯氨酸残基进行α-酰胺化而转化为TRH。我们使用特异性放射免疫分析法（RIA），测定了正常甲状腺、甲状腺功能减退以及经T4处理的250g雄性Sprague-Dawley大鼠生殖系统、胃肠道、肾上腺及其他内脏组织中的TRH-甘氨酸免疫反应性（TRH-Gly-IR）和TRH免疫反应性（TRH-IR）浓度。死亡时，肾上腺、胰腺、肠道和胃内的TRH-Gly-IR浓度比TRH-IR浓度高出2倍多。未经治疗的甲状腺功能减退和外源性TRH显著增加了肾上腺TRH-Gly-IR水平。甲状腺功能减退大鼠的胰腺TRH-甘氨酸水平增加了约2倍。60℃孵育显著增加了胰腺、肾上腺、肠道、胃和附睾中的TRH-Gly-IR水平，分别增加了14倍、3倍、6倍、6倍和6倍。同样，在60℃孵育后，胰腺、附睾和肠道中TRH-Gly-IR/TRH-IR比值分别增加了2.7倍、4倍和1.7倍。将合并的组织提取物通过阳离子交换和反相高效液相色谱法（HPLC）进行分离，以鉴定TRH-Gly-IR。两种色谱方法均显示，TRH-Gly-IR的一个主要峰与合成的TRH-甘氨酸共洗脱。与立即提取的对照等分试样相比，60℃孵育使肾上腺、胰腺、前列腺和甲状腺的TRH-Gly-IR分别增加了13.5倍、4.1倍、1.5倍和5倍。阳离子交换和反相HPLC还显示，60℃孵育4或20小时后产生了更高分子量的TRH前体肽。只有与pGlu-His-Pro-Gly共洗脱的TRH-Gly-IR峰被大鼠脑α-酰胺化酶转化为TRH。数据表明，TRH的生物合成发生在大鼠下丘脑外组织中，并且可能受到甲状腺状态、静脉注射TRH以及将前促甲状腺激素释放激素转化为TRH的酶的选择性热失活的调节。
