生长激素释放因子

人胰生长激素释放因子(hpGRF)为44肽酰胺,能特异地促进垂体细胞释放生长激素,在结构上属于胰高血糖紊族,尤其与PHI 最近似。人注射hpGRF后,血中生长激素浓度可升高10倍,无明显副作用;对血浆中其它垂体激素及肠活性肽水平也无影响。GRF 在临床上可用于垂体分泌GH 的功能试验,治疗GRF缺乏症,促进创伤及骨折愈合。     

新的生长激素异形体基因的发现及全长cDNA的克隆

在通过大规模 ESTS技术对垂体基因表达谱的研究中 ,从垂体组织产生了 72 2 2个 ESTS,有385个 ESTs是代表生长激素 (GH)基因的 ,其中 1个为中间缺失 1 38bp的 GH异形体基因 ,并经巢式 RT- PCR及测序证实 ;该基因编码 1 71个氨基酸的前肽 ,去除信号肽后 ,其成熟肽由 1 45个氨基酸组成 ;经生物信息学处理 ,其分子量大小约 1 7k D;与正常生长激素分子内有 2个 GH受体结合位点不同 ,该新的 GH异形体分子内仅有一个生长激素受体的结合位点 .研究结果揭示 :正常垂体内存在着新的 GH异形体基因 ,该基因可能编码外周血中 1 6k D的生长激素 ;其功能可能为 2 2k D GH的生理拮挤剂 .     

生长激素分泌促进剂及构效关系研究进展

生长激素分泌促进剂是一类作用于垂体和下丘脑的具有专一性促生长激素释放作用的寡肽及其类似物.由于其分子质量小、活性高、可口服、作用专一而有可能成为新的生长激素治疗药物.目前已经发展了很多具有此类活性的多种结构的化合物,如肽、环肽、肽醇及非肽类似物等.尽管这类化合物的作用机制尚未完全明确,但已有证据表明存在新的调节生长激素分泌的途径和新的调节因子.     

猪胰脏中胰岛素拮抗肽的分离纯化及其性质的初步研究

从猪胰脏的酸醇提取液中纯化了一个新的活性多肽——胰岛素拮抗肽,它在整体和细胞水平上对胰岛素都有明显的拮抗作用。猪胰脏的酸醇提取液经CM-52、BioGel P-6、DEAE-52及RP-HPLC纯化后,可得到纯的胰岛素拮抗肽。它能剂量相关地抑制胰岛素在离体大鼠脂肪细胞中的促脂合成活性,抑制50％胰岛素活性时所需的胰岛素拮抗肽为2.0×10~(-10)mol/L与被拮抗的胰岛素剂量在同一水平上。该肽含有较多的碱性氨基酸,分子量的3 000,其N-末端是封闭的。胰岛素拮抗肽的上述理化特征及其对胰岛素的拮抗活性均不同于目前已知的胰脏活性多肽。它对脂肪细胞中胰岛素的拮抗作用可能具有重要的生理意义。     

细胞因子对GH3细胞中人生长激素基因表达的影响

为了研究细胞因子IL 11、睫状神经营养因子 (CNTF)和转化生长因子 (TGF β)对大鼠垂体GH3 细胞中人生长激素 (hGH)的基因启动子活性的影响及其与垂体特异性转录因子Pit 1蛋白的关系 ,首先建立含hGH基因启动子 (- 4 84～ 30bp)和荧光素酶融合基因的稳定转化GH3 细胞系 ,然后用细胞因子刺激 ,检测细胞培养液和细胞裂解液中GH的含量 ,反映它们对GH分泌和合成的影响 ;检测GH3 细胞内荧光素酶的变化 ,说明细胞因子对hGH基因启动子活性的作用。将Pit 1蛋白表达质粒 (pcDNA pit 1 cDNA)单独转染或与Pit 1反义寡核苷酸 (Pit 1OND)共转染于稳定转化的GH3 细胞中 ,观察加入细胞因子后荧光素酶的变化 ,探讨细胞因子的作用与Pit 1蛋白的关系。结果表明 ,IL 11(2 0nmol/L)、CNTF(10nmol/L)能刺激大鼠垂体GH3 细胞中GH的分泌和合成 ,增强GH3 细胞中荧光素酶的表达 ,分别增加到对照组的 12 6 %、136 %。TGF β(5nmol/L)能减少GH的分泌和合成 ,抑制荧光素酶的表达到对照组的 77%。Pit 1蛋白过表达和表达被抑制对细胞因子的调节作用没有影响。这说明IL 11、CNTF和TGF β可通过调节大鼠垂体GH3 细胞中hGH基因启动子活性影响GH的合成 ,Pit 1蛋白可能不参与这些调节作用。     

活化素抑制大鼠垂体GH_3细胞中人生长激素基因启动子的活性

本研究旨在探讨活化素(activin)对大鼠垂体GH3细胞中人生长激素(hGH)基因启动子活性的影响及其可能的调节机制。采用荧光素酶报告基因方法。首先建立含hGH基因启动子(-484～+30bp)和荧光素酶融合基因的稳定转染GH3细胞株,然后加入活化素或同时加入活化素与相关信号转导途径的激动剂,通过检测细胞培养液和细胞裂解液中GH的含量,以及GH3细胞内荧光素酶的变化,反映活化素对GH分泌、合成和hGH基因启动子活性的影响。将含不同长度hGH基因启动子序列的荧光素酶表达质粒分别转染GH3细胞,观察它们对活化素的反应,寻找活化素影响hGH基因启动子活性的关键DNA序列。结果表明,活化素(5,50nmol/L)能抑制大鼠垂体GH3细胞中GH的分泌和合成,活化素(5,50nmol/L)还能够抑制GH3细胞中hGH基因启动子的活性,使之仅达对照组的77%和69%;在胞内信号转导激动剂中,丝裂原活化蛋白激酶激酶(MAPKK/MEK)特异性激动剂C6ceramide(1μmol/L)完全取消了活化素对hGH基因启动子活性的抑制作用;活化素发挥抑制作用所需要的hGH基因启动子关键序列位于-132～-66bp之间。上述研究表明,活化素能抑制大鼠垂体GH3中hGH基因启动子的活性,它可能是通过抑制细胞内依赖MAPK的信号转导途径来完成的,同时hGH启动子上-132～-66bp的序列在其中发挥重要的作用。     

湖南烙铁头蛇毒中一个新的舒缓激肽增强肽（TmF）

通过 70 %冷甲醇抽提、SephadexG 15分子筛和反相高效液相色谱C1 8层析 ,从湖南产烙铁头蛇毒 (Trimeresurusmucrosquamatus)冻干粉中纯化得到一个新的舒缓激肽增强肽 (BPP) ,命名为TmF。该小肽的氨基酸序列为pGlu Gly Arg Pro Leu Gly Pro Pro Ile Pro Pro (pGlu表示焦谷氨酸 )。序列结果分析表明 ,TmF和已经分离得到的BPPs有很高的序列同源性。MSI MS测定其分子量为 1.110 7kD。TmF的生物学活性和药理学活性检测的结果表明 ,它增强舒缓激肽 (BK) ( 1mg L)诱导的离体豚鼠回肠纵行肌收缩的活性为 ( 1.13± 0 .3)单位 (mg L) ;TmF ( 5 .0× 10 - 4mg kg)可以增强约 ( 14± 2 )mmHg的由BK( 5 .0× 10 - 5mg kg)诱导的舒张压下降 ;在抑制剂试验中 ,不同剂量的TmF和 5× 10 - 2 mg的血管紧张素转化酶保温 30min ,结果表明大约2 .0× 10 - 3mg的TmF表现出对ACE水解活性的半数抑制率 (IC50 )。     

生长抑素的生物合成

1968年Krulin等观察到大鼠下丘脑含有抑制生长激素(GH)释放的物质;1973年Braz-eau等确定其是含14个氨基酸的多肽,定名为GH释放抑制激素或生长抑素(SS)。在脊椎动物SS选择性地分布于全身的细胞内,脑、胃肠和胰腺内含量最高,占25%、70%和5%。SS抑制腺垂体分泌GH、促甲状腺素等,可能也抑制神经垂体激素的释放、抑     

鲑鱼生长激素基因分泌型表达质粒的构建

生长激素(GH)是动物垂体前叶分泌的一种多肽类激素.应用分子重组及PCR等技术,构建了一种鲑鱼生长激素基因分泌型表达质粒pOsGH153,使编码鲑鱼生长激素成熟肽的序列克隆在大肠杆菌分泌型表达载体PIN-Ⅲ-ompA内,直接位于编码大肠杆菌外膜蛋白A信号肽序列的下游,在Lpp-Lac杂合启动子控制下,经IPTG诱导,分子量约23 000的鲑鱼生长激素在大肠杆菌中获得高效表达,该产物具有天然鲑鱼生长激素的免疫活性,直接分泌到细胞周质,而信号肽被自动剪除.     

下丘脑生长抑素神经元的研究

1967年,Krulich等发现,下丘脑提取物有抑制生长激素(GH)释放的作用,首先提出,下丘脑存在生长激素释放抑制因子(SRIF)。10年后,Brazeau等成功地分离、提纯、鉴定了SRIF,它为14个氨基酸组成的多肽,对GH释放抑制作用具有剂量反应关系。之后,人们又陆续发现了12肽、28肽、25肽的SRIF,它们和14肽的SRIF有相同的氨基酸顺序片段,并对GH有较强的抑制作用。随着基因工程的发展,学者们得以推断出SRIF的前体和前肽原。研究表明,各种哺乳动物具有生物活性的部分差别不大,其羧基端14个氨基酸序列为保守区。目前,将SRIF基因克隆并合成SRIF已成为现实,将SRIF广泛药用于临床已为期不远。     

Structure and function relationships of three novel hGHRH-GGC analogs

Growth hormone releasing hormone (GHRH) is one of the hypothalamus hormones. For its potential applications in agriculture and medicine, GHRH analog with higher activity and longer half-life has been looked for. By using the fusion expression with unique acid labile linker Asp-Pro and biochemical purification, the three novel GHRH peptides, Pro-Pro-hGHRH(1–44)-Gly-Gly-Cys, Pro-hGHRH(1–44)-Gly-Gly-Cys, and ¹Pro-GHRH(2–44)-Gly-Gly-Cys, were obtained. The peptide molecular weight with 5,455, 5,373 or 5,210 Da measured by EIS-MS is coincident with the actual values. The peptides at 0.1–10 μg/ml increased rat pituitary GH releases in a dose-dependent manner and at 5 μg/ml increased human pituitary GH releases. The activity comparisons showed that at 10 μg/ml there were significant between ¹Pro-hGHRH(2–44)-Gly-Gly-Cys and Pro-Pro-hGHRH(1–44)-Gly-Gly-Cys or Pro-hGHRH(1–44)-Gly-Gly-Cys, ¹Pro-hGHRH(2–44) (P < 0.05). The ¹Pro-hGHRH(2–44)-Gly-Gly-Cys showed the highest GH release from rat pituitary. The activity results showed that the N-terminal Pro modulations and the C-terminal Gly-Gly-Cys extension regulate GH release from pituitary. The results showed that the three peptides had good GH release, function-selectivity and species specificity.     

Study on the constructions and activities of three novel hGHRH analogs with N-terminal prolyl modulation+

Growth hormone releasing hormone is one of the hormones secreted by the hypothalamus. Because of its potential applications in agriculture and medicine, its short half-life and its expensive chemical synthesis, an analog with high GHRH activity and prolonged half-life has been looked for. The fusion partner gene with 127 amino acid residues of the C-terminus from L-asparaginase was recombined respectively with asp-pro-pro-hGHRH(1-44), asp-pro-hGHRH(1-44) or asp-1pro-GHRH(2-44) genes synthesized by PCR method to form three kinds of fusion proteins with unique acid labile linker Asp-Pro. The Pro-Pro-hGHRH(1-44), Pro-hGHRH(1-44), and 1Pro-GHRH(2-44) peptides were purified to homogeneity by means of cell disruption, washing of inclusion body, ethanol fraction precipitation, acid hydrolysis, SP-Sephadex C-25 and Sephadex G-10 column chromatography. The peptide molecular mass of 5235, 5139 or 4975 Da was determined by ESI mass spectroscopy and purity was determined by SDS-PAGE. In the study of in vitro activity, the antiserum kit against human GH and peptide doses of 0.1, 1.0 and 10 microg/ml were used. These peptides obviously increased GH releases both from human pituitary and from rat pituitary. The activity comparisons showed that there was significant difference between Pro-Pro-hGHRH(1-44)-Gly-Gly-Cys and Pro-Pro-hGHRH(1-44) at 1.0 microg/ml, or between 1Pro-hGHRH(2-44) and Pro-Pro-hGHRH(1-44) or Pro-hGHRH(1-44) at 10 microg/ml. The structure-activity relationships showed that at the original C-terminus, for rat pituitary the activity of the GHRH analog with 1Tyr-->Pro was more than that of Pro-Pro-hGHRH(1-44) or Pro-hGHRH(1-44). The results showed that the analogs had good GH-releasing activity and species specificity.     

Production and enhanced biological activity of a novel GHRH analog, hGHRH with an N-terminal Pro-Pro extension

Growth Hormone Releasing Hormone (GHRH) is one of the most important hormones in life. Because of its potential clinical importance, its short half-life, and its expensive chemical synthesis, an analog of hGHRH with a prolonged half-life and better activity has been studied for clinical application, especially for the treatment of muscle wasting, type II diabetes, or sleep disorders. The Pro-Pro-hGHRH(1-44) peptide has better activity. The fusion partner gene with 127 amino acid residues of the C-terminus from l-asparaginase was recombined with asp-pro-pro-hGHRH(1-44) gene synthesized by PCR method to form a fusion protein with the unique acid labile linker Asp-Pro. The recombinant protein was expressed to high levels in Escherichia coli BL21 (DE3). The Pro-Pro-hGHRH(1-44) peptide was purified to homogeneity by means of cell disruption, washing, ethanol precipitation, acid hydrolysis, and SP-Sephadex C-25, and Sephadex G-25 column chromatography. The fold of the purification was about 88 times and the yield was 1.1% of the total protein weight of the inclusion body. The peptide molecular mass of 5235.25 Da was determined by ESI mass spectroscopy. Its purity was determined by SDS-PAGE. In the study of the activity, we measured GH release of rat pituitary by using the antiserum kit against human GH. The peptide doses of 0.01, 0.1, 1.0, 7.72, and 20.9 microg/ml used, respectively, released the GH values of 0.1+/-0.1, 12.5+/-7.3, 16.6+/-5.8, 49.8+/-7.6, and 79.5+/-5.7 ng/ml whereas their blank controls, respectively, were 0.5+/-0.8, 4.1+/-2.6, 3.1+/-3.1, 4.7+/-1.8, and 1.2+/-0.3 ng/ml. The activity results of all dose groups except 0.01 microg/ml Pro-Pro-hGHRH(1-44) group and hGHRH(1-40) group showed that there were significant differences between GH released by the peptide and that by its blank control. With the increase of dosage, the differences were more significant. hGHRH(1-40) showed no measured GH release when the dose was up to 2 microg/ml. The activity results show that the Pro-Pro-hGHRH(1-44) peptide is a potential GH releasing analog.     

Molecular cloning and expression of a pituitary-specific receptor for growth hormone-releasing hormone.

A novel cDNA was isolated from rat pituitary mRNA using the polymerase chain reaction to amplify sequences encoding G protein-coupled receptors. The human homolog of this cDNA was isolated and expressed in human kidney 293 cells, and membrane fractions from these cells were found to bind human GH-releasing hormone (GHRH) with high affinity and specificity. GHRH also stimulates intracellular cAMP production in these transfected cells. The encoded receptor protein contains seven potential membrane-spanning domains, a hallmark of G protein-coupled receptors, and is homologous to previously identified receptors for secretin and vasoactive intestinal peptide, ligands that are related to GHRH. The rat GHRH receptor mRNA is expressed predominantly, if not exclusively, in the anterior pituitary gland, the major target for GHRH action. These results define a mechanism for cellular signaling by GHRH and provide the opportunity to examine the role of the GHRH receptor in growth abnormalities that involve the GH axis.     

Effects of peripheral and central administration of GHRH on feeding in aging LOU rats

In aging LOU rats, a decreased protein intake is restored by GH administration. To study the contribution of GHRH to macronutrient selection, hGHRH NH(2) was administered sc. (1 mg/kg B.W./day/14 days) or icv. (4 and 40 pmol/rat) to 11-, 19-, 24- and 28-month-old rats. Sc. administration induced a decreased food and lipid intakes from 24 months of age and a transient stimulation of protein intake in 19-month-old and older low protein eaters (<10% protein/total intake). Icv. administration induced decreased food and lipid intakes in all age groups. These results suggest that GHRH may regulate feeding through pituitary and/or hypothalamic GHRH receptor mechanisms.     

Evaluation of the biological potency of new agmatine analogs of growth hormone-releasing hormone in the bovine.

Four new growth hormone-releasing hormone (GHRH) analogs with C-terminal agmatine were compared with the parent human GHRH(1-29)NH2 fragment to assess their abilities to increase serum concentrations of growth hormone (GH) in the bovine. The four analogs were: [D-Ala2, Nle27] GHRH(1-28)Agm (JG-73); [desNH2-Tyr1, Ala15, Nle27] GHRH(1-28)Agm (MZ-2-51); [desNH2-Tyr1, Ala15, D-Lys21, Nle27] GHRH(1-28)Agm (MZ-2-75); and [desNH2-Tyr1, D-Lys12,21, Ala15, Nle27] GHRH(1-28)Agm (MZ-2-87). The special characteristic of all four GHRH analogs is that arginine was replaced by agmatine (Agm) in Position 29. Five pregnant Holstein cows received these peptides subcutaneously at the following doses: 0.0156, 0.0625, 0.25, 1, and 4 micrograms/kg body wt. Each cow received each analog-dose combination according to a 5 x 5 Greco-Latin square design repeated for the 5-week treatment. Each cow also received saline vehicle only at the end of the 5-week treatment. Blood samples were collected from 30 min before until 360 min after treatment injection. Total area under the GH response curves for the 6-hr sampling period for each dose of each GHRH analog was compared. There was a linear dose-dependent GH release in response to hGHRH(1-29)NH2 and its four GHRH(1-28)Agm analogs. At the dose of 0.25 micrograms/kg, two GHRH analogs, JG-73 and MZ-2-75, stimulated greater GH release than hGHRH(1-29)NH2 (P less than 0.05). No differences were seen at the two lowest doses, 0.0625 and 0.156 micrograms/kg. When both total area under the GH response curves and GH peak amplitudes for each treatment were averaged for all doses, JG-73 and MZ-2-75 stimulated greater GH release than hGHRH(1-29)NH2 (P less than 0.05). In summary, three GHRH(1-28)Agm analogs, JG-73, MZ-2-75, and MZ-2-51, were found to be 11.8, 11.3, and 6.5 times more potent, respectively, on a weight basis, than hGHRH(1-29)NH2 in stimulating the release of GH in cows.     

Chronic orchidectomy does not influence the sensitivity of the pituitary somatotropes to varying doses of GHRH administered intravenously to the adult male rhesus monkey

In the present study, the pituitary growth hormone (GH) response to graded doses of GH-releasing hormone (GHRH) was determined in intact (n = 3) and chronically orchidectomized (n = 3) adult rhesus monkeys (Mucaca mulatta). GHRH in doses of 0, 6.25, 12.5 and 25 microg/kg BW was infused through a teflon cannula implanted in the saphenous vein. Blood samples were collected 60 min before and 90 min after the injection of the neurohormone at 15 min intervals. All bleedings were carried out under ketamine hydrochloride anesthesia. The plasma levels of GH were determined by using AutoDELFIA time-resolved flouroimmunoassay, whereas plasma levels of testosterone and estradiol were determined using specific radioimmunoassay systems. The GH responses to GHRH were not significantly different between intact and chronically orchidectomized monkeys at any of the dose levels tested (p > 0.05). The administration of GHRH resulted in a significant (p < 0.05) stimulation of GH secretion at all the doses tested and in both the groups studied. In both intact and orchidectomized animals, the greatest response was observed at 6.25 microg/kg and no further increase was noted with the higher doses of GHRH. In conclusion, the present study suggests that chronic orchidectomy does not influence the sensitivity of the pituitary somatotropes to GHRH stimulation implying that the responsiveness of the pituitary somatotropes to GHRH is independent of testicular steroid modulation.     

Growth hormone responses to growth hormone-releasing hormone (1-29)-NH2 and a D-Ala2 analog in normal men

Synthetic analogs of growth hormone-releasing hormone, GHRH(1-29)-NH2 and D-Ala2 GHRH(1-29)-NH2 were administered as a bolus intravenous injection to five normal men in a dose range of 0.015 to 0.5 micrograms/kg body weight. Vehicle only was administered in a control study. Peak responses to GHRH analogs occurred at 15 or 30 min. An increase in the integrated plasma growth hormone (GH) response was observed at each dose. The dose-response curve of GHRH(1-29)-NH2 indicated that it has a similar molar potency to GHRH(1-40) and GHRH(1-44). The potency of D-Ala2 GHRH(1-29)-NH2 was approximately twice that of GHRH(1-29)-NH2. Neither analog affected blood levels of PRL, TSH, LH, FSH, ACTH, insulin, glucagon, glucose, cortisol, free thyroxine, and free triiodothyronine. No side effects were noted other than transient flushing with the highest dose administered. The findings demonstrate GHRH(1-29)-NH2 and its D-Ala2 analog are potent stimulators of GH release and have potential application in clinical medicine.     

Model-projected mechanistic bases for sex differences in growth hormone regulation in humans

Models of physiological systems facilitate rational experimental design, inference, and prediction. A recent construct of regulated growth hormone (GH) secretion interlinks the actions of GH-releasing hormone (GHRH), somatostatin (SRIF), and GH secretagogues (GHS) with GH feedback in the rat (Farhy LS, Veldhuis JD. Am J Physiol Regul Integr Comp Physiol 288: R1649-R1663, 2005). In contrast, no comparable formalism exists to explicate GH dynamics in any other species. The present analyses explore whether a unifying model structure can represent species- and sex-defined distinctions in the human and rodent. The consensus principle that GHRH and GHS synergize in vivo but not in vitro was explicable by assuming that GHS 1) evokes GHRH release from the brain, 2) opposes inhibition by SRIF both in the hypothalamus and on the pituitary gland, and 3) stimulates pituitary GH release directly and additively with GHRH. The gender-selective principle that GH pulses are larger and more irregular in women than men was conferrable by way of 4) higher GHRH potency and 5) greater GHS efficacy. The overall construct predicts GHRH/GHS synergy in the human only in the presence of SRIF when the brain-pituitary nexus is intact, larger and more irregular GH pulses in women, and observed gender differences in feedback by GH and the single and paired actions of GHRH, GHS, and SRIF. The proposed model platform should enhance the framing and interpretation of novel clinical hypotheses and create a basis for interspecies generalization of GH-axis regulation.     

生长激素促释放剂受体配体的研究进展

生长激素促释放剂是一种合成的小分子化合物,它通过生长激素促释放剂受体而起作用,该受体是一种新的G蛋白偶联受体。以前曾认为生长激素促释放剂受体是一种孤儿受体,直到近年来从人和鼠的胃中鉴定到Ghrelin的存在,而改变了这种看法。Ghrelin是包含28个氨基酸残基的肽,在3号位的丝氨酸位点有辛酰化基团。该肽是在X／A样细胞分泌颗粒中发现的,Ghrelin的发现表明促垂体分泌生长激素可能不止受到来自下丘脑的生长激素释放激素的调节,同时还可能受到来自胃和下丘脑的Ghrelin的调节。