胃泌素C-端四肽的N-乙酰化和糖化及其对泌酸活性的影响

本文报道了胃泌素C—端四肽N—乙酰化和糖化的方法,并用大白鼠胃灌流技术研究了它们对泌酸活性的影响。结果表明,乙酰化和糖化均可提高其泌酸活性,其中葡萄糖—1—脱氧果糖四肽(Glc—1—deoxyfru—Trp—Met—Asp—Phe—NH_2)的泌酸活性超过了商品五肽胃泌素(Boc—α—Ala—Trp—Met—Asp—Phe—NH_2)     

胃泌素类似物的人工合成和泌酸活性研究

本文采用液相合成法合成了九种胃泌素类似物,并用大白鼠胃灌流技术测定其泌酸活性。若以五肽胃泌素(Boc-β-Ala-Trp-Met-Asp-phe-NH_2,Boc-五肽)的泌酸活性为100％,则胃泌其它类似物的活性分别为:Boc-四肽(Boc-Trp-Met-Asp-Phe-NH_2)30.32％,Boc-三肽(Boc-Met-Asp-phe-NH_2)0.13％;Fmoc-五肽(Fmoc-β-Ala-Trp-Met-ASP-Phe-NH_2)171％,Fmoc-四肽(Fmco-Trp-Met-Asp-Phe-NH_2)32.88％,Fmoc-三肽·(Fmoc-Met-Asp-Phe-NH_2)0.17％,五肽·TFA(β-Ala-Trp-Met-Asp-Phe-NH_2·TFA)17.45％,四肽·TFA(Trp-Met-Asp-phe-NH_2·TFA)5.58％。 实验结果显示四肽胃泌素类似物的活性这比三肽的高,表明胃泌素C-端片段中色氨酸残基对泌酸活性十分重要。在胃泌素类似物的N-端导入保护基同样提高它们的生物学活性,而Fmoc-保护基的作用还强于Boc-保护性。推测其主要原因是在N-式加上一个疏水性强的基团后有利于形成一个与受体相结合的活性构象。     

大鼠胃泌酸腺细胞膜和肝匀浆对五肽胃泌素及其类似物的降解

 本文用HPLC分离五肽胃泌素(Pentagastrin,Boc-β-Ala-Trp-Met-Asp-Phe·NH_2,PG_(1-5))及其N-端无保护基的TFA·五肽(β-Ala-Trp-Met-Asp-Pne·NH_2·TFA,TFA·PA_(1-5))被胃泌酸细胞膜和肝匀浆降解之产物,并对它们的氨基酸组成作了分析,又用薄层层析作了进一步验证,从而提出了酶促降解时可能的酶切位点。实验结果表明,N-端的Boc-基团能大幅度增加对酶降解的抵抗力,这从一个方面解释了为什么商品五肽胃泌素的泌酸活性大于其它胃泌素类似物的原因。     

分离的泌酸细胞的生理学研究

本文介绍近年来用分离的泌酸细胞,研究组织胺、乙酰胆碱和胃泌素以及组织胺、前列腺素(PG)和环一磷酸腺苷(cAMP)在盐酸分泌过程中的作用及其相互关系的一些设想。(1)组织胺、乙酰胆碱和胃泌素均可直接与泌酸细胞上各自的受体相结合而引起盐酸分泌,它们之间有着相互加强的作用;(2)组织胺通过增加泌酸细胞内的cAMP促进酸的分泌;(3)PG从两个方面对胃粘膜产生保护作用,即对抗依赖于组织胺的cAMP形成和促进另一细胞系统内依赖于PG的cAMP的形成。     

应激状态下NO的胃粘膜保护作用及其与壁细胞泌酸的关系

目的:探讨应激状态下一氧化氮(NO)的胃粘膜保护作用及其与壁细胞泌酸的关系.方法:采用水浸-束缚应激(WRS)方法制备应激性溃疡(SU)动物模型,检测胃粘膜溃疡指数(UI)、胃粘膜NO含量和壁细胞H ,K -ATPase活性,观察L-硝基精氨酸甲酯(L-NAME)和L-精氨酸(L-Arg)对应激后大鼠壁细胞H ,K -ATPase活性及胃粘膜损伤的影响.结果:L-NAME(20 mg·kg-1)可使胃粘膜NO含量减少(P＜0.01),壁细胞H ,K -AT-Pase活性增加(P＜0.05),并加重应激所致的胃粘膜损伤;L-Arg(300 mg·kg-1)则使胃粘膜NO含量增加(P＜0.01),壁细胞H ,K -ATPase活性下降(P＜0.05),减轻应激所致胃粘膜损伤.结论:NO对应激状态下大鼠胃粘膜具有保护作用,其机制与抑制壁细胞H ,K -ATPase活性有关.     

土壤杆菌固氮生理特性研究

对根癌土壤杆菌C58/pGV3850菌株的固氮生理特性研究结果表明,该菌具有自生固氮活性,其固氮活性的最适pH为8.0,温度为30℃.固氮活性在对数生长后期(14h)最高,延缓期和静止期乙炔还原活性较低.该菌株好氧,通过氧呼吸和吸氢酶的作用达到避氧固氮.当通入氧气超过呼吸耗氧时,对固氮活性产生抑制作用.在培养过程中增加NH_4~+浓度,固氮活性会降低,当达到15mmol/L时完全丧失固氮活性,表现NH_4~+阻遏固氮酶蛋白合成.在乙炔还原测定系统中加入NH_4~+并不影响乙炔还原反应,说明没有NH_4~+关闭现象.培养过程中加入MSX(2.5mg/ml)能解除10mmol/L NH_4~+对固氮酶合成的阻遏作用.固定的游离氮不能以NH_4~+形式分泌于胞外.固氮过程中放出大量氢气,培养16小时产氢量达65μmol H_2/mg蛋白.在限碳条件下(0.2%蔗糖)其吸氢酶活力可达520nmol H_2·ml~(-1)·h~(-1).     

铃蟾肽(Bombesin)对离体灌流大鼠胃的胃泌素、生长抑素、胰升糖素及胃酸释放的影响

本研究用离体大鼠胃灌流技术来观察铃蟾肽对胃-肠激素及胃酸分泌的影响。2×10~(?)mol/L铃蟾肽以0.3ml/min速度作动脉内输注,可刺激胃酸的分泌,自2.50±0.05×10~(-1)增至5.50±1.50×10~(-1)mEq/min,但与外源性五肽胃泌素无协同作用。铃蟾肽引起两次性的门脉中胃泌索及生长抑素的释放,但抑制胰升糖素释放。这三种激素的基础释放率分别为:胃泌素62±8pg,生长抑素5.9±1.1ng,胰升糖素0.40±0.03ng/min;2×10~(-8)mol/L铃蟾肽以0.3ml/min作动脉内输注,胃泌素及生长抑素的峰值分别为1,000±20pg及12.2±2.0ng/min,胰升糖素的最低值为0.17±0.05ng/min,三种激素的反应均与铃蟾肽的浓度成正比。在胃腔流出液中也可测到上述三种激素,但量要少得多。     

代谢工程改造酿酒酵母合成葡萄糖二酸

葡萄糖二酸是一种高附加值的有机酸,广泛用于食品、医药和化工领域。为获得生产葡萄糖二酸的微生物细胞工厂,通过共表达小鼠来源的肌醇加氧酶(MIOX)及恶臭假单胞菌来源的醛酸脱氢酶(Udh),在酿酒酵母Saccharomyces cerevisiae CEN.PK2-1C中构建了葡萄糖二酸合成途径,产量为(28.28±3.15)mg/L。在此基础上,通过调控前体肌醇的合成途径,发现肌醇-1-磷酸合成酶(INO1)是葡萄糖二酸合成途径的限速酶,过量表达INO1,葡萄糖二酸产量达到(107.51±10.87)mg/L,提高了2.8倍。进一步弱化竞争支路中磷酸果糖激酶(PFK1)的表达,最终葡萄糖二酸的产量达到(230.22±10.75)mg/L,为进一步获得高产葡萄糖二酸细胞工厂提供基础。     

消炎痛对蛋白胨灌流狗胃窦小胃的效应的影响

本工作的目的在于确定内源性前列腺素是否参与在胃泌素释放与作用的机制。用具有胃肠四通瘘的狗进行慢性实验,用蛋白胨溶液灌流隔离的有神经支配的胃窦小胃以引起胃泌素的释放。收集全部泌酸腺区的胃液分泌,测量其酸排出,作为衡量胃窦粘膜释放胃泌素的指标。用消炎痛作为抑制前列腺素合成的药理学工具。 结果指出,消炎痛明显地减低蛋白胨引起的胃酸排出,且随剂量的增加而减低的程度也增大。此外,消炎痛对四肽胃泌素引起的胃酸分泌并无影响。总结以上结果,我们认为,内源性前列腺素似乎参与胃泌素释放的生理机制,但它对胃泌素引起酸分泌的作用并无影响。     

光合细菌产氢条件的研究

从被有机物污染的土壤及水域中分离得到13株属于红螺菌科的光合细菌,对其在苹果酸、乳酸、葡萄糖、乙酸、丙酸及丁酸中的光致产氢现象进行了研究。最高产氢量为39.8ml·20ml菌液~(-1)·48h~(-1),产氢活性为7.8ml·g生物量~(-1)·h~(-1)”。底物对不同菌株的产氢量与产氢活性均具有影响,pH对产氢过程也有明显的作用,大于6000lx的光强度对提高产氢活性已无明显作用。固定化细胞在静态培养条件下也能提高产氢能力,但延长产氢时间的作用不明显。     

Ulcerogenic and healing impairing actions of monochloramine in rat stomachs: effects of zinc L-carnosine, polaprezinc.

Effects of a novel zinc compound (polaprezinc), N-(3-aminopropionyl)-L-histidinato zinc, on the mucosal ulcerogenic and healing impairing responses induced by monochloramine (NH2Cl) were examined in rat stomach. Oral administration of NH2Cl (> 60 mM) produced severe hemorrhagic lesions in unanesthetized rat stomachs with a marked increase of thiobarbituric acid reactants (TBAR). Pretreatment of the animals with polaprezinc (3 approximately 30 mg/kg, p.o.) showed a dose-dependent inhibition against gastric ulcerogenic and TBAR responses induced by NH2Cl (120 mM). Likewise, mucosal exposure to NH4OH (60 mM) in urethane anesthetized stomachs made ischemic by bleeding from the carotid artery (1 ml per 100 g body w.t.) resulted in severe gastric lesions. This ulcerogenic response caused NH4OH plus ischemia was also attenuated by prior application of polaprezinc as well as taurine (25 mg/ml, 1 ml). On the other hand, the healing of gastric mucosal lesions induced by NH2Cl occurred more slowly than of ethanol-induced lesions, and the latter was significantly delayed by the repeated administration of NH2Cl. Polaprezinc (> 10 mg/kg, p.o.) given twice daily for 7 days not only accelerated the healing of NH2Cl-induced gastric lesions but also antagonized the delayed healing of ethanol-induced lesions in the presence of NH2Cl as well. Polaprezinc showed a scavenging action against NH2Cl in vitro. These results suggest that NH2Cl caused deleterious action on the healing of pre-existing acute lesions as well as irritating action to the mucosa in the rat stomach. Polaprezinc not only protects the stomach against injury caused by NH2Cl but also promotes healing of NH2Cl-induced gastric lesions as well as the delayed healing of ethanol-induced lesions caused by NH2Cl. Although the detailed mechanisms underlying these actions of polaprezinc remain unknown, they may be partly attributable to a scavenging action of this agent against NH2Cl.     

Over-expression of 70-kDa heat shock protein confers protection against monochloramine-induced gastric mucosal cell injury

The major heat shock protein, HSP70, is known to be involved in cytoprotection against environmental stresses mediated by their function as a "molecular chaperone". Monochloramine (NH(2)Cl) is a potent cytotoxic oxidant generated by neutrophil-derived hypochlorous acid and Helicobacter pylori urease-induced ammonia. In this study, to evaluate the cytoprotective effect of HSP70 against NH(2)Cl-induced gastric mucosal cell injury, rat gastric mucosal cells (RGM-1) were stably transfected with pBK-CMV containing the human HSP70 gene (7018-RGM-1) or pBK-CMV alone (pBK-CMV-12) as control cells. These cells were treated with various concentrations of NH(2)Cl. Cell Viability was determined by MTT assay and the direct plasma membrane damage was analyzed by lactate dehydrogenase (LDH) release assay. Apoptosis was determined by DNA fragmentation analysis. NH(2)Cl caused injury to pBK-CMV-12 cells in a concentration-dependent manner. NH(2)Cl-induced gastric cell injury was significantly diminished in HSP70 over-expressing cell line (7018-RGM-1) both necrosis and apoptosis compared to the control cell line (pBK-CMV-12) transfected with CMV vector alone. These result suggest that overexpression of HSP70 plays an important role in protecting gastric cells against NH(2)Cl-induced injury.     

Central regulation of gastric acid secretion by platelet-activating factor in anesthesized rats

PAF has been implicated in the pathogenesis of acute gastric injury. When given peripherally, PAF induces severe gastric mucosal damage. PAF metabolizing enzymes are present in the brain but the central effects of PAF on the stomach are unknown. We have investigated in the rat the gastric secretion and gross mucosal integrity in response to intracerebroventricular (icv) PAF and compared it with that to icv TRH, a known central gastric secretagogue. Gastric acid output was markedly increased by TRH (171.6 +/- 26.3 mumol/h mean +/- SE) and by 20 micrograms/kg/h iv pentagastrin (107.6 +/- 23.6) when compared to controls receiving icv vehicle (20.2 +/- 7.5; p less than 0.01 for both). In contrast, acid output decreased after icv PAF (13.5 +/- 7.5). Furthermore, icv PAF markedly inhibited acid output stimulated by iv pentagastrin (45.1 +/- 7.03; p less than 0.05). Morphological studies showed acute gastric mucosal erosions after icv TRH and no damage was observed after icv PAF or vehicle. Thus, icv PAF reduces pentagastrin stimulated acid output and does not alter gastric mucosal integrity, whereas icv TRH stimulates acid secretion and induces gastric injury. The opposite effects of PAF and TRH suggests the existence of a gastric modulatory system at the central level.     

Stomach-brain communication by vagal afferents in response to luminal acid backdiffusion, gastrin, and gastric acid secretion

Vagal afferents play a role in gut-brain signaling of physiological and pathological stimuli. Here, we investigated how backdiffusion of luminal HCl or NH(4)OH and pentagastrin-stimulated acid secretion interact in the communication between rat stomach and brain stem. Rats were pretreated intraperitoneally with vehicle or appropriate doses of cimetidine, omeprazole, pentagastrin, dexloxiglumide (CCK(1) receptor antagonist), and itriglumide (CCK(2) receptor antagonist) before intragastric administration of saline or backdiffusing concentrations of HCl or NH(4)OH. Two hours later, neuronal activation in the nucleus of the solitary tract (NTS) and area postrema was visualized by c-Fos immunohistochemistry. Exposure of the rat gastric mucosa to HCl (0.15-0.5 M) or NH(4)OH (0.1-0.3 M) led to a concentration-dependent expression of c-Fos in the NTS, which was not related to gender, gastric mucosal injury, or gastropyloric motor alterations. The c-Fos response to HCl was diminished by cimetidine and omeprazole, enhanced by pentagastrin, and left unchanged by dexloxiglumide and itriglumide. Pentagastrin alone caused an omeprazole-resistant expression of c-fos, which in the NTS was attenuated by itriglumide and prevented by dexloxiglumide but in the area postrema was reduced by dexloxiglumide and abolished by itriglumide. We conclude that vagal afferents transmit physiological stimuli (gastrin) and pathological events (backdiffusion of luminal HCl or NH(4)OH) from the stomach to the brain stem. These communication modalities interact because, firstly, acid secretion enhances afferent signaling of gastric acid backdiffusion and, secondly, gastrin activates NTS neurons through stimulation of CCK(1) receptors on vagal afferents and of CCK(2) receptors on area postrema neurons projecting to the NTS.     

A pharmacological approach to cellular mechanisms of PGI2-induced gastric cytoprotection on ethanol-induced gastric mucosal damage in rats

The aims of this study were as follows: 1. to analyse the effects of drugs with different subcellular mechanisms on the PGI2-induced gastric cytoprotection in a non acid dependent (ethanol-induced) gastric ulcer model; 2. to identify the affinity and intrinsic activity curves on the PGI2-induced gastric cytoprotection; 3. to evaluate the main cellular mechanisms of PGI2-induced gastric mucosal defence. The observations were carried out on both sexes of CFY-strain rats, weighing 180 to 210 g. The gastric mucosal damage was produced by intragastric administration of 96% ethanol. The animals were killed at 1 hr after administration of ethanol, and the number and severity of gastric mucosal lesions (ulcers) was noted. Atropine, actinomycin D, cimetidine, mannomustine, dinitrophenol, epinephrine, pentagastrin, histamine, ouabain, tetracycline were given intraperitoneally (in different doses) at 30 min before administration of ethanol. The effects of these drugs were tested on the PGI2-induced (5 micrograms/kg was given intragastrically) gastric cytoprotection. It has been found that: 1. atropine, actinomycin D, cimetidine, epinephrine, ouabain, tetracycline and mannomustine inhibited the PGI2-induced gastric cytoprotection; 2. histamine, pentagastrin and 2,4-dinitrophenol enhanced the PGI2-induced gastric cytoprotection; 3. the molar concentrations of these drugs modifying the PGI2-induced gastric cytoprotection differed significantly. It has been concluded that: 1. the drugs stimulating or inhibiting the cell functions are capable to modify the extent of PGI2-induced gastric cytoprotection; 2. different subcellular mechanisms (oxidative phosphorylation, increased synthesis of proteins, ribonucleic and deoxyribonucleic acids, modifications of membrane-bound ATP-dependent energy systems) are involved in the development of PGI2-induced gastric cytoprotection.     

Pentagastrin and adenosine 3',5'-cyclic monophosphate stimulated secretion of somatostatin from perfused rat gastric mucosa.

Immunoreactive somatostatin is secreted by rat gastric mucosa perifused $\text{in vitro}$. Somatostatin release is stimulated by pentagastrin and cyclic AMP with theophylline. These results suggest that gastric mucosal somatostatin may have a paracrine action as feedback inhibitor of gastrin secretion.     

Participation of protein kinase C in hormonal regulation of pepsin secretion in the rat stomach]

It was shown that pentagastrin (0.5 micrograms/100 g of body mass) increases the activity of Ca2+ and phospholipid-dependent protein kinase C in the membrane fraction of rat gastric mucosa cells. This effect of pentagastrin is accompanied by a decrease of the protein kinase C activity in the cytosolic fraction. Chromatography of the membrane fraction revealed an additional peak of the enzyme activity. Analysis of isolated gastric mucosa cells demonstrated that pentagastrin (10(-8)-10(-6) M) (but not 10(-4) M histamine) added to the incubation mixture increased the protein kinase C concentration in the membranes. The pentagastrin effect was directly correlated with the amount of pepsin-producing chief cells in the cellular pools. Carbacholine, another well-known pepsin secretion stimulator, was able to activate, similar to pentagastrin, the protein kinase C activity. It is concluded that protein kinase C plays a prominent role in hormonal regulation of the chief gastric cell function.     

Inhibition of gastric acid secretion in dogs by neurotensin

Neurotensin is a tridacapeptide which has been isolated from bovine hypothalamus. The action of synthetic neurotensin was studied on gastric acid secretion in dogs provided with gastric pouches. Intravenously infused neurotensin, 50 ng × kg^?1 × min^?1, was found to produce a considerable inhibition of pentagastrin stimulated gastric acid secretion. On the other hand, there was no sign of inhibition of histamine induced gastric acid secretion. The experiments show that neurotensin, isolated from the central nervous system is a potent gastric secretory inhibitor and that it has a selective action in inhibiting gastric acid responses to pentagastrin but not to histamine.