鸡肠中血管活性肠肽的提取与测定

目的研究鸡肠不同部位血管活性肠肽的含量及其不同提取方法对血管活性肠肽含量的影响。方法分别取新鲜鸡的十二指肠、空肠、和回肠段,除去油脂及污物,用水冲洗干净,甩干水分,分别称取20.0g,采用不同方法提取,用酶联免疫吸附法测定血管活性肠肽的含量。结果鸡的十二指肠、空肠和回肠部位血管活性肠肽含量分别为25.53,25.01和23.27ng/g;热水提取含量较高,在酸性条件下加热容易破坏。结论鸡的全肠均含丰富的血管活性肠肽。     

Conformational study of vasoactive intestinal peptide by computational methods

The conformational profile of vasoactive intestinal peptide (VIP) was characterized using computational methods. The strategy devised included a close examination of the conformational profile of the first 11 residues fragment followed by a study that considered the compatibility of the different conformations found with a continuation of the polypeptide chain in a-helical conformation. Accordingly, a detailed analysis of the conformational preferences of the N-terminal fragment of VIP(1–11) was carried out within the framework of the molecular mechanics approach, using simulated annealing in an iterative fashion as the sampling technique. In a second step, low-energy structures of the fragment were fused to the remainder of the VIP chain in the form of two noninteracting α-helices, according to a model of the structure of the peptide proposed from NMR studies. After investigation for compatibility of each of the low-energy structures of VIP(1–11) with the two helical regions by energy minimization, only 5 of 35 structures were discarded. Analysis of the structures characterized indicates that most of the conformations of VIP(1–11), including the global minimum, can be described as bent conformations. Conformations exhibiting α-turns and ß-turns, previously proposed by NMR studies were also characterized. The conformational analysis also suggests that the common structural features found in VIP(1–11) should also be present in VIP. Finally, because of the sequence homology between VIP and Peptide T, and the fact that both are ligands of the CD4 receptor, both sets of low-energy conformations were compared for similarity. The relevance of these results as guidance of the design of new peptide analogs targeted to the CD4 receptor is also discussed.     

Structure-activity studies of vasoactive intestinal peptide (VIP): cyclic disulfide analogs

Analogs of vasoactive intestinal peptide with cysteine residues incorporated at selected sites within the sequence were prepared by solid phase methods, oxidized to the corresponding cyclic disulfides and purified to homogeneity by preparative HPLC. The cyclic compounds were assayed as smooth muscle relaxants on isolated guinea pig trachea, as bronchodilators in vivo in guinea pigs, and for binding to VIP receptors in guinea pig lung membranes. Of the analogs prepared at the N-terminus, one compound, Ac-[D-C～S⁶,D-Cys¹¹,Lys¹²,Nle¹⁷,Val²⁶,Thr²⁸]-VIP, was found to be a full agonist with slightly more than one tenth the potency of native VIP. Most other cyclic analogs in the N-terminal region were found to be inactive. A second analog, Ac-[Lys¹²,Cys¹⁷,Val²⁶,Cys²⁸]-VIP, was also found to be a full agonist with potency about one third that of native VIP. Furthermore, this compound was active as a bronchodilator in vivo in guinea pig, but with somewhat diminished potency as compared to native VIP. Strikingly, this cyclic compound was found to have significantly longer duration of action (>40 min) when compared to an analogous acyclic compound (5 min). The conformational restrictions imposed by formation of the cyclic ring structures may have stabilized the molecule to degradation, thus enhancing the effective duration of action. Analysis of this series of cyclic analogs has also yielded information about the requirements for the receptor–active conformation of VIP.     

Effect of vasoactive intestinal peptide on pulmonary surfactants phospholipid synthesis in lung explants

AIM: To investigate the effect of vasoactive intestinal peptide (VIP) on pulmonary surfactants (PS) phospholipidsynthesis in cultured lung explants. METHODS: Lung explants were cultured with serum-free medium, [methyl^3H]choline incorporation, total phospholipid, phosphatidylcholine, activity of choline-phosphate cytidylyltransferase(CCT) and CCTα mRNA level in lung explants were determined. RESULTS: (1) VIP (10^-10-10^-7 mol/L) for 16 hpromoted [methyl-^3H]choline incorporation in dose dependence and VIP (10.8 mol/L) for 2 h-16 h promoted [methyl-^3H]choline incorporation in time dependence. (2) VIP (10-8 mol/L) enhanced the contents of total phospholipidsand phosphatidylcholine in lung explants. (3) VIP (10^-10-10-7 mol/L) elevated microsomal CCT activity of lungexplants in dose dependence. (4) VIP (10.8 mol/L) increased expression of CCTα mRNA in lung explants andalveolar type II cells (ATII). (5) [D-P-C1-Phe(6)-Leu(17)]-VIP (10^-6 mol/L), a VIP receptors antagonist, abolishedthe increase of [^3H]choline incorporation, microsomal CCT activity and CCTα mRNA level induced by VIP (10-8mol/L) in lung explants. CONCLUSION: VIP could enhance synthesis of phosphatidylcholine, the major compo-nent of pulmonary surfactants by enhancing microsomal CCT activity and CCTα mRNA level via VIP receptor-mediated pathway.     

藿香正气液对大鼠血管活性肠肽的影响

目的：观察藿香正气液对大鼠血管活性肠肽（VIP）的影响。方法：80只Wistar大鼠随机分为藿香正气液组和时照组，分别给大鼠灌服藿香正气液或生理盐水1，6h后：①观察大鼠胃肠动力的变化；②运用放射免疫分析法测定血浆、胃窦和空肠组织匀浆中VIP含量的变化；③运用免疫组化法显示胃窦、空肠组织中VIP阳性产物的分布情况。结果：①应用藿香正气液后1，6h，大鼠胃肠动力显著增强，以用药后1h更为明显。②藿香正气液明显降低血浆、胃窦及空肠组织匀浆中VIP的含量，减少胃窦和空肠组织中VIP的阳性产物。结论：藿香正气液促胃肠动力作用可能与其对VIP的影响有关。     

Spontaneous hydrolysis of vasoactive intestinal peptide in neutral aqueous solution

Hydrolysis of radioiodinated vasoactive intestinal peptide (VIP) was observed in buffered aqueous solution at neutral pH and 38 °C. The reaction displayed apparent first-order kinetics at initial peptide concentrations below 3 nM (K_obs= 1.5 × 10^?5s^?1), but the rate deviated below predicted values at higher peptide concentrations. The rate constant derived from the reaction progress curve over three half lives, starting at a concentration of 82 PM peptide, was also consistent with a first-order process. The reaction results in several products that were isolated and characterized as peptide fragments. Based on the identity of these fragments, we deduced hydrolysis at five different peptide bonds clustered between residues 17–25 of VIP. Control experiments were devised to eliminate trivial explanations for the peptide hydrolysis. Peptides representing the C-terminal segment 15–28 and the internal segment 14–22 assayed by analogous methods and under identical conditions were not degraded at a measurable rate. Sodium dodecyl sulfate and acetonitrile, agents known to influence the secondary structure of VIP, inhibited its spontaneous hydrolysis, as did chloride salts of sodium and calcium, albumin and a peptide unrelated to VIP. The rate and product distribution are inconsistent with known pathways of peptide degradation involving cyclic imide or anhydride formation at asparagine or aspartate residues. We suggest that the breakdown of VIP in dilute solutions represents an autolytic process.     

血管活性肠肽化学及生物学稳定性研究

目的:考察血管活性肠肽(vasoactive intestinal peptide,VIP)的化学及生物学的稳定性,为VIP的制剂学研究提供依据。方法:考察VIP在不同pH值(2.0,4.0,7.0,9.0,11.0,13.0)、不同离子强度溶液、不同温度以及人工胃液和人工肠液中的稳定性,用HPLC法检测VIP含量变化。结果:VIP的稳定性具有pH依赖性,VIP在酸性及中性条件下稳定,pH≤7时几乎无降解,但VIP在碱性条件下不稳定,pH=13时30min已完全降解;离子强度对其稳定性无影响;VIP在冷冻条件下稳定性良好,在冷藏条件下低浓度存在降解;VIP在人工胃液和人工肠液中降解迅速,0 min即完全降解,无法检测到主药峰。结论:VIP化学及生物学的稳定性差,口服无效。     

盐酸戊乙奎醚对小鼠血浆血管活性肠肽的影响

目的观察盐酸戊乙奎醚对小鼠胃肠动力及血浆血管活性肠肽的影响。方法健康昆明小鼠30只,体重18～24g,雌雄不限,随机分为对照组、阿托品组和盐酸戊乙奎醚组,每组10只。阿托品组腹腔注射阿托品0.3mg/kg,盐酸戊乙奎醚组腹腔注射盐酸戊乙奎醚0.3mg/kg,对照组给予等容量0.9%氯化钠溶液。3组给药15min后以营养性半固体糊0.8ml/只灌胃,30min后处死小鼠,采用放免法测定血管活性肠肽水平,计算胃内残留率。结果与对照组比较,阿托品组血管活性肠肽水平升高(P<0.05),盐酸戊乙奎醚组血管活性肠肽水平差异无统计学意义(P>0.05);与阿托品组比较,盐酸戊乙奎醚组血管活性肠肽水平下降(P<0.05)。与对照组比较,阿托品组胃内残留率升高,盐酸戊乙奎醚组胃内残留率差异无统计学意义(P>0.05);与阿托品组比较,盐酸戊乙奎醚组胃内残留率降低(P<0.05)。结论与阿托品比较,盐酸戊乙奎醚不抑制胃肠运动,其对血管活性肠肽的分泌无影响。     

Involvement of vasoactive intestinal polypeptide in nicotine-induced relaxation of the rat gastric fundus

1. Nicotine-induced relaxation and release of vasoactive intestinal polypeptide (VIP)- and peptide histidine isoleucine (PHI)-like immunoreactivity (LI) were measured in longitudinal muscle strips from the rat gastric fundus.
2. Under non-cholinergic conditions (0.3 μM atropine), nicotine (3–300 μM) produced concentration-dependent relaxations of the 5-hydroxytryptamine (3 μM)-precontracted strips. Under non-adrenergic non-cholinergic (NANC) conditions (0.3 μM atropine+1 μM phentolamine+1 μM nadolol), relaxations induced by sub-maximal nicotine concentrations (10 and 30 μM) were significantly smaller, while that produced by the highest concentration used (300 μM) was similar to that seen under non-cholinergic conditions.
3. Re-exposure to the same nicotine concentration 1 h later induced smaller relaxations, indicating desensitization. The reductions seen in the second responses were proportional to the concentration used.
4. Under non-cholinergic conditions, the relaxant response to 30 μM nicotine was abolished by hexamethonium (100 μM) and significantly reduced by tetrodotoxin (TTX, 3 μM). The TTX-resistant component was not observed under NANC conditions.
5. NANC relaxation induced by 30 μM nicotine was significantly reduced by a specific anti-VIP serum (approximately 35% less than that seen with normal rabbit serum).
6. Nicotine (30–300 μM) caused significant, concentration-dependent increases in the outflow of VIP- and PHI-LI from the strips; these effects were also diminished with re-exposure. The increases in both types of immunoreactivity evoked by nicotine (300 μM) were abolished by hexamethonium (300 μM), TTX (3 μM) and a calcium-free medium.
7. These findings indicate that VIP and possibly PHI are involved in NANC relaxation of the rat gastric fundus induced by nicotine.

     

Furanoid sugar amino acids as dipeptide mimics in design of analogs of vasoactive intestinal peptide receptor binding inhibitor

Abstract: In this study we describe the development of peptidomimetic analogs of the potent vasoactive intestinal peptide receptor binding inhibitor, Leu¹ ‐Met² ‐Tyr³ ‐Pro⁴ ‐Thr⁵ ‐Tyr⁶ ‐Leu⁷ ‐Lys⁸ ‐OH 1, by incorporating furanoid sugar amino acids (SAAs) 2‐4 into the molecule. The furanoid SAAs 2‐4 were used as dipeptide isosteres to replace Tyr³ ‐Pro⁴ or Pro⁴ ‐Thr⁵ in sequence 1 . The resulting analogs 5 ‐ 9 were tested for their anti‐cancer activities in vitro, following the standard MTT assay on a panel of human cancer cell lines. One of the potent analogs, 6a was tested in vivo for tumor regression on primary colon tumor xenografted nude mice. Our experimental results suggest that many of these analogs show either retention or enhancement of biological activity.     

Solid-phase synthesis of chicken vasoactive intestinal peptide by a mild procedure using Nα-9-fluorenylmethyloxycarbonyl amino acids

The octacosapeptide amide corresponding to the entire amino acid sequence of chicken vasoactive peptide (VIP) was assembled on a p-benzyloxybenzylamine resin support using the base-labile 9-fluorenylmethyloxycarbonyl as N^α-protecting group, cleaved by mild acid treatment, and purified by gel-filtration and ion-exchange chromatography. The symmetrical anhydride coupling was employed and monitored by two independent methods, and acetic anhydride termination was incorporated to minimize formation of deletion peptides. The homogeneity of the final product, obtained in 18% yield, was assessed by t.l.c., disc electrophoresis, amino-terminal amino acid analysis, and amino acid analyses of acid and enzyme hydrolysates. The purified chicken VIP was shown to be active on gastric acid secretion and on pancreatic blood flow. Previously reported ring closure of the Asp-Asn unit seemed to be at a minimum, owing to the mild basic and acid treatments.     

Influence of thiorphan and phosphoramidon on the relaxant effect of vasoactive intestinal polypeptide and non-adrenergac non-cholinergic neurone stimulation in the rat gastric fundus

Relaxations were induced in longitudinal muscle strips of the rat gastric fundus by exogenous administration of vasoactive intestinal polypeptide (VIP) and by transmural stimulation in the presence of atropine. These responses were not influenced by two neutral endopeptidase inhibitors, thiorphan (3 × 10⁻⁵ M) and phosphoramidon (10⁻⁵ M). This suggests that neutral endopeptidase is not involved in the breakdown of exogenous VIP and non-adrenergic non-cholinergic inhibitory transmitters of the rat gastric fundus.     

Effect of kainic acid injections and other brain lesions on vasoactive intestinal peptide (VIP)-stimulated formation of cAMP in rat brain

Summary In rat striatal slices, both intrastriatal kainic acid injection, which destroys striatal neurones, and intranigral injection of 6-hydroxydopamine (6-OHDA), which leads to a degeneration of dopamine nerve terminals in the striatum, reduced vasoactive intestinal peptide (VIP)-induced cAMP accumulation by approximately 60%. Cortical ablation, which leads to degeneration of cortico-striatal fibres, had no effect on striatal VIP-induced cAMP formation, Knife cut lesions transecting the stria terminalis, which destroy afferent fibres to the amygdala, decreased the VIP-induced increase in cAMP in amygdala slices by 40%, while kainic acid injection into the amygdala had no effect. Kainic acid injection into several other brain regions, including hippocampus, cortex and hypothalamus also failed to affect the VIP-elicited increase in cAMP in slices, despite reductions in choline acetyltransferase, glutamate decarboxylase, cyclic nucleotide phosphodiesterase and basal levels of cAMP. The results of a study of the effects of various VIP fragments on cAMP stimulation in striatal and cortical slices suggests that the entire sequence of VIP is necessary for full activity. The results suggest that VIP may be involved in neuromodulation or neurotransmission in the striatum and/or nigrostriatal pathway and also in the stria terminalis from the bed nucleus to the amygdala.     

Stimulation of retinal adenylate cyclase by vasoactive intestinal peptide (VIP)

The effects of vascoactive intestinal peptide (VIP), secretin and glucagon on retinal adenylate cyclase were studied in rabbit, rat and calf retinas. The results demonstrate the presence of a highly active VIP sensitive adenylate cyclase in mammalian retina. The effects are not enhanced by the guanine nucleotide analog 5′-guanylyl-imidodiphosphate (Gpp(NH)p). Glucagon and secretin also caused significant increases in retinal adenylate cyclase activity although, in general, the magnitude of these effects was less than that of VIP.     

Antidipsogenic action of vasoactive intestinal peptide in the rat

The effect of vasoactive intestinal peptide (VIP) on the intake of water was studied in the rat. Intracerebroventricular administration of vasoactive intestinal peptide strongly inhibited drinking in rats deprived of water, but peripheral administration had no effect, indicating that the site of action was central. Drinking induced by angiotensin II was also markedly blocked by simultaneous administration of vasoactive intestinal peptide. The results indicate that in the rat, vasoactive intestinal peptide may play a role in the control of intake of water as a neuropeptide thirst inhibitor.     

Inhalable liposomal formulation for vasoactive intestinal peptide

Inhalation of vasoactive intestinal peptide (VIP) was suggested as promising treatment option of various lung diseases like asthma and pulmonary hypertension. However, the medical use of peptides is limited by their short half-life due to rapid enzymatic degradation in the airways. For that reason, we recently developed unilamellar nano-sized VIP-loaded liposomes (VLL). Now we investigated their applicability for inhalation purposes.

After nebulisation by a mouthpiece ventilation inhaler we found the particle size almost unaffected, being in a size range appropriate for bronchiolar deposition; we observed no peptide release due to nebulisation. The VIP release kinetics from VLL were tested by an ex vivo vasorelaxation model. Exposure to target organs revealed an immediate response, which was significantly retarded for VLL as compared to free VIP (p = 0.001). Using vasorelaxation as endpoint, we observed a sustained release and an extended pharmacological effect compared to equimolar free VIP.

The liposomes have the potential to improve VIP inhalation therapy by providing a “dispersible peptide depot” in the bronchi. Thereby, the release of VIP from liposomes may be triggered by exposure to cells, i.e. directly by ligand–receptor interactions.     

肝硬变门静脉高压症患者血中血管活性肠肽水平的测定

作者应用放射免疫方法测定了,肝硬变门静脉高压症患者(n=32)和对照组病人(n=32)门静脉、周围静脉及动脉血中的血管活性肠肽水平。肝硬变组/对照组病人血管活性肠肽(ng/ml)在门静脉血为:103.64±41.32/71.46±19.34;周围静脉血:88.93±24.16/49.92±18.31;动脉血:86.19±21.33/46.37±15.43;肝硬变组血中血管活性肠肽水平均显著高于对照组(P<0.01)。本研究结果表明肝硬变门静脉高压症患者门静脉、周围静脉和动脉血中血管活性肠肽存在着明显的代射紊乱,其对患者全身和内脏血流动力学的影响以及对门静脉高压症发生、发展的关系值得深入研究。     

STIMULATION OF SODIUM AND WATER SECRETION WITHOUT INHIBITION OF GLUCOSE ABSORPTION IN THE RAT JEJUNUM BY VASOACTIVE INTESTINAL PEPTIDE (VIP)

1. Infusion of vasoactive intestinal peptide (VIP) into the arterial blood supply of the small intestine in anaesthetized rats did not alter either the perfusion pressure in the superior mesenteric artery or the active absorption of glucose from the jejunum, but did produce a large net secretion of Na⁺ and water into the lumen of the jejunum. 2. The results are compared to the effects of prostaglandin E₁ which stimulates Na⁺ and water secretion and inhibits glucose active absorption in the rat jejunum.     

脑出血患者血浆和颅内血肿液中血管活性肠肽的含量

作者测定了脑出血患者静脉血浆和颅内血肿液中血管活性肠肽(VIP)含量的变化,发现脑出血患者静脉血浆VIP含量无显著改变,但颅内血肿液VIP含量显著低于静脉血浆VIP含量;不同时期颅内血肿液VIP含量比较,6～72h含量最低,72h后又有增高趋势。本研究结果表明,VIP参与了脑出血后脑循环改变的病理生理过程,尤其是参与了占位初始所致的局部脑损害过程,若能增加脑出血患者中枢神经系统(CNS)中VIP含量可能为脑出血的治疗指出一个新的途径。     

Effects of neuropeptide Y and vasoactive intestinal peptide on human venous smooth muscle in vivo

The direct and noradrenaline-modulating effects of neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) on venous smooth muscle were studied in healthy volunteers employing the dorsal hand vein compliance technique. Local infusions of NPY had no measurable effect on venous tone, but coinfusion of a constant high dose of NPY (242 pmol/min) with noradrenaline caused a 2.9-fold increase in the mean ED₅₀ for noradrenaline. The dilating effect of VIP on preconstricted hand veins was weak, maximal venodilation could not be achieved, because systemic side effects occurred at submaximally venodilating doses. Coinfusion of noradrenaline with a weakly venodilating, constant dose of VIP (93.2 pmol/min) caused a 0.5-fold decrease in the sensitivity for noradrenaline. Although functional interactions between NPY or VIP and noradrenaline could be demonstrated, the dosages of the peptides required were high. Thus our results indicate that neither NPY nor VIP exert a major direct or noradrenaline-modulating effect on human veins. Correspondence to: H. G. Eichler at the above address