降钙素基因相关肽、血管活性肠肽在复合应激大鼠模型阴茎组织中表达及伊木萨克干预的研究

目的:检测复合应激大鼠模型阴茎组织中降钙素基因相关肽(CGRP)、血管活性肠肽(VIP)的表达,并观察伊木萨克片对二者表达的影响。方法:选用56只正常雄性SD大鼠,其中10只为正常对照组(N),余46只为造模组,采用富含环境雌激素饲料+寒冷环境的干预条件建立复合性应激大鼠模型(20 w),并随机将其分为模型组(B1)、自然恢复组(B2)和伊木萨克干预组(B3),药物干预2 w后,免疫组化及Western blot方法检测大鼠阴茎组织中CGRP、VIP的表达。结果:①大鼠阴茎组织中CGRP表达:B1、B2组较N组明显减少(P0.05);B3组较B1、B2组明显增多(P0.05)。②大鼠阴茎组织中VIP表达:B1、B2组较N组显著降低(P0.05);B3组较B1、B2组显著升高(P0.05)。结论:复合应激大鼠模型阴茎组织中CGRP、VIP明显减少,伊木萨克片干预可抑制此变化。     

Embryonic Expression of Vasoactive Intestinal Peptide (VIP) and VIP Receptor Genes

Abstract: Vasoactive intestinal peptide (VIP) exhibits pronounced effects on the growth rate of cultured mouse embryonic day (E) 9.5 embryos and acts in tissue culture as a potent glial mitogen and neuron survival factor. However, previous studies using immunohistochemistry or in situ hybridization in the rat have not revealed the presence and location of VIP or VIP mRNA in the early developing embryo CNS. Using a sensitive in situ hybridization assay with a ³³P-labeled riboprobe, we show here that the VIP gene is expressed at least as early as E11 in the mouse hindbrain. Northern blot analysis on RNA from brain dissected from mouse embryos beginning at E14 confirmed that a correct-size mRNA for VIP was present by E14 and at later time points. Expression of the VIP2 receptor gene was also detected by northern analysis in E14 mouse brains. These studies support the hypothesis that VIP produced by the embryo exerts important effects on embryonic nervous system development.     

试论一氧化氮递质作用的特点

通过对一氧化氮（Nitric Oxide）递质和传统递质的比较,阐明了一氧化氮递质作用的特点。     

Interaction of a bovine thymic peptide extract with vasoactive intestinal peptide (VIP) receptors

Bovine t hymic peptide extract (1–100 g/ml) is shown to completely inhibit the binding of [¹²⁵I]VIP to rat blood mononuclear cells, lymphoid cells of spleen, and liver plasma membranes. In the three models, the bovine thymic peptide extract inhibits [¹²⁵I]VIP binding with a potency that is 4000–7000 times lower than that of the native VIP, on a weight basis. In rat liver plasma membranes, the bovine thymic peptide extract stimulates adenylate cyclase with a maximal efficiency that is similar to that of VIP. At maximal doses, VIP and thymic peptide extract do not exert an additive effect on adenylate cyclase, suggesting that the activation of the enzyme by the bovine thymic peptide extract occurs through VIP receptors. Finally, no VIP-like immunoreactivity was detected in the thymic peptide extract using an antiserum raised against mammalian VIP. All these data suggest the presence in the bovine thymic peptide extract of a new substance which behaves as a VIP agonist in rat.     

Stimulatory effect of hormonal signaling factors on (Ca2+−Mg2+)-ATPase activity in rat liver plasma membranes: Cross talk with regucalcin

The effect of hormonal signaling factors on (Ca²⁺–Mg²⁺)-ATPase activity in rat liver plasma membranes was investigated. The presence of inositol-glycan (10^–7–10^–5M), dibutyryl cAMP (10^–4 and 10^–3M) or inositol 1,4,5-trisphosphate (IP₃; 10^–6 and 10^–5 M) in the enzyme reaction mixture produced a significant increase in (Ca²⁺–Mg²⁺)-ATPase activity. These effects were completely inhibited by the presence of vanadate (10^–4 M), an inhibitor of the enzyme phosphorylation, and N-ethylmaleimide (5×10^–3 M), a SH group modifying reagent. Meanwhile, regucalcin, a Ca²⁺-binding protein isolated from rat liver cytosol, increased the enzyme activity by binding to the SH groups of (Ca²⁺–Mg²⁺)-ATPase in liver plasma membranes. The presence of regucalcin (0.25 M) with an effective concentration completely inhibited the effect of inositol-glycan (10^–5 M) to increase (Ca²⁺–Mg²⁺)-ATPase activity, while the effect of dibutyryl cAMP (10^–3M) or IP₃ (10^–5M) was not altered. The inositol-glycan effect was not modulated by the presence of dibutyryl cAMP or IP₃. Now, the preincubation of the plasma membranes with regucalcin did not modify the effect of inositol-glycan on the enzyme activity, suggesting that regucalcin competes with inositol-glycan for the binding to the plasma membranes. The present results suggest that there may be a cross talk with regucalcin and hormonal signaling factors in the regulation of (Ca²⁺–Mg²⁺)-ATPase activity in liver plasma membranes.     

Characterization of Functional Receptors for Vasoactive Intestinal Peptide in Bovine Cerebral Arteries

This study reports the characterization of receptors for vasoactive intestinal peptide (VIP) on membranes prepared from bovine cerebral arteries. By use of HPLC we prepared two purified monoiodinated VIP radioligands with nearly equivalent cerebral vasorelaxant potency as native VIP, [Tyr(125I)10 )VIP and [Tyr(125I)22]VIP. The former resulted in a higher proportion of specific binding to arterial membranes than the latter and was therefore thought to be the superior radioligand for receptor characterization. The binding of [Tyr(125I)10]VIP to cerebral arterial membranes was saturable, specific, reversible, and dependent on time and temperature. Scatchard analysis suggested the presence of a high- and a low-affinity binding site with KD values of 0.2 and 11 nM and receptor concentrations of 79 and 737 fmol/mg of protein, respectively. The dose-response curves for binding to the VIP receptor by the VIP-homologous peptides PHI, PHM, and rat growth hormone-releasing factor (GRF) were very similar to their dose-response curves for relaxation of cerebral arteries. The order of potency was VIP greater than PHM greater than PHI greater than rat GRF. It is suggested that the characteristics of the vascular VIP binding sites and the close correlation between the binding and vasorelaxant properties of VIP and its related peptides argue for the vascular binding sites being functional receptors for VIP.     

Studies on protein kinases in two human rectocolic cell lines by polyacrylamide gel electrophoresis.Effect of the vasoactive intestinal peptide

Electrophoresis and subsequent assay of the enzyme directly onto the gel has allowed a rapid and quantitative characterization of the cyclic AMP-dependent and -independent histone kinases, protamine, phosvitin and casein kinases in HT 29 and HRT 18 cells. The technique has been applied to soluble extracts from cytoplasmic and nuclear fraction prepared in the presence and absence of neutral detergent. A more precise identification of these enzymes has been possible by analysing enzyme fractions obtained after ion-exchange chromatography of the above extracts. The protein kinase equipment of both cell lines was found to be identical (11 major components) but with different relative proportions of several enzymes. In cytoplasmic extracts: VIP activates only the type I, cytosolic, (band 4) and the type II, membrane-bound, (bands 6 and 8) cyclic AMP-dependent histone kinases. These enzymes account, respectively, for 34 and 55% of the total histone kinases in HT 29 and HRT 18 cells. The cyclic AMP-independent histone kinases (band 1,2,5 and 7) also phosphorylate protamine; band 5 was found 3o be much higher (4-fold) in HT 29 cells. In addition, two casein/phosvitin kinases have been identified in both cell lines with phosphorylating activity similar to the total histone kinases. In the nuclear extract two cyclic AMP-independent histone kinases have been found with electrophoretic mobility differing from the cytoplasmic enzymes. Also, two phosvitin/casein kinases specifically nuclear, due to their chromatographical and electrophoretical behaviour, have been characterized.     

Vasoactive intestinal peptide binding autoantibodies in autoimmune humans and mice

Autoantibodies capable of binding the immunoregulatory neuropeptide vasoactive intestinal peptide (VIP) were detected in the sera of a mouse strain prone to autoimmune disease due to the lpr mutation (MRL/lpr). The autoantibodies were not present in control wildtype MRL/lpr mice, but they were readily detected in humans without autoimmune disease. The binding was due to low affinity VIP recognition. Increased VIP binding activity was evident in patients with systemic lupus erythematosus but not systemic sclerosis, Sjögren’s syndrome (SS), rheumatoid arthritis or autoimmune thyroiditis. Recombinant VIP binding Fv clones (fragment variable; the variable domains of the light and heavy chains antibody subunits joined with a peptide linker) were isolated from a phage display library prepared from lupus patients. One Fv clone displaying VIP-selective binding and several clones displaying cross-reactivity with unrelated peptides were identified. Replacement mutations in the VIP-selective clone were preferentially localized in the regions known to make contacts with the antigen, i.e. the complementarity determining regions, suggesting that the selective binding activity is due to immunological maturation of the antibodies. Frequent occurrences of autoantibody responses to VIP indicate that immunological tolerance to this neuropeptide can be readily broken. The depletion of VIP by specific antibodies in autoimmune disease may interfere with VIP regulation of T cells and inflammatory cells and result in further amplification of autoreactive immunological responses.     

地塞米松对哮喘豚鼠肺内VIP分布的影响

为探讨哮喘豚鼠肺内血管活性肠肽（ＶＩＰ）的分布及地塞米松对其分布的影响,将豚鼠随机分成哮喘组、地塞米松组和对照组三组,采用兔抗ＶＩＰ多克隆血清,免疫组织化学ＡＢＣ法和葡萄糖氧化酶－ＤＡＢ－镍染色技术,发现对照豚鼠肺内各级气道壁均可见ＶＩＰ免疫反应（ＶＩＰ－ＩＲ）阳性纤维分布。在平滑肌层中,纤维成束走行,且有较多分枝;而在基底膜和气道上皮内,纤维成单根行走。随气道口径的变小,纤维分布密度也逐渐变小。哮喘豚鼠肺内ＶＩＰ－ＩＲ消失。地塞米松处理后使ＶＩＰ－ＩＲ部分恢复。上述结果提示,豚鼠经反复抗原攻击后,肺内ＶＩＰ－ＩＲ阳性纤维消失,这在实验性哮喘的发生过程中可能起一定作用。     

Activation of Choline Acetyltransferase by Vasoactive Intestinal Peptide

Addition of vasoactive intestinal peptide (VIP) to brain homogenates increased the activity of choline acetyltransferase (ChAT) but not that of acetylcholinesterase or glucose-6-phosphate dehydrogenase. Activity of ChAT was increased in the anterior hypothalamus and in the dorsal and ventral hippocampus, but not in the parietal cortex or posterior hypothalamus. Increased activity occurred rapidly after VIP addition to homogenates and was maximal at 10(-7)M concentration. Kinetic analysis indicates that the Vmax of the enzyme is increased and the Km for choline, but not acetyl-coenzyme A, is decreased in the presence of VIP. Results support a possible VIP-cholinergic interaction in the CNS.     

Synthesis of vasoactive intestinal peptide (VIP) via the mixed anhydride method

Porcine VIP was synthesized from three segments. The segments, VIP(1-6), VIP(7-13), and VIP(14-28), were synthesized via the Repetitive Excess Mixed Anhydride (REMA) method. The low solubility of the C-terminal segment was greatly improved by a temporary substitution of Asn28 by a beta-t-butyl aspartic acid ester. The segments VIP(1-6) and VIP(7-13) were purified by HPLC and coupled via the mixed anhydride method. The product was purified by gel filtration. VIP was synthesized from VIP(1-13) and VIP(14-28) by the same procedure. After deprotection, Met17-sulfoxide reduction, and purification by ion-exchange chromatography, the product was found to have the expected amino acid composition and biological potency. A HPLC purified sample was compared with several commercial preparations of varying purity.     

Interaction of secretin5–27 and its analogues with hormone receptors on pancreatic acini

The C-terminal tricosapeptide of secretin (S_5–27) and two analogues, one with asparagine replacing aspartic acid in position 15 (15-Asn-S_5–27) and one with lysine replacing aspartic acid in position 15 (15-Lys-S_5–27) were tested for their abilities to interact with hormone receptors on pancreatic acinar cells. In interacting with the receptors which prefer vasoactive intestinal peptide (vaso-active intestinal peptide-preferring receptors), the apparent affinity of 15-Asn-S_5–27 was equal to that of 15-Lys-S_5–27 and was greater than that of S_5–27. In interacting with secretin-preferring receptors, the apparent affinity of 15-Asn-S_5–27 was equal to that of S_5–27 and was greater than that of 15-Lys-S_5–27. In interacting with the secretin-preferring receptors each of the secretin fragments was approximately 2% as effective as secretin in causing an increase in cellular cyclic AMP. None of these fragments was able to cause a detectable increase in cyclic AMP mediated by the vasoactive intestinal peptide-preferring receptors. The dose vs. response curves for the action of secretin and vasoactive intestinal peptide on cellular cyclic AMP and on amylase secretion as well as the patetern of effects of secretin fragments on these actions indicated that the increase in amylase secretion caused by vasoactive intestinal peptide and secretin is mediated exclusively by the vasoactive intestinal peptide-preferring receptors. Furthermore, occupation of approximately 50% of the vasoactive intestinal peptide-preferring receptors is sufficient to cause maximal stimulation of amylase secretion.     

Interactions Between Vasoactive Intestinal Peptide and Dopamine in the Rabbit Retina: Stimulation of a Common Adenylate Cyclase

Although 3,4-dihydroxyphenylethylamine (dopamine, DA) and vasoactive intestinal peptide (VIP) have been reported to stimulate adenylate cyclase activity in the rabbit retina, possible interactions between VIP-sensitive and DA-sensitive adenylate cyclase systems have not been previously investigated. To elucidate the interactions between these two putative transmitter-stimulated cyclase systems, the effects of VIP, DA, and VIP + DA on the conversion of [alpha-32P]ATP to [32P]cyclic AMP in rabbit retinal homogenates were measured. VIP stimulated adenylate cyclase activity in a biphasic manner, suggesting that two classes of VIP receptors may be involved in the induction of cyclic AMP formation. DA was less potent than VIP, and stimulated cyclase activity with a monophasic dose-response curve. When assayed together, these stimulations were partially nonadditive, implying the existence of a common adenylate cyclase pool that may be stimulated by both putative neurotransmitters. The dopaminergic antagonist (+)-butaclamol completely blocked dopaminergic stimulation, but had no significant effect on VIP-induced stimulation, indicating that VIP interacts with specific VIP receptor sites, which are distinct from the dopaminergic receptor sites. Furthermore, the specific D-2 dopaminergic receptor agonist LY141865 demonstrated no inhibitory effect on adenylate cyclase activity, suggesting that the interaction between the VIP- and DA-sensitive adenylate cyclase systems does not result from a D-2 receptor-mediated cyclase inhibition in the rabbit retina. Finally, at maximally effective concentrations, DA and VIP were less potent than fluoride or forskolin in the stimulation of cyclic AMP formation, suggesting that adenylate cyclase pools that are not sensitive to DA and VIP may also be present in this retina.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in urine levels of substance P,vasoactive intestinal peptide and calcitonin-gene-related peptide in patients with urinary tract infections

Urinary tract infections (UTI) are important health problems and predisposing causes of UTI are not entirely known. Neuro-immune interactions play an important role in human health and disease. Capsaicin-sensitive sensory nerves which in nerve bladder extensively regulate immune system through neuropeptides such as substance P (SP), calcitonin-gene related peptide (CGRP) and vasoactive intestinal peptide (VIP). In addition these neuropeptides also have anti-bacterial effects. To determine how the levels of these peptides changes during UTI, 67 patients (50–90 years-old) diagnosed with UTI in Akdeniz University Faculty of Medicine Hospital were compared with 37 healthy people 50 years or older as the control group. Additionally, 7 patients with UTI symptoms (dysuria, urgency) but with sterile pyuria were also included in the study. Urine samples from 15 patients, whose symptoms regressed with control urine cultures being sterile, were taken after completion of the treatments. Urine neuropeptide levels were determined by ELISA. CGRP levels are significantly higher in patients with UTI, but did not associate with pyuria whereas SP and VIP levels were significantly lower in patients with sterile pyuria, indicating sensory nerve deficiency. Since CGRP exerts immunosuppressive effects, increased levels of the peptide may predispose to UTI. Furthermore, the connection between the observed sensory nerve deficiency and sterile pyuria warrants further studies.     

Localization of VIP-immunoreactive nerves in airways and pulmonary vessels of dogs,cats, and human subjects

Summary VIP-containing neurons were localized in lungs from dogs, cats, and human subjects by means of the indirect immunofluorescence technique. Nerve fibers and terminals were observed in the smooth muscle layer and glands of airways, and within the walls of pulmonary and bronchial vessels, especially at the medial-adventitial junction. VIP-positive nerve cell bodies were identified in ganglia located in the walls of bronchi. These findings provide an anatomic basis for the possible modulation of airway and pulmonary vascular function by this neuropeptide.Supported by National Research Service Award HL 5914 and by Lung Center Award HL 14187 from NHLBI, USPHS     

Vasoactive intestinal peptide as a bronchodilator in asthmatic subjects

Vasoactive intestinal peptide (V.I.P.) caused bronchodilatation in 7 asthmatic volunteers when given intravenously at the rate of 6 pmol kg-1 min-1 for 15 minutes during a double blind study. Mean baseline FEV1 was 2.8 (+/- 0.3 S.E.) which was 81% of predicted and increased by 0.21 (range 0.1-0.45) l after 15 minutes infusion (p greater than 0.02). Tachycardia and cutaneous flushing were also observed during the infusion. Subsequent induced bronchoconstriction with a predetermined dose of histamine was ameliorated at 180 seconds following challenge when compared with placebo. Mean fall in FEV1 0.26 compared with 0.741 when pre-infusion FEV1 was taken on baseline. Mean fall in FEV1 0.49 l compared with 0.75 l when the FEV1 immediately preceding challenge was used on baseline (p greater than 0.02). The demonstration that V.I.P. is a bronchodilator in asthmatics and ameliorates histamine induced bronchoconstriction has important implications for the pharmacology of asthma.     

A recombinant adenoviral vector encoding functional vasoactive intestinal peptide

Vasoactive intestinal peptide (VIP) is a small neuropeptide, which exerts pleiotropic functions. Based on its immunomodulatory, secretory, and possibly trophic effects, VIP is a valuable candidate molecule for the management of autoimmune disease. The purpose of this study was to develop a recombinant viral vector capable of directing the expression of functional VIP. The vector rAd5CMVhVIP was constructed and used to infect 293 cells. VIP expression was measured by an ELISA and function was evaluated by measurement of intracellular cAMP formation. rAd5CMVhVIP directed VIP expression and the transgenic VIP elicited a dose-dependent increase of intracellular cAMP, mediated through the VIP receptor VPAC(1). This is the first report showing the construction of a recombinant viral vector encoding biologically active VIP.