Cyclic AMP phosphodiesterases in human lymphocytes

The function of lymphocytes, like platelets, has been shown to be inhibited by agents which increase intracellular cyclic AMP. Two high-affinity cAMP phosphodiesterases (PDEs), the cyclic GMP-inhibited cAMP phosphodiesterase, PDE3, and the cAMP-specific phosphodiesterase PDE4, are known to regulate cAMP concentration in haemopoietic cells by degrading cAMP to AMP. We characterized the relative contribution of the two PDEs to total lymphocyte PDE activity. We then determined which of the different gene products, PDE3A, typical of myocardium and platelets, or PDE3B, typical of adipocytes, were present in lymphocytes. The PDE3-specific inhibitor, milrinone, and the PDE4 inhibitor, rolipram, suppressed hydrolysis by 70% and 30% respectively, which indicated that both PDE4 and PDE3 were present, and that PDE3 was predominant. RT-PCR yields the expected size fragment for the primer pair PDE3B and not for PDE3A. The DNA sequence obtained had > 95% identity with PDE3B. PDE3B appears to be the major cAMP PDE in lymphocytes. In contrast to human platelets, human lymphocytes appear to contain the PDE3B subtype. Since PDE3B in adipocytes is subject to hormonal regulation, lymphocytes may be similarly modulated. Understanding the role of cAMP regulation and the involvement of cAMP in lymphocyte function may have important implications in drug development.     

Vasoactive intestinal peptide stimulates N-acetyltransferase and hydroxyindole-O-methyltransferase activities and melatonin production in cultured rat but not in Syrian hamster pineal glands

The purpose of this study was to compare the responses of the Syrian hamster and rat pineal glands in organ culture to vasoactive intestinal peptide (VIP). The endpoints in these studies were the activities of pineal N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), as well as pineal and medium melatonin levels. When rat pineal glands were incubated with either VIP (1 microM) or isoproterenol (1 microM), a beta-adrenergic agonist, a significant increase in NAT and HIOMT activities and melatonin levels were observed within 3 hr. Conversely, during the day, VIP (1 microM) was ineffective in stimulating these parameters in hamster pineal gland after incubation times of either 2, 4, 6, or 8 hr. In another experiment, hamster pineal glands were collected from animals killed in the late dark period (after 30 min light exposure). In these glands, isoproterenol promoted NAT activity and melatonin production; however, VIP was ineffective in stimulating either NAT or HIOMT activities; likewise, VIP had no stimulatory effect on pineal melatonin levels at night. Finally, when hamster pineal glands at night were incubated with either 0, 10 nM, 100 nM, 10 microM, or 100 microM VIP, no changes in any parameter of melatonin synthesis were measured. The results indicate that the hamster pineal gland, unlike that of the rat, may not respond to VIP with an increased melatonin production.     

Interaction of vasoactive intestinal peptide with human blood mononuclear cells

The binding of vasoactive intestinal peptide (VIP) and the stimulation of adenylate cyclase were studied in mononuclear cells from human peripheral blood. The binding depended on time, temperature and pH, and was reversible, saturable and specific. Binding studies suggested the presence of 2 classes of binding site: a class with high affinity (Kd = 2.4 X 10(-10)M) and low capacity (8 fmoles/10(6) cells), and a class with low affinity (Kd = 8.0 X 10(-8)M) and high capacity (800 fmoles/10(6) cells) at 15 degrees C. Secretin displaced [125I]VIP from the cells with a 400-fold lower affinity than VIP, but glucagon, somatostatin and insulin did not show any effect. VIP was a potent and efficient stimulator of cyclic AMP production. The stimulation was observed at a concentration as low as 3 X 10(-11)M and depended on time, temperature and pH. Maximal cyclic AMP production (4-fold above basal levels) was observed with 10(-9) M at 15 degrees. Half-maximal response was obtained at 10(-10)M VIP. Secretin was an agonist of VIP but exhibited a 7000 times lower potency. Peripheral blood mononuclear cells constitute an easily accessible and suitable system for the study of VIP action in different physiological and pathophysiological conditions.     

Decreased vasoactive intestinal peptide levels and glutathione depletion in acquired megacolon

We reported decreased vasoactive intestinal peptide levels in acquired megacolon. The origin of altered neuropeptide levels is unknown, but recent work suggested that tissue antioxidants may function as neuroprotectants. Our hypothesis was that altered levels of inhibitory neurotransmitters in human colon are associated with depletion of the tripeptide thiol, glutathione. Normal colon samples (N=10; from patients 41–80 years old) and acquired megacolon samples (N=10; from patients 31–98 years old) were obtained at surgery. Vasoactive intestinal peptide levels were decreased in muscularis externa from acquired megacolon (P=0.01), while there was a modest increase in NADPH diaphorase activity in muscularis externa from megacolon (P=0.10). Glutathione in acquired megacolon was detectable in muscularis externa from only five specimens (P<0.05), but was not significantly different (P>0.05) in the mucosal-submucosal layer. The results supported the presence of vasoactive intestinal peptide and NADPH diaphorase in distinct subpopulations of nerves in human colon. The results also supported the hypothesis that glutathione functions as a neuroprotectant in a subset of patients with acquired megacolon.Supported by VA Medical Research Funds.     

Human intraepithelial lymphocytes

This study evaluates the immunomodulation and receptor binding of vasoactive intestinal peptide on human peripheral blood lymphocytes and intraepithelial lymphocytes. Vasoactive intestinal peptide (VIP, 10^–8 and 10^–12 M) had no effect on the concanavalin A-induced proliferation or the spontaneous cytotoxicity against K-562 targets by either lymphocyte type. Human peripheral blood lymphocytes had a mean of 927 vasoactive intestinal peptide receptors per cell with a K_d of 1.12×10^–10 M, as demonstrated by the competitive displacement of [¹²⁵I]peptide by unlabeled peptide using Scatchard analysis. In contrast, intraepithelial lymphocytes had no high-affinity receptors as shown by the negligible binding of 50 pM [¹²⁵I]VIP. Peptide binding by peripheral blood lymphocytes, although reduced by exposure to dithiothreitol and ethylenediamine tetraacetic acid, was still greater than binding by intraepithelial lymphocytes. As intraepithelial lymphocytes are mainly CD8⁺ T cells, the possibility that this phenotype may not bind VIP at all was tested by specifically depleting peripheral blood lymphocytes by antibody and complement lysis. Peripheral blood lymphocytes expressing CD8, CD4, and/or CD2 were responsible for most of the binding, indicating that CD8⁺ T lymphocytes in the peripheral blood and in the intestinal epithelium differ in their capacity to bind VIP.This work was supported by grants from the National Foundation for Ileitis and Colitis and from the National Institutes of Health (RO1DK42166).     

Motor responsiveness of proximal and distal human colonic muscle layers to acetylcholine,noradrenaline, and vasoactive intestinal peptide

There is an increasing body of evidence suggesting regional heterogeneity in human colonic function. Using circular and longitudinal muscle strips from proximal and distal human colon, the present study sought to determine whether such regional differences were apparent in smooth muscle responsiveness to neurohumoral agents. Both proximal and distal muscle gave quantitatively similar responses to acetylcholine, as they did for noradrenaline. However, the circular muscle of the distal colon was more sensitive to vasoactive intestinal peptide than was the circular muscle of the proximal colon. Longitudinal muscle from both regions was comparatively insensitive to VIP.     

电针对胃动力紊乱大鼠血管活性肠肽表达的影响

[目的]探讨电针调整胃动力与血管活性肠肽(VIP)的内在联系.[方法]采用浆膜法测定胃电数据,放射和免疫组化方法观察针刺足三里穴对束缚冷应激致胃动力紊乱大鼠VIP表达的影响.[结果]与正常组比较,束缚冷应激大鼠胃运动频率和波幅升高(P＜0.01);与模型组比较,电针足三里穴可降低胃运动频率和波幅(P＜0.01),同时可影响VIP的表达(P＜0.05,＜0.01).[结论]VIP参与了电针对胃动力的调整作用.     

正规抗结核治疗对初治肺结核患者血清中血管活性肠肽水平的影响

目的 探讨正规抗结核治疗对初治肺结核患者血清中血管活性肠肽水平的影响及其与Th1型细胞因子γ-干扰素和Th2型细胞因子白细胞介素(IL)-4的相关性.方法 选择45例初治肺结核患者,其中30例Ⅲ型肺结核患者进行正规治疗2个月,观察血管活性肠肽、白细胞介素-4、γ-干扰素的变化情况,并与15例Ⅱ型肺结核患者及健康对照组相比较.结果 肺结核组血清VIP和IL-4水平均高于对照组,γ-干扰素水平低于对照组,Ⅲ型肺结核患者抗结核治疗2个月后,血清VIP和IL-4均明显低于治疗前,γ-干扰素水平明显低于对照组.结论 正规抗结核治疗2个月后,肺结核患者血清VIP水平明显下降,提示VIP对评价抗结核疗效具有一定的参考意义.     

胆胃舒颗粒对胆囊血管活性肠肽受体基因表达的影响

[目的]观察胆胃舒颗粒对胆囊血管活性肠肽(vasoactive intestinal peptide,VIP)受体基因表达的影响及预防胆囊结石的作用.[方法]雄性豚鼠90只,随机分为3组,每组30只,空白组喂养普通饲料40 g/(d·只);模型组喂养胆固醇结石诱石饲料40 g/(d·只);治疗组喂养胆固醇诱石饲料40 g/(d·只),加胆胃舒颗粒溶液1.5ml(含300mg胆胃舒颗粒)灌胃.实验2个月后观察3组胆囊结石情况、胆囊收缩功能及胆囊壁VIP受体基因表达.[结果]胆囊结石形成率:空白组3.33％(1/30),模型组92.59％(25/27),治疗组10.71％(3/28);胆囊收缩功能:空白组胆囊收缩率为(66.83± 5.34)％,模型组(43.06±4.27)％,治疗组(67.93±6.82)％;胆囊壁VIP受体基因表达:空白组0.30±0.07,模型组0.45±0.12,治疗组0.33±0.06.差异均有统计学意义(P＜0.05).[结论]胆胃舒颗粒可降低胆囊结石的形成,其作用机制可能通过降低胆囊壁VIP受体基因表达,从而加强胆囊收缩功能,促使胆汁排泄,防止胆囊结石的发生.     

Interaction of melatonin with human lymphocytes: Evidence for binding sites coupled to potentiation of cyclic AMP stimulated by vasoactive intestinal peptide and activation of cyclic GMP

Melatonin binding sites were characterized in human blood lymphocytes. The specific binding 2-[125I]iodo-melatonin ([125I]MEL) to human lymphocytes was dependent on time and temperature, stability, saturation, and reversibility. Moreover, guanine nucleotides decreased the specific binding of [125I]MEL to crude membranes of human lymphocytes, suggesting the coupling of these binding sites to a guanosine nucleotide binding regulatory protein(s). In competition studies, the specific binding of [125I]MEL to lymphocytes was inhibited by increasing concentrations of native melatonin. Scatchard analysis showed that data were compatible with the existence of two classes of binding sites: a high-affinity site with a Kd of 5.20 +/- 0.79 nM and a binding capacity of 50.6 +/- 11.0 fmol/10(7) cells, and a low-affinity site with a Kd of 208.5 +/- 50.2 nM and a binding capacity of 2691 +/- 265 fmol/10(7) cells. However, concentration-dependent binding of [125I]MEL to lymphocytes was saturable and resulted in a linear Scatchard plot, suggesting binding to a single class of binding sites. The Kd for the single site was 1.02 +/- 0.34 nM with a binding capacity of 10.1 +/- 1.6 fmol/10(7) cells. Their affinities closely correlated with the production of cyclic nucleotides, suggesting a physiological role for the melatonin binding sites. Thus, melatonin potentiated the effect of vasoactive intestinal peptide (VIP) on cyclic AMP production (ED50 = 1.9 nM) and stimulated cyclic GMP accumulation (ED50 = 125 nM). Results demonstrate the existence of two binding sites for [125I]MEL in human blood lymphocytes, with a high-affinity binding site coupled to the potentiation of the effect of VIP on cyclic AMP production and a low-affinity binding site coupled to activation of cyclic GMP production.     

肠型胃腺癌及不典型增生组织中血管活性肠肽mRNA表达的研究

目的　血管活性肠肽 (VIP)与肿瘤关系密切 ,VIP可促进和 /或抑制一些肿瘤生长 ,而且一些肿瘤细胞有VIP的自分泌调节作用。VIP对胃癌生长的影响 ,胃癌细胞是否分泌VIP及存在VIP自分泌调节作用尚有争议。研究肠型胃腺癌及不典型增生组织中VIPmRNA的表达情况 ,试图探讨VIP在胃腺癌发生发展中的作用。方法　通过RT PCR方法 ,检测 1 5例正常胃窦黏膜、1 6例不典型增生胃黏膜及 2 0例肠型胃腺癌组织中VIPmRNA的表达量 ,通过与恒定表达的β actin(内参照 )mRNA表达量比较 ,计算出VIPmRNA与 β actinmRNA表达量的积分光密度比值 (V/B) ,以V/B值反映各组织中VIPmRNA表达量的高低。结果　肠型胃腺癌组织中V/B值明显高于不典型增生胃黏膜及正常胃窦黏膜 ,其V/B值分别为 :1 .452 2± 0 .32 82 ,0 .962 3± 0 .2 2 5 0 ,0 .951 3± 0 .2 66 9,差异有非常显著性 (P<0 .0 1 )。正常胃窦黏膜与不典型增生胃黏膜中 ,V/B值的差异无显著性 (P >0 .0 5)。结论　肠型胃腺癌组织VIPmRNA表达量明显高于不典型增生胃黏膜 ;VIP可能在肠型胃腺癌的发生发展中起重要的作用。     

Adenosine A2 receptor-mediated stimulation of cyclic AMP in cultured chicken pineal cells

Abstract: The pineal gland of vertebrates produces the time-keeping hormone melatonin in a rhythmic manner. Regulation of melatonin production is a multifactorial process. In the chicken, light, perceived through the skull, and norepinephrine, acting through α2-adrenergic receptors, synergistically inhibit day time melatonin production. In addition, adenosine exerts autocrine/paracrine modulatory effects on melatonin secretion. In an attempt to elucidate how these effects of adenosine are mediated, chicken pineal cells were cultured, in the dark during day time, in the presence of different analogs of adenosine. When the adenosine transmembranous carrier was inhibited, chloroadenosine stimulated cyclic AMP (cAMP) accumulation in a time- and dose-dependent manner. The effects were antagonized by 8-phenyltheophylline, an antagonist at the A1/A2 adenosine receptors. A dose-dependent stimulation of cAMP accumulation was also obtained with other adenosine agonists, with the following order of potency: N-ethylcarboxamidoadenosine > cyclopentyladenosine > R-phenyl-isopropylade-nosine. The stimulatory effect of the latter compound was still observed when basal cAMP levels were increased in the presence of forskolin. Under our experimental conditions no inhibition of cAMP content was observed. Our results are consistent with the idea that stimulation of melatonin secretion by adenosine analogs is mediated through A2 receptors.     

Actions of dopamine on prolactin secretion and cyclic AMP metabolism in ovine pituitary cells

Prolactin secretion from cultured sheep pituitary cells was inhibited by low concentrations of dopamine (0.1 nM-0.1 microM) with a half-maximal effect at 3 nM. At a maximally effective dose (0.1 microM) dopamine significantly inhibited prolactin secretion within 5 min. with an 80% inhibition of basal secretion over 2 h. Basal prolactin secretion was stimulated by the addition of methylisobutylxanthine (MIX) (0.3-1.0 mM) and 8-bromo-cyclic AMP (2 mM), but cholera toxin (3 micrograms/ml) and prostaglandin E2 (0.1-1.0 microM), which also raised cellular cyclic AMP levels, had no effect on prolactin release. The inhibition of prolactin release by dopamine (0.1 microM) was not affected by any of these compounds. Dopamine inhibited MIX-induced cyclic AMP accumulation over a similar concentration range to the inhibition of secretion, but had no effect on the changes in cyclic AMP concentration produced by cholera toxin and prostaglandin E2. Overall the results with sheep pituitary cells suggest that lowered cyclic AMP levels do not mediate the inhibitory effects of dopamine on basal prolactin secretion, but that changes in cellular cyclic AMP levels may alter the secretion of this hormone, and dopamine may affect pituitary cell cyclic AMP concentrations in some circumstances.     

胃腺癌细胞中血管活性肠肽自分泌调节作用及其机制

目的　研究胃腺癌细胞株SGC7901细胞中是否存在血管活性肠肽(VIP)的自分泌调节作用及其机制。方法　用RT PCR检测SGC7901细胞中VIP及其受体(VIPR1、VIPR2 )mRNA的表达,用免疫组化观察SGC7901细胞蛋白的表达,用放射免疫法测定细胞上清液中VIP含量,用MTT及细胞计数法观察外源VIP和VIP受体拮抗剂 [D p Cl Phe6, Leu17 ] VIP对SGC7901细胞增殖的影响。同时用RT PCR检测外源VIP及其受体拮抗剂孵育前后细胞c myc、鸟氨酸脱羧酶(ODC)mRNA的表达量变化。结果　SGC7901细胞不仅表达VIPmRNA,而且是VIP免疫反应阳性细胞,VIP免疫反应阳性颗粒主要分布在细胞质,少数在细胞核。细胞培养上清液中可检测到VIP蛋白,平均每 106 个细胞可分泌 (13. 15±8. 54)pg的VIP。SGC7901细胞也能表达VIPR1 和VIPR2 mRNA。10-5 ~10-12 mol/L外源性VIP孵育 24h后,细胞增殖无显著影响 (P>0. 05),而 10-5 ~10-8 mol/L拮抗剂显著促进SGC7901细胞增殖(P<0. 05)。10-6 mol/L外源性VIP孵育 24h后,SGC7901细胞c myc及ODCmRNA的表达量显著下降(RM /β: 0. 816±0. 049比 0. 901±0. 054,P<0. 01;RO/β: 0. 737±0. 060比 0. 830±0. 051,P<0. 01);而10-6 mol/L[D p Cl Phe6, Leu17 ] VIP使细胞c myc及ODCmRNA的表达量显著升高 (RM/β: 0. 950±0     

Pancreatic vasoactive intestinal polypeptide-oma as a cause of secretory diarrhoea

A 42-year-old woman presented with a 4-year history of worsening diarrhoea that was watery, profuse and confirmed to be secretory in nature. She had tested positive for phenolphthalein on urinary laxative screening but continued to deny laxative usage. Her vasoactive intestinal polypeptide (VIP) level was subsequently found to be markedly elevated. Despite a normal abdominal ultrasound, a computed tomography scan revealed a 5-cm pancreatic tail mass. Octreotide scanning was used to exclude metastatic disease and she went on to have surgical removal of a localized pancreatic vasoactive intestinal polypeptide-oma which resulted in the complete resolution of her diarrhoea.     

Distribution of vasoactive intestinal polypeptide and substance P receptors in human colon and small intestine

Vasoactive intestinal polypeptide (VIP) and substance P are found in neurons in the lamina propria and submucosa and muscularis propria of human small intestine and colon. VIP receptors coupled to adenylate cyclase are present on epithelial, smooth muscle, and mononuclear cells. This study analyzes the distribution of[¹²⁵I]VIP binding and [¹²⁵]substance P in human colon and small intestine using autoradiographic techniques. [¹²⁵I]VIP binding was present in high density in the mucosal layer of colon and small intestine. [¹²⁵I]VIP binding was not significantly greater than nonspecific binding in smooth muscle layers or the lymphoid follicles. In contrast, [¹²⁵I]substance P binding was present in high density over the colonic muscle but was not present over the mucosal layer. In human colon cancer, [¹²⁵I]VIP grain density over the malignant tissue was only slightly higher than background. These autoradiographic studies of [¹²⁵I]VIP binding indicate that the highest density of VIP receptors was found in the small intestine and superficial colonic mucosa, whereas the density of substance P receptors was highest over the smooth muscle layers. These findings suggest a mismatch between immunochemical content of the peptide and autoradiographic density of the receptor.This work was supported by funds from the Research Service of the Veterans Administration.     

Vasopressin and vasoactive intestinal peptide infused in the paraventricular nucleus of the hypothalamus elevate plasma melatonin levels

Abstract: The connection between the suprachiasmatic nucleus (SCN) and the paraventricular nucleus of the hypothalamus (PVN) forms an important component of the melatonin rhythm-generating system. However, the chemical identity of this projection is not known. To test the possible implication of the SCN peptides vasopressin (VP) and vasoactive intestinal peptide (VIP) in this projection, we performed microinfusions in the PVN during the first half of the dark period and subsequently monitored resulting plasma melatonin levels. Infusions for 7 hr of either VP or VIP, but not oxytocin, caused increased plasma melatonin levels in the middle of the dark period. These observations confirm the role of the PVN in the melatonin rhythm-generating pathway and indicate that both VP and VIP released at the level of the PVN, and probably derived from the SCN, are able to influence peripheral plasma melatonin levels.