Corticosterone- or metapyrone-induced alterations in adrenal function and expression of the arginine vasotocin receptor VT2 in the pituitary gland of domestic fowl, Gallus gallus

The avian neurohypophyseal hormone arginine vasotocin (AVT) is known to be involved in the regulation of adrenocorticotropin (ACTH) release by interacting with the VT2 receptor (VT2R), which is homologous to the mammalian vasopressin V1b receptor (V1bR). To study the role of glucocorticoid in the expression and regulation of the VT2R, corticosterone (1 or 5 mg/bird/day) or metapyrone (10 or 50 mg/kg body weight/day) were administered daily for 8 days to white leghorn chickens. While low doses were ineffective, a high dose of corticosterone upregulated, while metapyrone downregulated, pituitary VT2R mRNA expression and ir-VT2 in the cephalic lobe of the anterior pituitary. Further, although no change was observed in the expression of POMC mRNA, adrenal activity (as judged by the changes in total cholesterol, 3β HSD, cortical cord width and cortico-medullary ratio) exhibited suppression or stimulation following treatment with corticosterone or metapyrone, respectively. In view of the classical negative feedback effect of glucocorticoids at the level of hypothalamic CRH neurons and pituitary corticotrophs, high corticosterone level-induced suppression of the CRH-ACTH axis may have been masked by VT2R-mediated stimulation of corticotrophs, and hence the POMC mRNA level did not change. The same argument could be used for metapyrone. It is concluded that expression of the VT2 receptor is regulated by glucocorticoids in chicken. These findings confirm a role for AVT, mediated by the VT2 receptor, in regulating ACTH secretion during stress and suggest that corticotroph VT2 receptor levels may be dynamically regulated depending on the HPA axis activity.     

Characterization and immunohistochemical visualization of the vasotocin VT2 receptor in the pituitary gland of the chicken, Gallus gallus

Arginine vasotocin is a neurohypophysial hormone in lower vertebrates including birds. Its actions are mediated through G-protein coupled receptors that belong to the vasopressin/oxytocin receptor family. Our laboratory recently cloned a vasotocin receptor, designated the VT2 receptor (VT2R), which shares high sequence identity at both the nucleotide and amino acid level with the mammalian V1b vasopressin receptor (V1bR). In the present study, we report development and use of an antibody to the VT2R to obtain anatomical evidence for testing the hypothesis that the VT2R is the avian homolog of the mammalian V1bR. Results verified the specificity of the antibody and demonstrated a receptor distribution occurring predominantly in the cephalic lobe of the pars distalis and co-localizing with adrenocorticotropin in corticotrophs. With respect to VT2R distribution and cell-type localization in pituitary gland, evidence presented support its similarity with the mammalian V1bR. In contrast to the mammalian V1bR, VT2R expression was not observed in chicken brain. Further research will be required to determine which receptor/s in the arginine vasotocin/mesotocin family are expressed in brain and mediate regulatory functions of vasotocin in the central nervous system.     

Bacterial lipopolysaccharide-induced coordinate downregulation of arginine vasopressin receptor V3 and corticotropin-releasing factor receptor 1 messenger ribonucleic acids in the anterior pituitary of endotoxemic steers

AVP and CRF are potent stimulators of pituitary ACTH secretion in cattle. Actions of AVP and CRF at the anterior pituitary are mediated by AVP receptor V3 (V3) and CRF receptor 1 (CRFR1). The primary objective of these studies was to determine the effect of systemic inflammatory stress on V3 and CRFR1 mRNAs in the bovine anterior pituitary. Holstein steers (n=20) were injected with 200 ng/kg bacterial lipopolysaccharide (LPS) and tissues collected 0, 2, 4, 12, and 24 h later. All animals responded to LPS administration with an increase in body temperature, plasma ACTH, and cortisol (p<0.05). Abundance of anterior pituitary V3 mRNA was decreased at 2, 4, and 12 h following LPS administration (p<0.05) and returned to basal by 24 h. A similar temporal regulation of pituitary CRFR1 mRNA (p<0.05), but not pituitary pro-opiomelanocortin (POMC) mRNA, was observed following LPS administration. Similar downregulation of CRFR1 mRNA was not observed in other brain regions following LPS administration (cerebellum, hypothalamus). Our results indicate that V3 and CRFR1 mRNAs are coordinately downregulated in the anterior pituitary during systemic inflammatory stress. Decreased AVP and CRF receptor expression may help regulate the pituitary-adrenal response to stress.     

Kidney function and the role of arginine vasotocin in three agamid lizards from habitats of differing aridity in Western Australia

Western Australian agamid lizards are diverse and inhabit mesic to very arid areas of the state. Although reptilian kidneys are unable to elaborate hyperosmotic urine, we hypothesised that the renal system of lizards inhabiting arid areas would display an enhanced ability to conserve water under the control of the antidiuretic peptide hormone, arginine vasotocin (AVT). To examine this, the renal physiological and endocrine responses to osmotic challenge in three closely-related Australian agamid lizards inhabiting arid, semi-arid, and mesic environments were studied. The species studied were Pogona minor (mesic), Ctenophorus salinarum (semi-arid), and Ctenophorus nuchalis (arid). Circulating AVT was assayed and renal variables such as glomerular filtration rate (GFR), urine flow rate (V), and fractional reabsorption of filtrate FRH2O were measured in response to hypernatraemia, water load, and dehydration. Hypernatraemia and dehydration induced antidiuresis in all three species through similar mechanisms involving both glomerular and tubular responses. However, in salt-loaded P. minor the response was largely glomerular in nature, as FRH2O did not increase relative to the hydrated condition. The magnitude of the antidiuretic response was also greater in P. minor, indicating a greater sensitivity to osmotic challenge. Plasma concentrations of AVT were significantly correlated with FRH2O in P. minor (r2=0.38, P=0.025), but with GFR in C. nuchalis (r2=0.16, P=0.041). We found that the control and mechanisms of renal function among these lizards were largely similar, and there was little support for the hypothesis that arid lizards possess physiological adaptations not present in closely-related mesic lizards. Yet, differences remain in their response to hypernatraemia which may reflect the aridity of their different environments, or their varying habits.     

大肠杆菌O157VT2 B亚基基因的克隆与表达

目的对大肠杆菌O157VT2毒素B亚基基因进行克隆、表达与鉴定,为VT2毒素的抗原、抗体大规模制备及疾病的预防、诊断和疫苗研究打下基础。方法设计寡核苷酸引物,利用PCR技术从出血性大肠杆菌O157的染色体基因组中扩增出VT2毒素B亚基基因,连接到表达载体pET-28a以构建重组质粒。将重组质粒转入表达宿主菌BL21感受态细胞,通过IPTG诱导进行表达,对表达蛋白进行SDSPAGE电泳分析和Western检测。结果用PCR方法扩增出了预期大小的VT2B亚基基因,克隆得到重组质粒pET28aVT2B,转化后宿主菌成功表达分子量为8kD的目的蛋白,用特异抗体经Western检测该条带为阳性反应。结论目的基因成功克隆到宿主菌内,目的蛋白得到高效表达,最佳诱导时间为4h,目的蛋白表达量可达总蛋白量45.72%。     

Angiotensin II type 2 receptor inhibits expression and function of insulin receptor in rat renal proximal tubule cells

Both renin–angiotensin systems and insulin participate in kidney-involved blood pressure regulation. Activation of angiotensin II type 2 receptor (AT₂R) decreases sodium reabsorption in renal proximal tubule (RPT) cells, whereas insulin produces the opposite effect. We presume that AT₂R has an inhibitory effect on insulin receptor expression in RPT cells, which may affect renal sodium transport and therefore be of physiological or pathological significance. Our present study found that activation of AT₂R inhibited insulin receptor expression in a concentration and time-dependent manner in RPT cells from Wistar-Kyoto (WKY) rats. In the presence of a protein kinase C (PKC) inhibitor (PKC inhibitor peptide 19–31, 10^?6 mol/L) or a phosphatidylinositol 3 kinase inhibitor (wortmannin, 10^?6 mol/L), the inhibitory effect of AT₂R on insulin receptor was blocked, indicating that both PKC and phosphatidylinositol 3 kinase were involved in the signaling pathway. There was a linkage between AT₂R and insulin receptor which was determined by both laser confocal microscopy and coimmunoprecipitation. However, the effect of AT₂R activation on insulin receptor expression was different in RPT cells from spontaneously hypertensive rats (SHRs). Being contrary to the effect in WKY RPT cells, AT₂R stimulation increased insulin receptor in SHR RPT cells. Insulin (10^?7 mol/L, 15 minutes) enhanced Na⁺-K⁺-ATPase activity in both WKY and SHR RPT cells. Pretreatment with CGP42112 decreased the stimulatory effect of insulin on Na⁺-K⁺-ATPase activity in WKY RPT cells, whereas pretreatment with CGP42112 increased it in SHR RPT cells. It is suggested that activation of AT₂R inhibits insulin receptor expression and function in RPT cells. The lost inhibitory effect of AT₂R on insulin receptor expression may contribute to the pathophysiology of hypertension.     

Dopamine receptor D2S gene transfer improves the sensitivity of GH3 rat pituitary adenoma cells to bromocriptine

Bromocriptine, a dopamine agonist (DA), has been used in the treatment of prolactinomas. Recent studies have indicated that dopamine 2 receptor short isoform (D2S) may play an important role in suppressing PRL synthesis and prolactinoma cell growth under DA treatment. In the current study, we investigated the role of D2S in the therapeutic action of bromocriptine in GH3 using both in vitro and in vivo approaches. Infection of adenovirus-D2S increased D2S expression in GH3 cells (P < 0.05). D2S expression significantly decreased the GH3 cell viability subjected to bromocriptine treatment in vitro (P < 0.05). In nude mice, adenovirus-D2S transfection sensitized GH3 xenograft to bromocriptine treatment evidenced by the significant inhibition of D2S expressed tumor growth as compared with vector control. Furthermore, decrease of Bcl-2 expression, increase of Bax, and active Caspase-3 were found in D2S expressed GH3 xenograft subjected to bromocriptine treatment. In summary, our study indicates that D2S expression plays a critical role in the therapeutic action of bromocriptine in pituitary adenomas and that adenovirus-mediated D2S gene transfer combined with bromocriptine may provide a novel treatment for DA-resistant prolactinomas.     

甲泼尼龙对慢性咳嗽患者诱导痰巨噬细胞中Toll样受体2表达的影响

目的 探讨Toll样受体2(TLR2)在慢性咳嗽患者诱导痰巨噬细胞中的表达与气道炎症的关系以及甲泼尼龙对TLR2表达的影响.方法 收集2008年9月至2010年3月泸州医学院附属医院呼吸科门诊的慢性咳嗽患者86例,其中咳嗽变异性哮喘(CVA)组31例,上气道咳嗽综合征(UACS)组14例,嗜酸细胞性支气管炎(EB)组16例,胃食管反流性咳嗽(GERC)组25例.对照组为20名健康志愿者.各组均行痰液诱导检查,HE染色,免疫荧光染色测定TLR2表达,酶联免疫吸附法(ELISA)测定诱导痰上清液中细胞因子的浓度.结果 (1)CVA、UACS、EB和GERC各组患者诱导痰中细胞总数[分别为(3.4±1.1)×106/L、(2.6±0.6)×106/L、(2.6±0.6)×106/L、(2.7±0.5)×106/L]均明显高于对照组[(1.4±0.6)×106/L],差异均有统计学意义(均P＜0.01);分类计数显示以上4组痰中巨噬细胞比例(分别为0.35±0.04、0.40±0.06、0.38±0.04、0.43±0.05)均明显低于对照组(0.63±0.08);中性粒细胞比例(分别为0.52±0.06、0.58±0.06、0.52±0.04、0.54±0.06)明显高于对照组,差异均有统计学意义(均P＜0.01).CVA和EB组患者嗜酸细胞比例(分别为0.115±0.054和0.099±0.034)明显高于其他各组,差异有统计学意义(均P＜0.01),但两组间差异无统计学意义(t=1.18,P＞0.05).(2)CVA、UACS、EB和GERC各组患者诱导痰上清液白细胞介素-8浓度[分别为(7.0±1.2)μg/L、(10.2±3.1)μg/L、(6.3±1.9)μg/L、(7.7±2.5)μg/L]和肿瘤坏死因子-α浓度[分别为(30±11)ng/L、(46±16)ng/L、(2l±5)ng/L、(33±15)ng/L]明显高于对照组[分别为(1.6±0.8)μg/L和(13±6)ng/L],差异均有统计学意义(均P＜0.01).(3)CVA、UACS、EB和GERC各组患者诱导痰上清液中白细胞介素-8与痰中中性粒细胞数呈正相关(r值分别为0.628、0.816、0.769、0.733,均P＜0.05);CVA、UACS、EB和GERC各组患者诱导痰上清液中肿瘤坏死因子-α与痰中中性粒细胞数呈正相关(r值分别为0.579、0.865、0.730、0.755,均P＜0.05).(4)CVA、UACS、EB和GERC各组患者诱导痰巨噬细胞TLR2表达量(56±15、38±7、65±17、27±4)明显低于对照组(135±14),甲泼尼龙可上调以上各组患者诱导痰巨噬细胞中TLR2(75±17、53.29±16、94±30、41±5)的表达.结论 CVA、UACS、EB和GERC等慢性咳嗽患者诱导痰巨噬细胞中TLR2的表达下降,甲泼尼龙可上调巨噬细胞TLR2的表达.

Abstract：

Objective To explore the relationship between airway inflammation and Toll-like receptor 2 ( TLR2 ) expression on macrophages in induced sputum from chronic cough patients, and to observe the effect of methylprednisolone on this receptor. Methods Eighty-six outpatients with chronic cough were enrolled in this study from the respiratory department in the Affiliated Hospital of Luzhou Medical College from September 2008 to March 2010. These diseases were diagnosed according to the guideline of Chinese Society of Respiratory Diseases in 2005, excluding severe liver, heart and kidney pathology and pregnant women. Among 86 cases, 31 were cough variant asthma ( CVA), 14 upper airway cough syndrome (UACS), 16 eosinophilic bronchitis (EB) and 25 gastroesophageal reflux (GERC). Twenty healthy volunteers were included as controls. Induced sputum was collected and cell counts were analyzed. TLR2 expression on macrophages was determined by immunofluorescence staining. Cytokine levels were measured with enzyme linked immunosorbent assay (ELISA). Results (1) The total inflammatory cells [(3.4±1.1) ×106/L, (2.6 ±0.6)×106/L, (2.6±0.6)×106/L, (2.7±0.5) × 106/L] and neutrophils (0.52±0.06, 0.58 ±0.06, 0.52±0.04, 0.54 ±0.06) in induced sputum from CVA, UACS, EB and GERC were significantly increased(t value = 5.27, 4.09, 3. 16, 3.92 and 3.62, 3.49, 4.82, 6.17respectively, all P＜0.01 ), with decreased macrophages (0.35±0.04, 0.40±0.06,0.38±0.04,0.43 ±0. 05) in comparison with the control group (t value =4. 76,5.24,4. 57,3. 18, all P ＜0. 01 ). There was a significant increase in eosinophils(0.118±0.054,0.099±0.034) in CVA and EB group (t value =4.46,3.87,5. 19 and 3.51,4. 06,4. 38 respectively, all P＜0.01). (2) The concentration of IL-8[(7.0±1.2)μg/L,(10.2±3.1)μg/L,(6.3±1.9)μg/L,(7.7±2.5)μg/L] and TNF-α[(30 ± 11)ng/L, (46±16)rng/L, (21 ±5)ng/L, (33 ± 15)ng/L] from induced sputum in CVA, UACS, EB and GERC groups were significantly increased, compared with the control group( t value =4. 58,3.67,4. 26,5. 39 and 5.16,3.97,4. 59,3.34 respectively, all P ＜ 0.01 ). (3) The concentration of IL-8 was positively correlated with neutrophils in each group( R value = 0.628,0. 816,0. 769 and 0. 733 respectively, all P＜0. 05 ). The concentration of TNF-o was also positively correlated with neutrophils in each group ( R value = 0. 579,0. 865,0. 730 and 0. 755 respectively,all P ＜ 0. 05 ). (4) The expression of TLR2 ( 56 ± 15,38 ± 7,65 ±17,27 ±4) on macrophages in CVA, UACS,EB and GERC was significantly decreased. The expression of TLR2 (75±17, 53±16,94±30,41±5 ) on macrophages was enhanced by methylprednisolone.Conclusions The expression of TLR2 on macrophages from induced sputum in CVA, UACS, EB and GERC was decreased, but could be enhanced by methylprednisolone, suggesting that TLR2 might play a regulatory role in airway inflammatory response.     

Shell gland responsiveness to prostaglandins F2α and E1 and to arginine vasotocin during the laying cycle of the domestic hen (Gallus domesticus)

Contractile responses of isolated shell gland (SG) strips from laying hens displayed no significant differences 6 hrs before oviposition, at oviposition, and 6 hrs after oviposition when stimulated with arginine vasotocin (AVT), prostaglandin E₁ (PGE₁), or prostaglandin F_2α (PGF_2α). Dose-response curves show that the sensitivity of the SG to these agents, in vitro, is: AVT > PGF₂ > PGE₁. PGF_2α, however, produces the largest contractile response, while PGE₁ appears to be a poor agonist of contractile activity in vitro. These results are discussed in relation to the known hormonal patterns during the ovulatory cycle of the hen and the physiological roles attributed to these oxytocics in the control of oviposition.     

Involvement of the corticotropin-releasing factor (CRF) type 2 receptor in CRF-induced thyrotropin release by the amphibian pituitary gland

Corticotropin-releasing factor (CRF) is considered to be a main adrenocorticotropin-releasing factor in vertebrates. In non-mammalian species, CRF and related peptides cause the release of thyroid-stimulating hormone (TSH) from the anterior pituitary. The actions of CRF peptides are mediated by two G protein coupled receptors (CRF1 and CRF2) that have different ligand specificities. Using ligands that bind preferentially or selectively to the CRF2 we tested the hypothesis that TSH release by the amphibian pituitary gland is mediated by the CRF2. Injection of frog CRF, urocortin 1 or the CRF2-specific ligand urocortin 3 all produced significant, acute increases (by 2 h) in plasma thyroxine concentration in prometamorphic tadpoles. Chronic injections of CRF peptides accelerated tadpole metamorphosis, and the peptides with the highest affinity for the CRF2 (urocortin 1 and sauvagine) had the greatest potency. Ligands selective for the CRF2 (frog urocortin 3, mouse urocortins 2 and 3) all accelerated tadpole metamorphosis. We then tested frog urocortins 1 and 3, mouse urocortin 2 and sauvagine for their TSH-releasing activity using dispersed frog anterior pituitary cells in culture. All of the peptides tested markedly enhanced the release of TSH. Secretagogue-induced TSH release was completely blocked by the general CRF receptor antagonist astressin or the CRF2-specific antagonist antisauvagine-30. Conversely, the type 1 CRF receptor-specific antagonist antalarmin had no effect on TSH secretion. Our results support the hypothesis that CRF-induced TSH release by the amphibian pituitary gland is mediated by the CRF2.     

The glycine allele of a glycine/arginine polymorphism in the beta2-adrenergic receptor gene is associated with essential hypertension in a population of Chinese origin.

BACKGROUND: Several studies implicate polymorphisms in the human beta-adrenergic receptor gene (ADRB2) in the susceptibility to hypertension. We sought to replicate these results in a population of Chinese origin primarily from Taiwan and the San Francisco Bay area. METHODS: We genotyped >800 hypertensive subjects and individuals with low-normal blood pressure that were derived largely from the same families as the hypertensive patients for three polymorphisms in the ADRB2 gene: a C/T transition at position 47 (C-47T) in the 5' leader cistron; another C/T transition that results in a glycine/ arginine substitution at codon 16 (Gly16Arg), and a G/C transversion that causes a glutamate/glutamine substitution at codon 27 (Glu27Gln). RESULTS: The Gly16Arg was significantly associated with hypertension (P < .03). Under a dominant model, for hypertension the relative risk for the Gly/Gly and Gly/Arg genotypes versus the Arg/Arg genotype was 1.35 (95% confidence limits [CL] 1.08, 1.70); for low-normal blood pressure the relative risk was 0.79 (95% CL 0.66, 0.94). This polymorphism explained approximately 1% of the variance in systolic and diastolic blood pressures in our study population. There was no evidence of association between the C-47T and Glu27Gln polymorphisms and hypertension in this population. CONCLUSIONS: The Glyl6 allele in the beta2-adrenergic receptor gene is a susceptibility allele for essential hypertension in a population of Chinese origin.     

不同病程心肌缺血损伤大鼠心电图和心肌P2X3受体mRNA表达的对应变化

摘要：目的 观察异丙肾上腺素(ISO)诱发大鼠心肌缺血时,不同病程模型大鼠心电图和心肌P2X3受体mRNA表达的对应变化。方法 大鼠皮下注射60 mg /kg ISO（连续2次,间隔24h)的方法建立心肌缺血损伤组,选取心肌缺血损伤组50只,并以病程1、2、3、4、5日再分成5用心电图和qPCR检测方法观察同一病程下大鼠心电图波形S-T段和心肌P2X3受体mRNA的表达。结果 模型大鼠随病程进展,心电图波形中S-T段呈先抬高后下降趋势;心肌P2X3 受体mRNA的表达亦为先上升后下降,两者呈明显对应关系,且差异具有统计学意义。结论 心肌缺血损伤大鼠心肌P2X3受体mRNA的表达增加程度和心电图波形中S-T段抬高幅度明显相关,表明心肌缺血时P2X3受体敏感性显著增强,并参与了心肌缺血的病理过程。     

人Ⅱ型大麻受体真核表达体系构建及其在 HEK293细胞中的表达

目的：构建人Ⅱ型大麻受体（ hCB2）基因GV230真核表达质粒,并检测hCB2基因在HEK293细胞中的表达。方法利用人脑皮质细胞的总RNA为模板,RT-PCR获得cDNA,通过酶切、连接及测序鉴定正确后,再将目的片段插入真核表达载体GV230,构建重组表达质粒GV230-hCB2,阳性克隆用脂质体瞬时转染HEK293细胞。激光共聚焦扫描显微镜和Western blotting法检测hCB2基因表达产物在细胞的表达情况。结果扩增出hCB2基因片段,成功构建了重组表达质粒,并检测到目的蛋白在转染细胞中表达,观察到hCB2受体在胞膜分布和表达。结论成功构建GV230-hCB2质粒,该质粒在HEK293细胞中能表达hCB2蛋白,此为进一步研究hCB2生物学功能奠定了实验基础。     

Differential regulation of GHRH-receptor and GHS-receptor expression by long-term in vitro treatment of ovine pituitary cells with GHRP-2 and GHRH

GH secretion is regulated by GHRH and somatostatin via actions on their specific receptors in pituitary somatotropes. Ghrelin and synthetic analogs, GHRPs, also stimulate GH release via GHS-receptors (GHS-R). To examine the long-term effect of GHRH and/or GHRP on somatotropes, primary cultured ovine somatotropes were treated with GHRH (10⁻⁹ and 10⁻⁸ M) and GHRP-2 (10⁻⁸ and 10⁻⁷ M) for up to 2 d. After treatment, culture medium was collected for GH assay, and total RNA was extracted for RT-PCR analysis. To evaluate cell cultures used in this report, somatotrope-enriched pituitary cells were challenged by 6 h GHRH and dexamethasone (DEX) treatment. As expected, GHRH significantly decreased, whereas DEX increased, the levels of GHRH-R mRNA. Combined low doses of GHRH (10⁻⁹ M) and GHRP-2 (10⁻⁸ M) treatment for 24 h increased accumulated GH secretion, significantly more than that induced by high doses of GHRH (10⁻⁸ M) and GHRP-2 (10⁻⁷ M). While levels of GHRH-R mRNA increased, GHS-R mRNA levels were decreased by low doses of GHRH and GHRP-2 for 24 h. High doses of GHRH and/or GHRP-2 for 2 d did not increase GH secretion in the second day of treatment and reduced the level of GHRH-R mRNA. High doses of GHRP-2 treatment decreased the levels of both GHRH-R and GHS-R mRNA. Low doses of GHRH and/or GHRP-2 for 2 d increased the level of GHS-R mRNA without changing GHRH-R mRNA levels. Such treatment also increased ghrelin- (10⁻⁹ M) or ghrelin/GHRH (10⁻⁹ M)-induced GH secretion. These results suggest that low doses of GHRP-2 and GHRH prime somatotropes for stimulation by GHRH and ghrelin.