Suramin is a Competitive But Slowly-Equilibrating Antagonist at P2x-Receptors in the Rabbit Ear Artery

Abstract

Antagonist effects of suramin at the P_2x-receptor in the rabbit ear artery were associated with agonist curve depression and a steep Schild plot. Kinetic analysis revealed a problem of slow equilibration and established conditions under which suramin fulfilled all criteria for simple competition.     

Regional Difference of Purinergic Modulation on Adrenergic Neurotransmission in Isolated Rabbit Pulmonary Artery

Abstract

Effects of 2-Chloroadenosine on different parts of pulmonary artery isolated from rabbit were studied, and it was suggested that there was a significant regional difference of purinergic modulation on noradrenaline release between proximal and distal parts of the artery.     

Tissue Interactions in the Regeneration of Rabbit Ear Holes

SYNOPSIS It a hole is cut through the ear of a rabbit it willfill in by the comergent growth of new tissue from around themargins Centimeter wide holes are usually obliterated in 6 to8 weeks The regenerated tissues include newly differentiatedcartilage and occasional tufts of hair To determine the specificityof skin in this kind of growth that on both sides of the eaiwas replaced by grafts of belly sVin When holes were subsequentlypunched through the transplant regions limited regenerationensued but no new cartilage was produced If sheets of cartilagewere removed fiom poitions of rabbit eais and the resultantaieas perforated through the remaining two layers of skin noregeneration whatever occurred Not even the missing cartilagegieu back These remits indicate that replacement of the ciitilaginoussheet requnes the proximity of a healing wound in the oveilyingear skin, ind that skin from clscwhcie on the body canno' supportits regeneiation     

咪喹莫特抑制兔耳瘢痕增生的机制研究

目的:研究咪喹莫特抑制兔耳瘢痕增生的作用及可能机制.方法:选取10只新西兰大耳白兔,雌雄不限,根据本实验室的改良方法建立兔耳瘢痕模型,每只兔耳腹侧做六个直径为1cm的圆形创面,每个相距1.5cm,双侧对称,左耳为实验组涂抹5％咪喹莫特软,右耳为对照组涂抹等量凡士林软膏,待术后14天上皮化均完全后开始涂抹,一日一次.分别于术后14天、21天、28天、35天及42天同一时间点空气栓塞随机处死2只兔子,后沿每个瘢痕边缘外周的0.5cm处环形切下,经瘢痕最凸点直径一切为二,一半固定后行苏木精-伊红(HE)染色及Masson三色法染色,观察形态学差异,测量并计算瘢痕增生指数(Scar elevation index,SEI):另一半用于提取RNA,反转录后行Real-time PCR检测细胞外基质I型胶原(Collagen-I,Col-I),细胞因子IFN-γ及IL-4的表达.结果:HE及Masson染色可见实验组胶原沉积较对照组明显减少,SEI显示空白对照组于术后第21天增生程度达到高峰,其增生程度明显高于实验组;Real-time PCR结果可见实验组Col-I及IL-4的表达在各时间点明显降低,且IFN-γ的表达较对照组增高.结论:咪喹莫特可能通过调节IFN-γ及IL-4的表达来发挥抑制瘢痕增生的作用.     

Lysine Transport across Isolated Rabbit Ileum

Lysine transport by in vitro distal rabbit ileum has been investigated by determining (a) transmural fluxes across short-circuited segments of the tissue; (b) accumulation by mucosal strips; and (c) influx from the mucosal solution across the brush border into the epithelium. Net transmural flux of lysine is considerably smaller than that of alanine. However, lysine influx across the brush border and lysine accumulation by mucosal strips are quantitatively comparable to alanine influx and accumulation. Evidence is presented that the "low transport capacity" of rabbit ileum for lysine is due to: (a) a carrier-mediated process responsible for efflux of lysine out of the cell across the serosal and/or lateral membranes that is characterized by a low maximal velocity; and (b) a high "backflux" of lysine out of the cell across the mucosal membrane. A possible explanation for the latter observation is discussed with reference to the relatively low Na dependence of lysine transport across the intestinal brush border.     

Sodium Transport in Triads Isolated from Rabbit Skeletal Muscle

Triads and transverse tubules isolated from mammalian skeletal muscle actively accumulated Na+ in the presence of K+ and Mg-ATP. Active Na+ transport exhibited a fast single-exponential phase, lasting 2 min, followed by slower linear uptake that continued for 10 minutes. Valinomycin stimulated Na+ uptake, suggesting it decreased a pump-generated membrane potential gradient (Vm) that prevented further Na+ accumulation. At the end of the fast uptake phase transverse tubule vesicles incubated in 30 mM external [Na+] attained a ratio [Na+]in/[Na+]out=13.4. From this ratio and the transverse tubule volume of 0.35 microl/mg protein measured in this work, [Na+]in=400 mM was calculated. Determinations of active K+ transport in triads, using 86Rb+ as tracer, showed a 30% decrease in vesicular 86Rb+ content two minutes after initiating the reaction, followed by a slower uptake phase during which vesicles regained their initial 86Rb+ content after 10 minutes. Transverse tubule volume increase during active Na+ transport-as shown by light scattering changes of isolated vesicles--presumably accounted for the secondary Na+ and 86Rb+ uptake phases. These combined results indicate that isolated triads have highly sealed transverse tubules that can be polarized effectively by the Na+ pump through the generation of significant Na+ gradients.     

Characterisation of P2Y12 Receptor Responsiveness to Cysteinyl Leukotrienes

Leukotriene E₄ (LTE₄), the most stable of the cysteinyl leukotrienes (cysLTs), binds poorly to classical type 1 and 2 cysLT receptors although in asthmatic individuals it may potently induce bronchial constriction, airway hyperresponsiveness and inflammatory cell influx to the lung. A recent study has suggested that the purinergic receptor P2Y₁₂ is required for LTE₄ mediated pulmonary inflammation in a mouse model of asthma and signals in response to cysLTs. The aim of the study was to characterise the responsiveness of human P2Y₁₂ to cysteinyl leukotrienes. Models of human CysLT₁, CysLT₂ and P2Y₁₂ overexpressed in HEK293, CHO cells and human platelets were used and responsiveness to different agonists was measured using intracellular calcium, cAMP and β-arrestin recruitment assays. CysLTs induced concentration dependent calcium mobilisation in cells overexpressing CysLT₁ and CysLT₂ but failed to induce any calcium response in cells expressing P2Y₁₂ or P2Y₁₂+ Gα₁₆. In contrast, selective P2Y₁₂ agonists ADP and 2-MeS-ADP induced specific calcium flux in cells expressing P2Y₁₂+ Gα₁₆. Similarly, specific response to 2-MeS-ADP, but not to cysLTs was also observed in cells expressing P2Y₁₂ when intracellular cAMP and β-arrestin signalling were analysed. Platelets were used as a model of human primary cells expressing P2Y₁₂ to analyse potential signalling and cell activation through P2Y₁₂ receptor or receptor heterodimers but no specific LTE₄ responses were observed. These results show that LTE₄ as well as other cysLTs do not activate intracellular signalling acting through P2Y₁₂ and suggest that another LTE₄ specific receptor has yet to be identified.     

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously^1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.     

Effect of Inhibitors on Alanine Transport in Isolated Rabbit Ileum

The effects of metabolic inhibitors and ouabain on alanine transport across rabbit ileum, in vitro, have been investigated. Net transport of alanine and Na across short-circuited segments of ileum is virtually abolished by cyanide, 2,4-dinitrophenol, iodoacetate, and ouabain. However, these inhibitors do not markedly depress alanine influx from the mucosal solution, across the brush border, into the intestinal epithelium, and they do not significantly affect the Na dependence of this entry process. The results of this investigation indicate that: (a) the Na dependence of alanine influx does not reflect a mechanism in which the sole function of Na is to link metabolic energy directly to the influx process; and (b) the inhibition of net alanine transport across intestine is, in part, the result of an increased rate coefficient for alanine efflux out of the cell across the brush border. Although these findings do not exclude a direct link between metabolic energy and alanine efflux, the increased efflux may be the result of the increased intracellular Na concentration in the presence of these inhibitors. The results of these studies are qualitatively consistent with a model for alanine transport across the brush border which does not include a direct link to metabolic energy.     

Characteristics of Single, Large-Conductance Calcium-Dependent Potassium Channels (BKCa) from Smooth Muscle Cells Isolated from the Rabbit Mesenteric Artery

Smooth muscle cells isolated from the secondary and tertiary branches of the rabbit mesenteric artery contain large Ca²⁺-dependent channels. In excised patches with symmetrical (140 mm) K⁺ solutions, these channels had an average slope conductance of 235 ± 3 pS, and reversed in direction at −6.1 ± 0.4 mV. The channel showed K⁺ selectivity and its open probability (P _o ) was voltage-dependent. Iberiotoxin (50 nm) reversibly decreased P _o , whereas tetraethylammonium (TEA, at 1 mm) reduced the unitary current amplitude. Apamin (200 nm) had no effect. The channel displayed sublevels around 1/3 and 1/2 of the mainstate level. The effect of [Ca²⁺] on P _o was studied and data fitted to Boltzmann relationships. In 0.1, 0.3, 1.0 and 10 μm Ca²⁺, V _1/2 was 77.1 ± 5.3 (n= 18), 71.2 ± 4.8 (n= 16), 47.3 ± 10.1 (n= 11) and −14.9 ± 10.1 mV (n= 6), respectively. Values of k obtained in 1 and 10 μm [Ca²⁺] were significantly larger than that observed in 0.1 μm [Ca²⁺]. With 30 μm NS 1619 (a BK_Ca channel activator), V _1/2 values were shifted by 39 mV to the left (hyperpolarizing direction) and k values were not affected. TEA applied intracellularly, reduced the unitary current amplitude with a K _d of 59 mm. In summary, BK_Ca channels show a particularly weak sensitivity to intracellular TEA and they also display large variation in V _1/2 and k. These findings suggest the possibility that different types (isoforms) of BK_Ca channels may exist in this vascular tissue. Received: 22 December 1997/Revised: 27 March 1998     

Amppcp Acts Via P1 and Not P2 Receptors in Some Isolated Smooth Muscle Preparations

Abstract

Isolated tissue studies using the P₁-purinoceptor antagonist 8-SPT show that AMPPCP acts entirely via P₁ receptors in the rat duodenum and by P₁ and P_2Y receptors in the guinea-pig taenia caeci.     

经隐动脉建立糖尿病兔股动脉血管狭窄模型

目的探讨建立糖尿病兔股动脉损伤后血管狭窄的模型方法。方法采用四氧嘧啶静脉注射建立糖尿病兔模型。建模成功后分为手术组、对照组,手术组接受了经隐动脉途径行球囊损伤股动脉术,对照组不行球囊损伤。术后均给予普食喂养,4周处死,行HE染色后观察血管狭窄情况。结果所有兔均完成了手术,24只存活至实验结束,平均手术时间（26±7）min,成功造成糖尿病兔股动脉狭窄模型,平均相关狭窄达（88.11±1.9）%。结论成功建立了糖尿病兔股动脉损伤后狭窄的实验模型,手术成功率高,操作时间少,存活率高,模型稳定,重复性好,为糖尿病外周血管病变的实验研究提供了借鉴方法。     

Complete Genomic Sequences of Rabbit Hemorrhagic Disease Virus G1 Strains Isolated in the European Rabbit Original Range

The complete genomic sequences of rabbit hemorrhagic disease virus (RHDV) strains isolated in 1995 (CB137) and 2006 (CB194) from wild European rabbits from Portugal are described. The strains were isolated in the original range of the European rabbit and assigned to genogroup 1 (G1), which is known to have persisted only in the Iberian Peninsula. ORF2 of isolate CB137 might encode a shorter minor structural protein, VP10.     

Beta Adrenergic Overstimulation Impaired Vascular Contractility via Actin-Cytoskeleton Disorganization in Rabbit Cerebral Artery

Background and Purpose

Beta adrenergic overstimulation may increase the vascular damage and stroke. However, the underlying mechanisms of beta adrenergic overstimulation in cerebrovascular dysfunctions are not well known. We investigated the possible cerebrovascular dysfunction response to isoproterenol induced beta-adrenergic overstimulation (ISO) in rabbit cerebral arteries (CAs).

Methods

ISO was induced in six weeks aged male New Zealand white rabbit (0.8–1.0 kg) by 7-days isoproterenol injection (300 μg/kg/day). We investigated the alteration of protein expression in ISO treated CAs using 2DE proteomics and western blot analysis. Systemic properties of 2DE proteomics result were analyzed using bioinformatics software. ROS generation and following DNA damage were assessed to evaluate deteriorative effect of ISO on CAs. Intracellular Ca²⁺ level change and vascular contractile response to vasoactive drug, angiotensin II (Ang II), were assessed to evaluate functional alteration of ISO treated CAs. Ang II-induced ROS generation was assessed to evaluated involvement of ROS generation in CA contractility.

Results

Proteomic analysis revealed remarkably decreased expression of cytoskeleton organizing proteins (e.g. actin related protein 1A and 2, α-actin, capping protein Z beta, and vimentin) and anti-oxidative stress proteins (e.g. heat shock protein 9A and stress-induced-phosphoprotein 1) in ISO-CAs. As a cause of dysregulation of actin-cytoskeleton organization, we found decreased level of RhoA and ROCK1, which are major regulators of actin-cytoskeleton organization. As functional consequences of proteomic alteration, we found the decreased transient Ca²⁺ efflux and constriction response to angiotensin II and high K⁺ in ISO-CAs. ISO also increased basal ROS generation and induced oxidative damage in CA; however, it decreased the Ang II-induced ROS generation rate. These results indicate that ISO disrupted actin cytoskeleton proteome network through down-regulation of RhoA/ROCK1 proteins and increased oxidative damage, which consequently led to contractile dysfunction in CA.     

水痘-带状疱疹病毒地方分离株在兔脑神经细胞中的形态与形态发生特征

为阐明水痘-带状疱疹病毒济南分离株（VZVJ1）在兔脑神经细胞（RNC）中的形态与形态发生特征,我们利用超薄切片电子显微镜技术对感染VZVJ1的RNC进行了观察研究。结果表明RNC在感染VZVJI6h后核内可见散在的核衣壳,12h后细胞核和细胞质内核衣壳明显增多,24h达高峰,而细胞核和细胞质内的成熟病毒颗粒较少见。病毒大小、形态基本一致,呈圆形或椭圆形,核心直径30～50nm,核衣壳74～96nm,成熟病毒110～180nm。核衣壳内有3种类型的核心,即电子致密核心、部分致密核心和电子透明核心。细胞核和细胞质内均可见核心样电子致密体和布纹样结构。在细胞质内还可见少量“繁殖复合体”,由膜性结构包绕多个囊泡构成。提示VZVJ1在RNC中的形态发生不同于其它性质的细胞。     

Characterisation of the R276A gain-of-function mutation in the ectodomain of murine P2X7

The cytolytic P2X7 purinoceptor is widely expressed on leukocytes and has sparked interest because of its key role in the activation of the inflammasome, the release of the pro-inflammatory cytokine IL-1β and cell death. We report here the functional characterisation of a R276A gain-of-function mutant analysed for its capacities to induce membrane depolarisation, calcium influx and opening of a large membrane pore permeable to YO-PRO-1. Our results highlight the particular sensitivity of R276A mutant to low micromolar adenosine triphosphate (ATP) concentrations, which possibly reflect an increased affinity for its ligands, and a slower closing kinetics of the receptor channel. Our findings support the notion that evolutionary pressures maintain the low sensitivity of P2X7 to ATP. We also believe that the R276A mutant described here may be useful for the generation of new animal models with exacerbated P2X7 functions that will serve to better characterise its role in inflammation and in immune responses.     

Characterisation of the Plasmidome within Enterococcus faecalis Isolated from Marginal Periodontitis Patients in Norway

The present study aimed to identify and characterize plasmids in a national collection of oral Enterococcus faecalis (n = 106) isolated from patients with marginal periodontitis. Plasmid replicon typing was performed by multiplex-PCR and sequencing with specific primers for 18 rep-families and 1 unique sequence. Additional plasmid analysis by S1-PFGE was performed for comparison. Totally 120 plasmid replicon amplicons of seven rep-families were identified in 93 E. faecalis strains, e.g. rep9 (prototype pCF10), rep6 (prototype pS86), rep2 (prototype pRE25/pEF1), and rep8 (prototype pAM373). Rep9 was the most predominant rep-family being detected in 81 (76.4%) strains. Forty of these strains were tetracycline resistant and three were erythromycin resistant. Rep6 was the second predominant rep-family being detected in 22 (20.8%) strains. Rep2 was detected in eight (7.5%) strains. All rep2-positive strains were resistant to tetracycline and/or erythromycin and six of them contained Tn916/Tn1545 genes. The rep-positive E. faecalis exhibited divergence in multilocus sequence types (STs). There was a significant correlation between rep9 and ST21, while multiple rep-families appeared in ST40. Totally 145 plasmid bands were identified in 95 E. faecalis strains by S1-PFGE, 59 strains carrying one plasmid, 27 carrying two, five carrying three, three carrying four, and one strain carrying five plasmids. Plasmid sizes varied between 5–150 kbp. There was a significant correlation between the number of plasmids identified by PCR rep-typing and by S1-PFGE. The results indicate that the majority of E. faecalis of marginal periodontitis are likely to be a reservoir for diverse mobile genetic elements and associated antimicrobial resistance determinants.     

Use of a Hanging-weight System for Isolated Renal Artery Occlusion

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia ^1-3. A recent study of hospitalized patients revealed that only a mild increase in serum creatinine levels (0.3 to 0.4 mg/dl) is associated with a 70% greater risk of death than in persons without any increase ¹. Along these lines, surgical procedures requiring cross-clamping of the aorta and renal vessels are associated with a renal failure rates of up to 30% ⁴. Similarly, AKI after cardiac surgery occurs in over 10% of patients under normal circumstances and is associated with dramatic increases in mortality. AKI are also common complications after liver transplantation. At least 8-17% of patients end up requiring renal replacement therapy ⁵. Moreover, delayed graft function due to tubule cell injury during kidney transplantation is frequently related to ischemia-associated AKI ⁶. Moreover, AKI occurs in approximately 20% of patients suffering from sepsis ⁶.The occurrence of AKI is associated with dramatic increases of morbidity and mortality ¹. Therapeutic approaches are very limited and the majority of interventional trials in AKI have failed in humans. Therefore, additional therapeutic modalities to prevent renal injury from ischemia are urgently needed ^{3, 7-9}. To elucidate mechanisms of renal injury due to ischemia and possible therapeutic strategies murine models are intensively required ^7-13. Mouse models provide the possibility of utilizing different genetic models including gene-targeted mice and tissue specific gene-targeted mice (cre-flox system). However, murine renal ischemia is technically challenging and experimental details significantly influence results. We performed a systematic evaluation of a novel model for isolated renal artery occlusion in mice, which specifically avoids the use of clamping or suturing the renal pedicle ¹⁴. This model requires a nephrectomy of the right kidney since ischemia can be only performed in one kidney due to the experimental setting. In fact, by using a hanging-weight system, the renal artery is only instrumented once throughout the surgical procedure. In addition, no venous or urethral obstruction occurs with this technique. We could demonstrate time-dose-dependent and highly reproducible renal injury with ischemia by measuring serum creatinine. Moreover, when comparing this new model with conventional clamping of the whole pedicle, renal protection by ischemic preconditioning is more profound and more reliable. Therefore his new technique might be useful for other researchers who are working in the field of acute kidney injury.     

Ion Transport in Isolated Rabbit Ileum : I. Short-circuit current and Na fluxes

The transmural potential difference, short-circuit current, and Na fluxes have been investigated in an in vitro preparation of isolated rabbit ileum. When the tissue is perfused with a physiological buffer, the serosal surface is electrically positive with respect to the mucosal surface and the initial potential difference in the presence of glucose averages 9 mv. Unidirectional and net Na fluxes have been determined under a variety of conditions, and in each instance, most if not all of the simultaneously measured short-circuit current could be attributed to the active transport of Na from mucosa to serosa. Active Na transport is dependent upon the presence of intact aerobic metabolic pathways and is inhibited by low concentrations of ouabain in the serosal medium. A method is described for determining whether a unidirectional ionic flux is the result of passive diffusion alone, in the presence of active transport of that ion in the opposite direction. Using this method we have demonstrated that the serosa-to-mucosa flux of Na may be attributed to passive diffusion with no evidence for the presence of carrier-mediated exchange diffusion or the influence of solvent-drag.