Taurine preserves gap junctional intercellular communication in rat hepatocytes under oxidative stress

Gap junctional intercellular communication (GJIC) between hepatocytes is important for the maintenance of differentiated liver functions. Taurine is known to be cytoprotective, and is used clinically to improve liver functions. We evaluated the effect of taurine on GJIC in hepatocyte doublets under oxidative stress. Hepatocyte doublets were isolated from female Wistar rats, using a collagenase perfusion technique, and cultured in Leibovitz-15 medium containing fetal bovine serum (10%). H₂O₂ (2 mM) and/or taurine (0.1–1 mM) were added 2 h after inoculation, and the culture was incubated for 3 h. Fluorescent dye (Lucifer Yellow CH) coupling between adjacent cells was evaluated by microinjection. The distribution and quantity of connexin 32 (Cx32) in hepatocytes were detected using indirect immunofluorescence analysis and Western blotting. Steady state mRNA levels of Cx32 were detected by Northern blotting. The percentage of dye coupling 5 h after inoculation was 88 ± 6.3% in the control, however, this was decreased to almost half the control value by H₂O₂. Taurine prevented the decrease caused by H₂O₂ in a dose-dependent manner. Immunofluorescence analysis for Cx32 demonstrated numerous punctate fluorescent spots along the intercellular plasma membrane in controls, which were significantly decreased by H₂O₂. Taurine prevented the decrease of Cx32. Western blot analysis also showed the decrease of Cx32 protein levels by H₂O₂ treatment, which decrease was prevented by taurine. Interestingly, H₂O₂ and/or taurine treatments did not affect Cx32 mRNA levels. Our findings indicated that H₂O₂ treatment decreased GJIC between hepatocytes, most likely due to augmenting the degradation of Cx32 proteins, whereas taurine prevented this process. This effect of taurine is beneficial for the preservation of differentiated functions in the liver under oxidative stress. Received: August 12, 1999 / Accepted: November 26, 1999     

Melatonin effects on intercellular junctional communication in MCF-7 human breast cancer cells

Melatonin exerts a direct antiproliferative effect on estrogen-responsive MCF-7 cells in culture. Recently, the importance of the anti-invasive actions of melatonin as a part of the oncostatic action of this indolamine has been reported. Gap junctional intercellular communication is known to be involved in controlling cell proliferation and differentiation, and a decrease in intercellular junctional communication has been described in highly invasive mammary cancer cells. Because melatonin at physiological doses (1 nM) shifts MCF-7 cells to a lower invasive status, we postulate that melatonin could modulate the levels of gap junctional intercellular communication in these tumor cells. To test our hypothesis, we studied gap junctional intercellular communication in MCF-7 human breast cancer cells previously (7-8 days) treated, or not, with melatonin (10 microM or 1 nM). Using the scrape-loading assay dye-transfer technique to introduce 0.05% Lucifer yellow into cells, we measured the ability of the tumor cells to transfer dye to adjacent cells. Rhodamine dextran (0.05%) was used as a control dye to verify that dye-transfer occurs through intercellular junctions. The presence of melatonin (10 microM or 1 nM) in the culture medium significantly increased (P < 0.01) the transfer of the dye to adjacent cells through gap junctions. This increase was greater at 10 microM melatonin, and averaged scan profiles of cells treated with melatonin 10 microM showed a statistically significant increase (P < 0.01) in the integrated optical density values, and a broadening of the densitometric scan. These findings suggest that melatonin could exert its antitumor action, at least in part, by increasing regulatory signals that are passed between adjacent epithelial cells through intercellular junctions.     

ZP123 increases gap junctional conductance and prevents reentrant ventricular tachycardia during myocardial ischemia in open chest dogs

INTRODUCTION: The aim of this study was to determine if the stable antiarrhythmic peptide (AAP) analogue ZP123 increases gap junctional intercellular conductance and prevents reentrant ventricular tachycardia (VT) during coronary artery occlusion. METHODS AND RESULTS: Voltage clamp experiments demonstrated that 10 nM ZP123 improved gap junctional intercellular conductance by 69% +/- 20% in pairs of guinea pig ventricular myocytes. VT was induced by programmed stimulation in alpha-chloralose anaesthetized open chest dogs 1 to 4 hours after coronary artery occlusion. Three-dimensional activation mapping was done using six bipolar electrograms on each of 23 multipolar needles in the risk zone. When VT was reproducibly induced, dogs were randomly assigned to receive either saline or ZP123 cumulatively at three dose levels (intravenous bolus followed by 30-min infusion per dose). Attempts to induce VT were repeated in each infusion period. Mass spectrometry was used to measure ZP123 plasma concentrations. Twenty-six dogs with reentrant VT were included. ZP123 significantly prevented reentrant VT at all plasma concentrations vs saline: 1.0 +/- 0.2 nM: 6/12 vs 0/12; 7.7 +/- 0.6 nM: 7/13 vs 1/12; and 69.2 +/- 5.4 nM: 9/13 vs 1/13. The preventive effect of ZP123 on reentrant VT was closely correlated to reversal of functional, unidirectional conduction block. ZP123 did not affect effective refractory period, surface ECG parameters, mean arterial pressure, or infarct size. CONCLUSION: The stable AAP analogue ZP123 increased gap junctional intercellular conductance and specifically prevented the induction of reentrant VT during ischemia in a broad dose range without proarrhythmic or hemodynamic side effects. ZP123 is a promising candidate for use in preventing ischemia-induced VT.     

Longitudinal shift in diabetic wound microbiota correlates with prolonged skin defense response

Diabetics frequently suffer from chronic, nonhealing wounds. Although bacterial colonization and/or infection are generally acknowledged to negatively impact wound healing, the precise relationship between the microbial community and impaired wound healing remains unclear. Because the host cutaneous defense response is proposed to play a key role in modulating microbial colonization, we longitudinally examined the diabetic wound microbiome in tandem with host tissue gene expression. By sequencing 16S ribosomal RNA genes, we show that a longitudinal selective shift in wound microbiota coincides with impaired healing in diabetic mice (Lepr^db/db; db/db). We demonstrate a parallel shift in longitudinal gene expression that occurs in a cluster of genes related to the immune response. Further, we establish a correlation between relative abundance of Staphylococcus spp. and the expression of cutaneous defense response genes. Our data demonstrate that integrating two types of global datasets lends a better understanding to the dynamics governing host–microbe interactions.     

Aberrant expression,function and localization of connexins in human esophageal carcinoma cell lines with different degrees of tumorigenicity

We have analyzed the level of mRNA expression and protein localization of the gap-junction protein connexins (Cx) 26, 32, and 43, as well as gap-junctional intercellular communication (GJIC) in seven human esophageal careinoma cell lines (TE series). These cell lines exhibited various degrees of tumorigenicity in nude mice; two (TE-1 and TE-8) formed progressively growing tumors, four (TE-2. TE-3, TE-9, and TE-13) developed non-progressing tumors and one (TE-10) showed no tumorigenicity. We found that normal human esophageal tissue expressed both Cx26 and Cx43 and that most of the cell lines expressed lower amounts of Cx26 and Cx43 mRNAs than normal human esophageal tissues or none at all. The co-expression of Cx26 and Cx43 mRNAs and proteins was observed only in two cell lines (TE-3 and TE-9) that showed a high level of GJIC and non-progressive tumor development. However, the non-tumorigenic cell line TE-10 did not express either connexin. A possible regulator of GJIC, E-cadherin, was expressed in all cell lines. These results suggest that aberrant expression and function of connexins are common among human esophageal carcinoma cell lines, but there is no quantitative relationship between connexin expression and tumorigenic properties of these cell lines.Abbreviations Cx connexin - GJIC gap-junctional intercellular communication     

Electrical interaction of mechanosensitive fibroblasts and myocytes in the heart

Fibroblasts in the heart can respond to mechanical deformation of the plasma membrane with characteristic changes of their membrane potential. Membrane depolarization of the fibroblasts occurs during the myocardial contractions and is caused by an influx of cations, mainly of sodium ions, into the cells. Conversely, application of mechanical stretch to the cells, i.e., during diastolic relaxation of the myocardium, will hyperpolarize the membrane potential of the fibroblasts due to reduced sodium entry. Thus, cardiac fibroblasts can function as mechano–electric transducers that are possibly involved in the mechano–electric feedback mechanism of the heart. Mechano–electric feedback refers to the phenomenon, that the cardiac mechanical environment, which depends on the variable filling pressure of the ventricles, modulates the electrical function of the heart. Increased sensitivity of the cardiac fibroblasts to mechanical forces may contribute to the electrical instability and arrhythmic disposition of the heart after myocardial infarction. Novel findings indicate that these processes involve the intercellular transfer of electrical signals between fibroblasts and cardiomyocytes via gap junctions. In this article we will discuss the recent progress in the electrophysiology of cardiac fibroblasts. The main focus will be on the intercellular pathways through which fibroblasts and cardiomyocytes communicate with each other.     

卡维地洛对大鼠心肌间隙连接通讯的影响及作用机制研究

目的研究卡维地洛对大鼠心肌缺血再灌注损伤、心肌间隙连接通讯（GJIC）及连接蛋白43（CX43）的影响,验证卡维地洛可通过改变CX43磷酸化状态抑制GJIC起到防止心肌再灌注损伤的假设.方法将大鼠随机分为假手术组、缺血再灌注组和卡维地洛组.结扎左冠状动脉前降支致缺血30 min后复灌4 h,建立心肌缺血再灌注损伤模型,于再灌注4 h末测定心肌酶及心肌梗死范围变化;制作Langendorf心脏灌流模型,随机分为假手术组、缺血再灌注组、卡维地洛组和庚醇组,于整体缺血30 min末期用改良的划痕标记染料示踪技术测定GJIC;用Western blot技术检测缺血30 min CX43磷酸化状态的改变.结果与假手术组相比,缺血再灌注组心肌酶及心肌梗死范围明显增加,GJIC无明显变化,非磷酸化CX43升高.与缺血再灌注组比较,卡维地洛组心肌酶及心肌梗死范围显著减少,同间隙连接抑制剂庚醇作用相似,GJIC也明显受到抑制,伴非磷酸化CX43水平明显上升.结论卡维地洛具有使CX43去磷酸化进而抑制GJIC防止心肌再灌注损伤的作用.     

大蒜素对胃上皮细胞Cx37、Cx43 mRNA表达及细胞间隙连接通讯功能的影响

目的观察体外实验中大蒜素对胃上皮细胞系MGC-803Cx37 mRNA、Cx43 mRNA表达及细胞间隙连接通讯功能的影响。方法终浓度为3μg／ml、6μg／ml、9μg／ml、12μg／ml大蒜素分别加入胃上皮细胞MGC-803培养24h、48h、72h，同时设立不加药物的阴性对照，用RT，PCR法检测细胞cx37mRNA、Cx43 mRNA表达，LY染料传输方法检测细胞间隙连接通讯功能。结果MGC-803细胞cx37mRNA有表达，cx43mRNA无表达，GJIC功能弱，加入不同浓度大蒜素后，cx37mRNA表达和细胞间隙连接通讯功能增强，cx37mRNA表达随大蒜素浓度和培养时间的增加而增强（均P〈0．05），Cx43 mRNA仍无表达。结论大蒜素在转录水平上调胃上皮细胞MGC-803 Cx37基因的表达，改善细胞间隙连接通讯功能。     

Up-regulation of gap junctional intercellular communication and connexin43 expression by retinoic acid in human endometrial stromal cells

CONTEXT: Gap junctions, made up of connexins (Cxs), play fundamental roles in coordinating a number of cellular processes through their ability to directly regulate cell-cell communication. Cx43 is the most widely expressed Cx in the endometrium and is known to be important in a variety of physiological and pathological processes in this tissue. OBJECTIVE: In this study, we investigated the ability of the retinoid, all-trans-retinoic acid (RA), to regulate Cx43 expression in human endometrial stromal cells. DESIGN: Primary endometrial stromal cells obtained from patients undegoing surgery for infertility workup were treated in vitro with RA and control compounds for different time periods, up to 48 h. Cx43 mRNA and protein levels, protein phosphorylation, and gap junctional intercellular communication (GJIC) were analyzed. RESULTS: Treatment of the cells with RA showed a dose-dependent increase in Cx43 expression at both the mRNA and protein levels. In addition, RA induced a relative decrease in the phosphorylated species of Cx43 while causing a corresponding increase in the nonphosphorylated form. Concomitant with these changes, RA-treated cells demonstrated up to a 250% enhancement of GJIC as assessed by dye transfer experiments. Augmentation of GJIC and alterations of Cx43 expression were observed over the same range of RA concentrations. Treatment of cells with the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate increased the phosphorylated species of Cx43 and correspondingly inhibited GJIC. CONCLUSIONS: Phosphorylation of Cx43 is inversely related to GJIC in endometrial stromal cells. Retinoids increase GJIC in endomentrial stromal cells through upregulation of Cx43 expression while inducing a decrease in the phosphorylated species of the protein. The data suggest a novel mechanism by which retinoids can influence endometrial cell biology.     

Extracellular matrix in human diabetic nephropathy: reduced expression of heparan sulphate in skin basement membrane

Summary In diabetic nephropathy, expression of glycosaminoglycan side chains of heparan sulphate proteoglycan in the glomerular basement membrane is reduced proportionally to the degree of proteinuria. We performed a cross-sectional study to evaluate whether non-vascular basement membranes also show a decrease in heparan sulphate side chain staining in patients with diabetic nephropathy. We evaluated the skin basement membrane for extracellular matrix components in the following groups: control subjects (n = 16); patients with Type 1 diabetes and normoalbuminuria (n = 17), microalbuminuria (n = 7), and macroalbuminuria (n = 16); patients with Type 1 diabetes and diabetic nephropathy undergoing renal replacement therapy (n = 13); and non-diabetic patients undergoing renal replacement therapy (n = 21). The following antibodies were used for this immunohistochemical study: monoclonal antibodies against the heparan sulphate side chain (JM403) and core protein (JM72) of the glomerular heparan sulphate proteoglycan; polyclonal antibodies against the core protein (B31); polyclonal antibodies against collagen types I, III, and IV, fibronectin, and laminin; and monoclonal antibodies against the non-collagenous domain of α1(collagen IV) and α3(collagen IV), against transforming growth factor β(2G7), and against advanced glycosylation end products (4G9). Expression of heparan sulphate side chains was reduced in the skin basement membrane of patients with overt diabetic nephropathy, of those with Type 1 diabetes undergoing renal replacement therapy, and those with non-diabetic renal failure. Increased intensity of staining was found for collagen type I and advanced glycosylation end products in patients with diabetic nephropathy. Changes in the extracellular matrix of the skin basement membrane seem to be similar to those in the glomerular basement membrane. These findings support the suggestion that patients with diabetic nephropathy also have altered heparan sulphate and collagen staining in extrarenal basement membranes. However, patients with non-diabetic renal failure also had reduced expression of heparan sulphate in the skin basement membrane, suggesting that this finding is not specific for diabetic nephropathy. [Diabetologia (1998) 41: 791–798] Received: 13 November 1997 and in revised form: 10 February 1998     

Aging of diabetic and nondiabetic skin fibroblasts in vitro: life span and sequential growth curves

Skin fibroblasts from age-matched normal and juvenile diabetic (IDDM) subjects were studied throughout their life span in vitro. The number of mean population doublings (MPD) attained at senescence was determined. Sequential 5-day growth curves were constructed. Normal cells were compared to cells from diabetics with recent onset of disease, 1-5 years, 6-10 years, and greater than 10 years of insulin therapy. A trend of decreasing MPD in vitro with increasing duration of diabetes in vivo was found. The growth potential of all cells (sequential growth curves) decreased with increasing time in vitro. The confluent density (Day 5) of diabetic (10 years' duration) cells was significantly lower than that for normal cells at Stage 2 of growth (passage 6-10). The in vitro aging of cells from diabetics (recent onset) was essentially the same as that of normal cells.     

人脐动脉内皮细胞和平滑肌细胞体外培养鉴定及缝隙连接通讯功能测定

目的:应用双光子激光扫描共聚焦显微镜鉴定体外培养的脐动脉内皮细胞(ECs)和平滑肌细胞(SMCs),应用荧光光漂白恢复技术(FRAP)测定血管ECs、SMCs之间的缝隙连接通讯(GJIC)功能。方法:人脐动脉ECs、SMCs分离培养,Ⅷ因子和SMα-actin相关抗原鉴定ECs和SMCs,应用FRAP技术测定血管内皮细胞、平滑肌细胞之间的GJIC功能,记录实时成像结果,应用动态比(M)计算漂白区域内标记荧光的分子中动态分子的比例。结果:第一组ECs和SMCs单独培养,选择漂白细胞与周围至少3个同种细胞相连接,SMCs被漂白后平均M值为31.79±5.69;ECs被漂白后平均M值为23.43±2.11;第二组ECs和SMCs混合培养,选择ECs和SMCs独立相连的2个细胞,SMCs被漂白后平均M值为14.47±3.28,ECs被漂白后平均M值为6.41±0.80。结论:FRAP实时动态恢复曲线可直接观察荧光恢复强度及速度,参照FRAP恢复曲线,M值可做为组间GJIC比较相对定量的可靠指标,通过检测证实ECs和SMCs之间存在GJIC,且荧光由ECs向SMCs方向的传递大于由SMCs向ECs方向的传递。     

Insulin binding and activation of glycogen synthase in fibroblasts from type 1 (insulin-dependent) diabetic patients

Summary ¹²⁵I-Insulin binding and insulin stimulation of glycogen synthase were examined in fibroblasts cultured from nine Type 1 (insulin-dependent) diabetic patients with age of onset of <42 years. In all cases specific insulin binding was qualitatively and quantitatively normal. Total ¹²⁵I-insulin binding was elevated in cells from three patients with early onset diabetes (two with onset before age 1 year) due to an increase in non-specific binding. When the ability of insulin to stimulate the conversion of the glucose-6-phosphate dependent to the glucose-6-phosphate independent form of glycogen synthase was measured, all cell lines responded, albeit to differing degrees. In general, the response of cells from diabetic donors was more variable than that of control fibroblasts. A slightly lower level of cellular glycogen was evident in the cells of the diabetic patients, and this was mirrored in slightly higher levels of the independent form of the enzyme. The average maximal level of the independent form of the enzyme also was higher in the diabetic patients' cells. Fibroblasts from one of the patients with very early onset diabetes had glycogen synthase levels that were markedly lower than in any other cell line examined. In summary, fibroblasts cultured from Type 1 diabetic patients do not show major defects in either insulin binding or action. A suggestion of subtle differences in the cells from the diabetic patients, particularly those with very early onset, is evident, however. Whether these are secondary to some primary genetic defect or represent some selection during culture remains to be determined.     

细胞间隙连接通讯及其通路蛋白与大肠癌发生关系的研究进展

细胞间隙连接通讯(gap junctional intercellular communication,GJIC)是多细胞生物体内普遍存在的一种通讯方式,他参与离子和其他小分子信号物质的转运.GJIC对细胞的生长、增殖和分化起重要的调控作用,他的改变与肿瘤的发生密切相关.大肠癌是人类常见的恶性肿瘤之一,目前研究表明,大肠癌的发生是一个多步骤、多基因、多阶段的过程,包括癌基因激活、抑癌基因失活、DNA转录表达失控、DNA损伤等.不论何种原因的细胞转化,其最终表现为细胞周期失控、细胞无限增殖.本文就GJIC及其通路蛋白、细胞因子与大肠癌的发生的研究进展作一综述.     

酪氨酸蛋白激酶抑制剂对缺氧复氧时内皮细胞连接通讯功能的影响

目的:缺氧复氧刺激能损伤内皮细胞间隙连接通讯功能,本研究观察了酪氨酸蛋白激酶抑制剂genistein对这种损伤的影响。方法:将人脐静脉内皮细胞株ECV304分3组给予不同处理。①常氧对照组:常氧状态下培养12～18小时;②缺氧复氧组:氧浓度低于1％状态下培养12～14小时,再置于常氧状态复氧0～6小时;③genistein组:加入不同浓度genistein(2μM、10μM、50μM)后,给予缺氧复氧组相同的处理。在试验各时段应用荧光漂白回复技术对细胞间隙连接通讯功能进行分析,结果用荧光回复率表示。结果:不同浓度genistein对复氧2小时的内皮细胞荧光回复率作用不同。低浓度genistein(2μM)对荧光回复率没有作用;中、高浓度genistein(10μM、50μM)能使荧光回复率上升(P<0.05)。结论:gensetein能保护缺氧复氧时内皮细胞间隙连接通讯功能。提示缺氧刺激通过酪氨酸蛋白激酶途径损伤内皮细胞的间隙连接通讯功能。     

糖尿病大鼠全层皮肤缺损创面愈合过程中皮肤神经末梢再生的研究

目的观察糖尿病大鼠与正常大鼠全层皮肤缺损刨面愈合过程中神经末梢的再支配情况与创面愈合的关系。方法非糖尿病大鼠（n=12）与糖尿病大鼠（n=12）制成相同的全层皮肤缺损创面愈合模型。于伤后3d、7d和14d采取创面皮肤标本，用组织病理HE和SP抗体免疫组织化学染色技术检测创面肉芽再生与再上皮化情况，并对肉芽创面内周围神经末梢再生情况进行观察。结果糖尿病大鼠全层皮肤缺损创面愈合的时间延长，其周围神经末梢的数量较正常组明显减少，再生速度缓慢，肉芽组织中微血管和胶原形成的能力明显降低。结论糖尿病大鼠创面的愈合障碍与神经组织再生延迟相关。     

Formation and growth of gap junctions in mouse myocardium during ontogenesis: quantitative data and their implications on the development of intercellular communication.

The results of a study on the formation and growth of gap junctions in the mouse heart during ontogenesis was subjected to a statistical analysis in relation to their density and junctional surface area. A mathematical method was developed to calculate the density of gap junctions (average of gap junctions per unit area) in gap junction-containing regions. The results show that there is no mathematically significant increase in gap junction density in the gap junction-containing regions. However, a significant increase during the heart development in the ratio of gap junction to plasma membrane area is demonstrated. Results are criticized in relation to the validity of the sampling process. Implications of the results in terms of the development of non-electrical and electrical communication are discussed.     

Remodeling of myocardial sleeve and gap junctions in canine superior vena cava after rapid pacing

Objective We studied the response of the superior vena cava (SVC) myocardial sleeve to atrial fibrillation (AF). Methods and results We examined adult male dogs without pacing (N=6) and after rapid atrial pacing (600 bpm) for 2 weeks (P2w; N=5) and 6–8 weeks (P6–8w; N=5). After pacing, the sleeve was increased in thickness (non-paced vs. either paced group, both P<0.05). This was associated with an increase in proliferative activity, which was higher in the P2w than the P6–8w animals (P < 0.05). In addition, collagen content increased, and the component cardiomyocytes become more unevenly oriented and shorter and narrower in shape (non-paced vs. either paced group, both P < 0.05). Pacing had different effects on connexin40 (Cx40) and Cx43 gap junctions. There was a 98% increase in Cx43 signal in P2w, and a 74% increase in P6–8w animals (non-paced vs. each paced group, both P < 0.05). In contrast, Cx40 signal decreased 47% in P2w but increased 44% in P6–8w animals (non-paced vs. each paced group, both P < 0.05). Conclusions Rapid atrial pacing results in a specific pattern of remodeling of the canine SVC sleeve, including changes in size and shape, spatial orientation, and gap junction expression profile of the component cardiomyocytes. These changes may co-operatively affect the electrical properties and contribute to the formation and maintenance of the arrhythmogenic substrate of AF.     

载洛伐他汀纤维膜促糖尿病大鼠皮肤修复的研究

目的 探讨载洛伐他汀纤维膜（Lov/PDLGA）对糖尿病大鼠皮肤创伤的促修复作用. 方法 采用高压静电纺丝技术制备Lov/PDLGA,扫描电子显微镜观察纤维形貌,高效液相色谱法检测纤维膜载药量、包封率和体外药物释放行为.建立糖尿病大鼠皮肤损伤模型,分别覆盖空白纤维膜（PDLGA）和Lov/PDLGA,另以自然愈合创面为对照,观察各组创面愈合速度. 结果 Lov/PDLGA纤维直径（2.66±0.73）μm.载药量、包封率分别为（7.39±0.40）％、（80.45±4.39）％.4h体外药物突释量（33.86±7.07）％,随后5d缓慢持续释放药物（79.72±1.11）％.Lov/PDLGA组创面20 d愈合（99.98±0.61）％,愈合速度快于其余组（P＜0.05）. 结论 Lov/PDLGA可通过持续释放载洛伐他汀促进糖尿病大鼠皮肤创面愈合,有望应用于糖尿病皮肤溃疡治疗.