Acute internalization of gap junctions in vascular endothelial cells in response to inflammatory mediator-induced G-protein coupled receptor activation

During the inflammatory response, activation of G-protein coupled receptors (GPCRs) by inflammatory mediators rapidly leads to inhibition of gap junction intercellular communication (GJIC); however, the steps that lead to this inhibition are not known. Combining high-resolution fluorescence microscopy and functional assays, we found that activation of the GPCRs PAR-1 and ET_A/B by their natural inflammatory mediator agonists, thrombin and endothelin-1, resulted in rapid and acute internalization of gap junctions (GJs) that coincided with the inhibition of GJIC followed by increased vascular permeability. The endocytosis protein clathrin and the scaffold protein ZO-1 appeared to be involved in GJ internalization, and ZO-1 was partially displaced from GJs during the internalization process. These findings demonstrate that GJ internalization is an efficient mechanism for modulating GJIC in inflammatory response.     

Cisplatin-induced premature senescence with concomitant reduction of gap junctions in human fibroblasts

To examine the role of gap junctions in cell senescence, the changes of gap junctions in cisplatin-induced premature senescence of primary cultured fibroblasts were studied and compared with the replicative senescent human fibroblasts.Dye transfer assay for gap junction function and immunofluorescent staining for connexin 43 protein distribution were done respectively. Furthermore, cytofluorimetry and DAPI fluorescence staining were performed for cell cycle and apoptosis analysis, p53 gene expression level was detected with indirect immunofluorescence. We found that cisplatin(10mM) treatment could block cell growth cycle at G1 and induced premature senescence. The premature senescence changes included high frequency of apoptosis, elevation of p53 expression, loss of membranous gap junctions and reduction of dye-transfer capacity. These changes were comparable to the changes of replicative senescence of human fibroblasts. It was also concluded that cisplatin could induce premature senescence concomitant with inhibition of gap junctions in the fibroblasts. Loss of functional gap junctions from the cell membrane may account for the reduced intercellular communication in the premature senescent fibroblasts. The cell system we used may provide a model useful for the study of the gap junction thus promoting agents against premature senescence.     

Chronopharmacology of simvastatin on hyperlipidaemia in high‐fat diet‐fed obese mice

The chronopharmacology refers to the utilization of physiological circadian rhythms to optimize the administration time of drugs, thus increasing their efficacy and safety, or reducing adverse effects. Simvastatin is one of the most widely prescribed drugs for the treatment of hypercholesterolaemia, hyperlipidemia and coronary artery disease. There are conflicting statements regarding the timing of simvastatin administration, and convincing experimental evidence remains unavailable. Thus, we aimed to examine whether different administration times would influence the efficacy of simvastatin. High‐fat diet‐fed mice were treated with simvastatin at zeitgeber time 1 (ZT1) or ZT13, respectively, for nine weeks. Simvastatin showed robust anti‐hypercholesterolaemia and anti‐hyperlipidemia effects on these obese mice, regardless of administration time. However, simvastatin administrated at ZT13, compared to ZT1, was more functional for decreasing serum levels of total cholesterol, triglycerides, non‐esterified free fatty acids and LDL cholesterol, as well as improving liver pathological characteristics. In terms of possible mechanisms, we found that simvastatin did not alter the expression of hepatic circadian clock gene in vivo, although it failed to change the period, phase and amplitude of oscillation patterns in Per2::Luc U2OS and Bmal1::Luc U2OS cells in vitro. In contrast, simvastatin regulated the expression of Hmgcr, Mdr1 and Slco2b1 in a circadian manner, which potentially contributed to the chronopharmacological function of the drug. Taken together, we provide solid evidence to suggest that different administration times affect the lipid‐lowering effects of simvastatin.     

VEGF reduces astrogliosis and preserves neuromuscular junctions in ALS transgenic mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease resulting from motor neuron loss in the spinal cord and brain stem. In the present study, we found that systemic administration of recombinant vascular endothelial growth factor (VEGF) significantly diminished astrogliosis and increased the number of neuromuscular junctions in a Cu/Zn superoxide dismutase (SOD1) transgenic mouse model of ALS. Our results thus demonstrate a novel regulatory role of VEGF on astrocytes and are suggestive of protective effects of VEGF both in the peripheral and central nervous system in the SOD1 transgenic mouse model. These findings warrant further evaluation of the mechanism(s) of regulatory effects of VEGF on neuronal and non-neuronal cells, and the relation of these events to motor neuron degeneration and the onset and progression of ALS.     

Molecular and morphological approach to study the innexin gap junctions in Rhynchosciara americana

Gap junctions mediate communication between adjacent cells and are fundamental to the development and homeostasis in multicellular organisms. In invertebrates, gap junctions are formed by transmembrane proteins called innexins. Gap junctions allow the passage of small molecules through an intercellular channel, between a cell and another adjacent cell. The dipteran Rhynchosciara americana has contributed to studying the biology of invertebrates and the study of the interaction and regulation of genes during biological development. Therefore, this paper aimed to study the R. americana innexin-2 by molecular characterization, analysis of the expression profile and cellular localization. The molecular characterization results confirm that the message is from a gap junction protein and analysis of the expression and cellular localization profile shows that innexin-2 can participate in many physiological processes during the development of R. americana.     

Notch receptor expression in Trypanosoma cruzi‐infected human umbilical vein endothelial cells treated with benznidazole or simvastatin revealed by microarray analysis

Chagas disease is a vector‐borne disease caused by the protozoan parasite Trypanosoma cruzi. Current therapy involves benznidazole. Benznidazole and other drugs can modify gene expression patterns, improving the response to the inflammatory influx induced by T. cruzi and decreasing the endothelial activation or immune cell recruitment, among other effects. Here, we performed a microarray analysis of human umbilical vein endothelial cells (HUVECs) treated with benznidazole and the anti‐inflammatory drugs acetylsalicylic acid or simvastatin and infected with T. cruzi. Parasitic infection produces differential expression of a set of genes in HUVECs treated with benznidazole alone or a combination with simvastatin or acetylsalicylic acid. The differentially expressed genes were involved in inflammation, adhesion, cardiac function, and remodeling. Notch1 and high mobility group B1 were genes of interest in this analysis due to their importance in placental development, cardiac development, and inflammation. Quantitative polymerase chain reaction confirmation of these two genes indicated that both are upregulated in the presence of benznidazole.     

Development changes in regulation of embryonic chick heart gap junctions

Summary Embryonic chick myocyte pairs were isolated from ventricular tissue of 4-day, 14-day, and 18-day heart for the purpose of examining the relationship between macroscopic junctional conductance and transjunctional voltage during cardiac development. The double whole-cell patch-clamp technique was employed to directly measure junctional conductance over a transjunctional voltage range of ±100 mV. At all ages, the instantaneous junctional current (or conductance=current/voltage) varied linearly with respect to transjunctional voltage. This initial response was followed by a time- and voltage-dependent decline in junctional current to new steady-state values. For every experiment, the steady-state junctional conductance was normalized to the instantaneous value obtained at each potential and the data was pooled according to developmental age. The mean steadystate junctional conductance-voltage relationship for each age group was fit using a two-state Boltzmann distribution described previously for other voltage-dependent gap junctions. From this model, it was revealed that half-inactivation voltage for the transjunctional voltage-sensitive conductance shifted towards larger potentials by 10 mV, the equivalent gating charge increased by approximately 1 electron, and the minimal voltage-insensitive conductance exactly doubled (increased from 18 to 36%) between 4 and 18 days of development. Decay time constants were similar at all ages examined as rate increased with increasing transjunctional potential. This data provides the first direct experimental evidence for developmental changes in the regulation of intercellular communication within a given tissue. This information is consistent with the hypothesis that developmental expression of multiple gap junction proteins (connexins) may confer different regulatory mechanisms on intercellular communication pathways within a given cell or tissue.     

Glial connexins and gap junctions in CNS inflammation and disease

Gap junctions facilitate direct cytoplasmic communication between neighboring cells, facilitating the transfer of small molecular weight molecules involved in cell signaling and metabolism. Gap junction channels are formed by the joining of two hemichannels from adjacent cells, each composed of six oligomeric protein subunits called connexins. Of paramount importance to CNS homeostasis are astrocyte networks formed by gap junctions, which play a critical role in maintaining the homeostatic regulation of extracellular pH, K⁺, and glutamate levels. Inflammation is a hallmark of several diseases afflicting the CNS. Within the past several years, the number of publications reporting effects of cytokines and pathogenic stimuli on glial gap junction communication has increased dramatically. The purpose of this review is to discuss recent observations characterizing the consequences of inflammatory stimuli on homocellular gap junction coupling in astrocytes and microglia as well as changes in connexin expression during various CNS inflammatory conditions.     

Vascular abnormalities in mice lacking the endothelial gap junction proteins connexin37 and connexin40

Cells within the vascular wall are coupled by gap junctions, allowing for direct intercellular transfer of low molecular weight molecules. Although gap junctions are believed to be important for vascular development and function, their precise roles are not well understood. Mice lacking either connexin37 (Cx37) or connexin40 (Cx40), the predominant gap junction proteins present in vascular endothelium, are viable and exhibit phenotypes that are largely non-blood vessel related. Since Cx37 and Cx40 are coexpressed in endothelial cells and could overlap functionally, some roles of junctional communication may only be revealed by the elimination of both connexins. In this study, we interbreed Cx37 and Cx40 knockout mice to generate Cx37-/- Cx40-/- animals and show that they display severe vascular abnormalities and die perinatally. Cx37-/- Cx40-/- animals exhibit localized hemorrhages in skin, testis, gastrointestinal tissues, and lungs, with pronounced blood vessel dilatation and congestion occurring in some areas. Vascular anomalies were particularly striking in testis and intestine. In testis, abnormal vascular channels were present, with these channels coalescing into a cavernous, endothelium-lined blood pool resembling a hemangioma. These results provide evidence of a critical role for endothelial gap junction-mediated communication in the development and/or functional maintenance of segments of the mouse vasculature.     

Quantitative evaluation of gap junctions in rat brown adipose tissue after cold acclimation

Summary The decrease in the metabolic capacity of rat brown adipose tissue during the late postnatal period can be reversed by cold acclimation of the animals. In order to find out whether a parallel decrease in capability for intercellular communication observed during this period is also reversed by cold acclimation, gap junction size and number per unit area of cell surface have been quantified in freeze-fracture replicas; cell diameters have been measured in semi-thin sections. It was found that the specific number of gap junctions remains unchanged during cold acclimation. However, the mean gap junction size increases by 75% and the ratio of gap junctional area per cell volume, an index for intercellular exchange capacity, is doubled. This result illustrates further the parallelism between metabolic capacity and cell communication in brown fat.     

Fate of annular gap junctions in the papillary cells of the enamel organ in the rat incisor

Summary To investigate the mechanisms whereby annular gap junctions in the papillary cells of the enamel organ are degraded intracellularly, continuously growing rat incisors were examined by electron microscopy of routine thin sections as well as for the cytochemical localization of inorganic trimetaphosphatase activity. Routine thin-section analysis revealed small flat or undulated gap junctions, hemi-annular gap junctions between an invaginated cell process and a cell body, and fully internalized cytoplasmic annular gap junctions. Both hemi-annular and annular gap junctions usually contain various organelles and/or inclusions, such as mitochondria, endoplasmic reticulum, ribosomes, vesicles, and lysosomes in the cytoplasm confined by the junctional membranes. Annular gap junctions are sometimes fused with vesicular or tubulovesicular structures. Cytochemistry of inorganic trimetaphosphatase activity revealed an intense enzymatic reaction within a system of tubular structures and round or oval dense bodies. Both structures are believed to correspond to primary lysosomes. A part of the Golgi apparatus also shows a weak reaction. Although hemi-annular gap junctions never show enzymatic reaction, annular gap junctions sometimes contain reaction products throughout their interior cytoplasm and inclusions. Fusion of annular gap-junctional membranes with reaction-positive tubular structures is also observed. In one instance, revealed in serial sections, an annular gap junction was encircled entirely by a reaction-positive structure. These results suggest that cytoplasmic annular gap junctions are formed by endocytosis of hemi-annular gap junctional membranes from the cell surface and then degraded intracellularly by lysosomal enzymes.     

Internalized gap junctions in ciliary epithelium of rabbit and rat

Summary Transmission electron microscopy was used to examine internalized gap junctions (IGJ) in rabbit and rat ciliary epithelial cells. A prominent feature of all the specimens studied was the presence of different images of IGJ membrane that entrapped a portion of an adjoining cell. We documented and analyzed more than 500 gap junction (GJ) vacuoles and invaginations, the latter comprising less than 20% of all the structures examined. With ten exceptions found in non-pigmented cells, all the IGJ were unidirectionally internalized within the cytoplasm of pigmented epithelial cells. Morphological signs of autophagic degradation of GJ vacuoles were observed. An essential finding was that once a GJ membrane started to invaginate, a lucidation of a part of the protruding cytoplasm occurred; no planar GJ membranes exhibited such an alteration. The present findings suggest that IGJ derived from the epithelium of ciliary processes arise through an invagination-endocytosis mechanism and are degraded autophagically. This phenomenon may be relevant to aqueous humor production.     

Protein kinase C affects reformation of endothelial junctions in xenopus XTH-2 cells.

Endothelial cells can reversibly be forced to suppress the formation of endothelial junctions (EJ) by cultivation in a low calcium medium. The authors localized vinculin and cadherin as marker proteins of EJ and actin as a cytoskeletal component by fluorescence microscopy, and used this cell model to study the reformation of endothelial junctions under conditions of activation and inhibition of protein kinase C (PKC). Inhibition of PKC by H-7 leads to an acceleration of EJ reformation, while constitutive activation by TPA inhibits the reformation process.     

Immunological characterization of rat cardiac gap junctions: Presence of common antigenic determinants in heart of other vertebrate species and in various organs

Summary Antibodies to the following synthetic peptide, SALGKLLDKVQAY, were purified by affinity chromatography and characterized by ELISA and immunoblotting. These antibodies, shown to be specific to the major protein constitutent of isolated rat heart junctions: connexin 43, cross-reacted with a homologous protein in immunoreplicas of whole heart fractions of trout, frog, chicken, guinea pig, mouse and rat, suggesting a phylogenic conservation of connexin 43 in vertebrates. By immunoblotting of whole organ fractions it was also demonstrated that these antibodies cross-reacted with major proteins ofM _r 32 and 22 kD in rat and mouse liver, ofM _r 41 kD in rat cerebellum, ofM _r 43 kD in uterus, stomach and kidney of rat, ofM _r 46 and 70 kD in rat lens, suggesting that these proteins share common or related epitopes with the synthetic peptide and connexin 43.     

Differential regulation of intracellular calcium oscillations by mitochondria and gap junctions

Fluctuations of intracellular Ca²⁺ ([Ca²⁺]_i) regulate a variety of cellular functions. The classical Ca²⁺ transport pathways in the endoplasmic reticulum (ER) and plasma membrane are essential to [Ca²⁺]_i oscillations. Although mitochondria have recently been shown to absorb and release Ca²⁺ during G protein-coupled receptor (GPCR) activation, the role of mitochondria in [Ca²⁺]_i oscillations remains to be elucidated. Using fluo-3-loaded human teratocarcinoma NT2 cells, we investigated the regulation of [Ca²⁺]_i oscillations by mitochondria. Both the muscarinic GPCR agonist carbachol and the ER Ca²⁺-adenosine triphosphate inhibitor thapsigargin (Tg) induced [Ca²⁺]_i oscillations in NT2 cells. The [Ca²⁺]_i oscillations induced by carbachol were unsynchronized among individual NT2 cells; in contrast, Tg-induced oscillations were synchronized. Inhibition of mitochondrial functions with either mitochondrial blockers or depletion of mitochondrial DNA eliminated carbachol—but not Tg-induced [Ca²⁺]_i oscillations. Furthermore, carbachol-induced [Ca²⁺]_i oscillations were partially restored to mitochondrial DNA-depleted NT2 cells by introduction of exogenous mitochondria. Treatment of NT2 cells with gap junction blockers prevented Tg-induced but not carbachol-induced [Ca²⁺]_i oscillations. These data suggest that the distinct patterns of [Ca²⁺]_i oscillations induced by GPCR and Tg are differentially modulated by mitochondria and gap junctions.     

Communicating (gap) junctions between chloride cells in the gill epithelium of the lamprey,Geotria australis

Summary Freeze-fracture replicas show that communicating (gap) junctions are present between chloride cells in the gill epithelium of young adults of the Southern Hemisphere lamprey, Geotria australis, acclimated to full-strength sea water. The junctions, which were already present when these lampreys were migrating downstream, may help coordinate the secretory activities of the chloride cells during the marine phase of the lamprey life cycle.     

Deciphering the functional role of endothelial junctions by using in vivo models

Endothelial cell-to-cell junctions are vital for the formation and integrity of blood vessels. The main adhesive junctional complexes in endothelial cells, adherens junctions and tight junctions, are formed by transmembrane adhesive proteins that are linked to intracellular signalling partners and cytoskeletal-binding proteins. Gene inactivation and blocking antibodies in mouse models have revealed some of the functions of the individual junctional components in vivo, and are increasing our understanding of the functional role of endothelial cell junctions in angiogenesis and vascular homeostasis. Adherens-junction organization is required for correct vascular morphogenesis during embryo development. By contrast, the data available suggest that tight-junction proteins are not essential for vascular development but are necessary for endothelial barrier function.     

Structure and distribution of gap junctions in lens epithelium and fiber cells

Summary We report a comparative study of gap junctions in lens epithelia of frog, rabbit, rat and human, using a double mounting method for freeze-fracture electron microscopy. The gap junctions on the narrow sides of hexagonal cortical fiber cells of various species were also studied with the same technique. Gap junctions were commonly present between epithelial cells of the entire undifferentiated epithelium, between fiber cells on both wide and narrow sides, and between epithelial cells and fiber cells. Structural diversity of gap junctions, based on connexon arrangements, was evident in lens epithelia among the four species studied. Gap junctions with random arrays of connexons were found predominantly in frog lens epithelium, while the crystalline and striated configurations were mainly observed in the epithelia of human and rat, and of rabbit, respectively. On the other hand, there was no structural variation of gap junctions observed on either wide or narrow sides of lens fiber cells from any species studied. Only the random-type gap junction was found. However, the distribution of gap junctions was unique on the narrow sides. There was a single row of junctional plaques along the middle of the narrow sides, whereas the wide sides showed an uneven distribution pattern. The gap junctions between epithelial cells and fiber cells had a random packing of connexons.     

Hepatic gap junctions in the hepatocarcinogen-resistant DRH rat

Although the gap junction or connexin (Cx) is considered to be a tumor-suppressor, it is also required for tumor promotion. Therefore, we examined hepatic gap junctions in hepatocarcinogen-resistant (DRH) rats. Specifically, we investigated gap junction structure and Cx32 expression during normal conditions and in response to a hepatocarcinogen, 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB). On a basal diet without 3'-MeDAB, hepatic gap junctions and Cx32 protein expression were greater in DRH rats than in control Donryu rats, as evidenced by morphometry, immunohistochemistry and immunoblotting. On a diet containing 3'-MeDAB, gap junctions and expressed Cx32 were increased significantly in Donryu rats, but not in DRH rats. In this condition, Donryu rats lost weight but DRH rats increased relative liver weight. After 3'-MeDAB treatment, cathepsin D expression in hepatocytes was significantly increased only in Donryu rats, indicating that DRH rats were less susceptible to 3'-MeDAB. The abundance of mitogen-activated protein kinase, some constituent of which might be associated with the degree of Cx protein phosphorylation, was reduced to a greater extent in Donryu than in DRH rats after 3'-MeDAB treatment. The resistance of DRH rats to carcinogenesis may be due partially to their stabilized gap junctions, which could coordinate metabolic coupling to evade 3'-MeDAB toxicity.