Number Of Gap Junction Plaques Research Articles

Novel GJA1/Cx43 Variant Associated With Oculo-Dento-Digital Dysplasia Syndrome: Clinical Phenotype and Cellular Mechanisms

Oculodentodigital dysplasia syndrome is associated with numerous pathogenic variants in GJA1, the gene encoding connexin43 gap junction protein. A novel in-frame deletion (p.Lys134del) was found in our clinic. The patient showed all the typical dysmorphic features of the syndrome. The functional consequences of this variant were also studied in an in vitro system. Cells expressed significantly less number of gap junction plaques with a great number of them retained intracellularly.

Alterations at Arg76 of human connexin 46, a residue associated with cataract formation, cause loss of gap junction formation but preserve hemichannel function.

The connexins are members of a family of integral membrane proteins that form gap junction channels between apposed cells and/or hemichannels across the plasma membranes. The importance of the arginine at position 76 (Arg76) in the structure and/or function of connexin 46 (Cx46) is highlighted by its conservation across the entire connexin family and the occurrence of pathogenic mutations at this (or the corresponding homologous) residue in a number of human diseases. Two mutations at Arg76 in Cx46 are associated with cataracts in humans, highlighting the importance of this residue. We examined the expression levels and macroscopic and single-channel properties of human Cx46 and compared them with those for two pathogenic mutants, namely R76H and R76G. To gain further insight into the role of charge at this position, we generated two additional nonnaturally occurring mutants, R76K (charge conserving) and R76E (charge inverting). We found that, when expressed exogenously in Neuro2a cells, all four mutants formed membrane hemichannels, inducing membrane permeability at levels comparable to those recorded in cells expressing the wild-type Cx46. In contrast, the number of gap-junction plaques and the magnitude of junctional coupling were reduced by all four mutations. To gain further insight into the role of Arg76 in the function of Cx46, we performed homology modeling of Cx46 and in silico mutagenesis of Arg76 to Gly, His, or Glu. Our studies suggest that the loss of interprotomeric interactions has a significant effect on the extracellular domain conformation and dynamics, thus affecting the hemichannel docking required for formation of cell-cell channels.

Connexin30 mediated intercellular communication plays an essential role in epithelial repair in the cochlea

A role for connexin (Cx)30 in epithelial repair following injury was examined in the organ of Corti, the sensory epithelium of the cochlea. In this tissue, lesions caused by loss of the sensory hair cells are closed by the supporting cells that surround each one. Gap junctions in which Cx30 is the predominant connexin are large and numerous between supporting cells. In mice carrying a deletion in the gene (Gjb6) that encodes Cx30, the size and number of gap junction plaques, and the extent of dye transfer, between supporting cells was greatly reduced compared with normal animals. This corresponded with unique peculiarities of the lesion closure events during the progressive hair cell loss that occurs in these animals in comparison with other models of hair cell loss, whether acquired or as a result of a mutation. Only one, rather than all, of the supporting cells that contacted an individual dying hair closed the lesion, indicating disturbance of the co-ordination of cellular responses. The cell shape changes that the supporting cells normally undergo during repair of the organ of Corti did not occur. Also, there was disruption of the migratory activities that normally lead to the replacement of a columnar epithelium with a squamous-like one. These observations demonstrate a role for Cx30 and intercellular communication in regulating repair responses in an epithelial tissue.

Density and distribution of connexin 43 in corpus cavernosum tissue from diabetic and hypogonadal patients with erectile dysfunction.

Altered expression of connexin 43 (Cx43) has been postulated to be involved in the development and progression of various diseases including erectile dysfunction (ED). The aim of this study was to determine whether distribution and density of the gap junction protein Cx43 are altered in human corpus cavernosum (HCC) tissue samples derived from diabetic or hypogonadal patients with ED compared to those from normal subjects. HCC tissue sections derived from normal, diabetic and hypogonadal subjects were fixed in 4% formaldehyde, embedded in paraffin and immunostained with a monoclonal mouse anti-rat Cx43 antibody. Cx43 density was expressed as the cumulative number of gap junction plaques per unit area of tissue corrected for number of 4',6-diamidino-2-phenylindole, dihydrochloride-labeled smooth muscle cells (dots per unit area corrected for number of cells). The distribution of Cx43 plaques in smooth muscle was not affected in tissues derived from diabetic or hypogonadal subjects with ED compared with those from normal subjects. However, the number of Cx43 plaques was significantly reduced in HCC tissues derived from diabetic or hypogonadal subjects (73 ± 8% and 68 ± 11% of normal, respectively), indicating reduced Cx43 gap junctions in diabetic and hypogonadal subjects with ED. Cx43 density in the HCC was diminished in tissue samples derived from diabetic or hypogonadal patients with ED compared to tissue samples from normal non-diabetic subjects. This marked decrease in Cx43 gap junction channels may contribute to attenuated gap junction function and to diminished erectile physiology.

Endocytosis of Connexin Protein in Adrenal Cells

The ability of adrenocorticotropin (ACTH) to affect gap junctions was examined in adrenal cells in vivo and in vitro. Treatment with ACTH increased the size and number of gap junction plaques on the cell membranes in hypophysectomized animals and in adrenal culture. Intracellular (cytoplasmic) annular gap junctions were observed in cells of the inner adrenal cortical zones and in adrenal cell cultures. To investigate the relationship of annular gap junctions to surface junctions, adrenal cells in culture were transfected with cDNA encoding a green fluorescent protein tagged connexin 43 construct (Cx43‐GFP), and subsequently studied by time‐lapse video microscopy, immunocytochemistry, and transmission electron microscopy (TEM). Internalization of part or all of a surface gap junction plaque resulted in annular gap junction formation. These studies support the hypothesis that cytoplasmic vesicles, initially described with TEM methods, can result from removal of gap junction plaques from the cell surface. It is suggested that hormones can play a regulatory role in cell‐cell communication by influencing the availability of gap junction protein at the cell surface and that hormonally‐sensitive processes might serve as a means of altering intercellular communication.

Hypophysectomy Results in a Loss of Connexin Gap Junction Protein from the Adrenal Cortex

To test the hypothesis that gap junctions are dependent on the tropic state of the adrenal gland, the effect of hypophysectomy on connexin 43 (α1-Cx43) gap junction protein occurrence and distribution was examined in mice. Gap junction protein occurrence was assessed with immunohistochemical techniques. In the adrenal gland from intact animals, α1- Cx43 gap junction protein was detected in the zonae fasciculata(ZF) and reticularis (ZR) while only a few α1- Cx43 gap junction plaques were found connecting zona glomerulosa(ZG) cells. Hypophysectomy led to a profound atrophy of the cortex which was more marked in the inner zones (zonae fasciculata and reticularis) than in the zona glomerulosa. There was a time dependent loss of α1- Cx43 gap junction protein in the adrenal cortex after hypophysectomy. At 33 day following hypophysectomy there was a two fold decrease in gap junctions in the zona fasciculata while the average gap junction plaque size was not different than the size seen in control animal adrenal glands. ACTH (lU/gm body weight) treatment in hypophysectomized animals increased the number of gap junction plaques in the zona fasciculata. Hypophysectomy led to diminished α1-Cx43 gap junction expression in the zona fasciculata which could be restored by ACTH treatment. Because altering the tropic state of the adrenal glands via hypophysectomy leads to a reduction in gap junction number, it can be suggested that control of gap junction expression in the adrenal gland is hormone dependent and linked to adrenal gland function.

Progesterone, oestradiol and oxytocin and their in vitro effect on maintaining the number of gap junction plaques in human myometrium at term.

Previous studies by electron microscopy and laser confocal microscopy of immunohistochemically stained sections have shown that during pregnancy the extent of gap junction formation in human myometrium is low, but that an increase occurs in the active stages of labour despite a high concentration of progesterone in maternal blood. The present investigation focused on the effect of in vitro exposure of isolated myometrial tissue to progesterone, oestradiol and oxytocin, on the number of gap junction plaques in human myometrium at term. Myometrial biopsies were obtained at term from 13 pregnant women who had an elective caesarean section in the 37th or 40th week of pregnancy. The biopsies were immersed immediately in Hepes buffer and buffer containing 0.5, 5.0 micrograms mL-1 of progesterone, and 0.1 micrograms mL-1 of oestradiol. The muscle biopsies were trimmed under a stereo microscope into strips along the bundles of smooth muscle cells and mounted in tissue baths, superfused with Hepes buffer supplemented with glucose (0.01 mM); subsequently the strips were exposed to buffer containing different concentrations of progesterone, oestradiol and oxytocin. Incubation of the muscle strips for 180 min resulted in a significant decrease of the number of gap junctions (P < 0.01). Neither oestradiol or oxytocin, alone or in combination, had a significant effect on the maintenance of the number of gap junctions. The progesterone concentration of 5.0 micrograms mL-1, combined with oxytocin, and with or without oestradiol has a significantly positive effect on the number of gap junction plaques in strips of human myometrium at term (P < 0.05 vs. buffer alone). The high concentration of progesterone in the superfusion medium during in vitro experiments may be responsible for the maintenance of high numbers of gap junction complexes in term human myometrium. This finding is of interest in the light of findings of persisting high progesterone levels in maternal blood during labour.

Inhibition of gap junction intercellular communications of cultured rat hepatocytes by ethanol: role of ethanol metabolism

Background/Aims: In a previous study, we reported that in cultured rat hepatocytes, ethanol inhibits intercellular communication which is known to play a central role in the regulation of cell growth and differentiation. This work was designed to find out if ethanol exerts a direct action on cell membranes, comparable to other long-chain (C6–C9) alcohols, or an indirect action. Methods: Intercellular communication was measured on short-term cultured rat hepatocytes by the fluorescent Lucifer-Yellow CH transfer method. Intracellular pH was measured by spectrofluorimetry and membrane expression of connexin 32 by indirect immunofluorescence. Results: Under our conditions, ethanol (20 mM) inhibited intercellular communication of hepatocytes to the same extent as did octanol at 1 mM. Immunofluorescence semi-quantitative studies of connexin 32 suggested that the observed inhibition was not related to a decrease in the number of gap junction plaques. In contrast with those of octanol, the inhibitory effects of ethanol appeared to be indirect because the inhibition of ethanol metabolism by 4-methyl pyrazole abolished its effects on intercellular communication, while 4-methyl pyrazole did not influence the effects of octanol. Acetaldehyde, the main metabolite of ethanol was without effect on gap junctions. Conclusions: This suggests that the inhibition of intercellular communication induced by ethanol may be included among the consequences of intermediary cell metabolism disturbances indirectly due to ethanol oxidation. This may be one of the mechanisms by which ethanol metabolism exerts a hepatotoxic and possibly carcinogenic action.