Neonatal hypothyroidism alters the localization of gap junctional protein connexin 43 in the testis and messenger RNA levels in the epididymis of the rat

The objectives of this study were to determine the effects of propylthiouracil (PTU)-induced neonatal hypothyroidism on the gap junctional protein Cx43 in rat testis and epididymis. PTU (0.02%) was administered via lactation from birth to Day 30, and the rats were sampled at 14, 18, 22, 26, 30, and 91 days of age. Testicular Cx43 was localized along the plasma membranes and cytoplasm of Sertoli cells until Day 22. At Day 30, the immunostaining was localized exclusively along the plasma membrane of Sertoli cells. In PTU-treated rats, Cx43 did not localize to the plasma membrane and was still cytoplasmic at 30 days of age. Occludin was present in tubules of treated rats, but was not localized to the blood-testis barrier in 30-day-old rats, as in controls. There were no differences in Cx43 immunostaining in the adult testis. In the proximal epididymis (initial segment, caput, corpus), Cx43 mRNA levels were lower in PTU-treated rats at 14, 18, and 22 days of age, but no differences were observed in the distal (cauda) epididymis at these ages. In 22- and 30-day-old rats, Cx43 was localized along the plasma membrane between principal and basal cells throughout the epididymis. In PTU-treated rats, Cx43 was not detectable in initial segment, caput, or corpus epididymidis. In the cauda epididymidis, however, Cx43 immunostaining in PTU-treated rats was similar to controls. These data suggest that thyroid hormones regulate Cx43-dependent gap junctional communication in the testis and epididymis.     

Histochemical demonstration of zinc in rat epididymis using a sulphide-silver method

We studied the histochemical distribution of zinc in rat epididymis using a sulphide-silver method. In the supranuclear cytoplasm of the principal cells that line the epididymis of rats, varying amounts of sulphide-silver-reactive zinc were visualized. In adult mating rats, significant amounts of zinc were found in the proximal portion of the epididymis, whereas in non-mating, mature and immature young rats, this heavy metal was most prominent in the distal portion of this organ. In all of the rats studied, zinc was sparsely distributed in the intermediate portion of the epididymis. From these results, it can be assumed that the zinc present in the epithelial lining of rat epididymis plays an important role in the maturation of spermatozoa. The present results represent a useful contribution to our understanding of the functional morphology of rat epididymis.     

Histochemical demonstration of zinc in rat epididymis using a sulphide-silver method

Summary We studied the histochemical distribution of zinc in rat epididymis using a sulphide-silver method. In the supranuclear cytoplasm of the principal cells that line the epididymis of rats, varying amounts of sulphide-silver-reactive zinc were visualized. In adult mating rats, significant amounts of zinc were found in the proximal portion of the epididymis, whereas in non-mating, mature and immature young rats, this heavy metal was most prominent in the distal portion of this organ. In all of the rats studied, zinc was sparsely distributed in the intermediate portion of the epididymis. From these results, it can be assumed that the zinc present in the epithelial lining of rat epididymis plays an important role in the maturation of spermatozoa. The present results represent a useful contribution to our understanding of the functional morphology of rat epididymis.Dedicated to Professor Dr. T.H.Schiebler on the occasion of his 65th birthday     

Identification of Cells Expressing Cx43, Cx30, Cx26, Cx32 and Cx36 in Gap Junctions of Rat Brain and Spinal Cord

We have identified cells expressing Cx26, Cx30, Cx32, Cx36 and Cx43 in gap junctions of rat central nervous system (CNS) using confocal light microscopic immunocytochemistry and freeze-fracture replica immunogold labeling (FRIL). Confocal microscopy was used to assess general distributions of connexins, whereas the 100-fold higher resolution of FRIL allowed co-localization of several different connexins within individual ultrastructurally-defined gap junction plaques in ultrastructurally and immunologically identified cell types. In >4000 labeled gap junctions found in >370 FRIL replicas of gray matter in adult rats, Cx26, Cx30 and Cx43 were found only in astrocyte gap junctions; Cx32 was only in oligodendrocytes, and Cx36 was only in neurons. Moreover, Cx26, Cx30 and Cx43 were co-localized in most astrocyte gap junctions. Oligodendrocytes shared intercellular gap junctions only with astrocytes, and these heterologous junctions had Cx32 on the oligodendrocyte side and Cx26, Cx30 and Cx43 on the astrocyte side. In 4 and 18 day postnatal rat spinal cord, neuronal gap junctions contained Cx36, whereas Cx26 was present in leptomenigeal gap junctions. Thus, in adult rat CNS, neurons and glia express different connexins, with “permissive” connexin pairing combinations apparently defining separate pathways for neuronal vs. glial gap junctional communication.     

Four novel members of the connexin family of gap junction proteins. Molecular cloning, expression, and chromosome mapping.

We have used low stringency hybridization and polymerase chain reaction (PCR) amplification with degenerate oligonucleotides to identify four new members of the rat connexin gene family. On the basis of their predicted molecular mass, these proteins have been designated connexin (Cx) 40 (Cx40), Cx37, Cx33, and Cx31.1. The new connexins exhibit all of the conserved structural features of the connexin family, including highly similar extracellular and transmembrane domains but divergent major cytoplasmic domains. On the basis of primary sequence similarity, the connexin family may be divided into two classes. Cx40, Cx37, and Cx33 are similar to the previously characterized Cx43 and Cx46. Cx31.1 is similar to Cx26, Cx31, and Cx32. Cx37 and Cx40 mRNAs are expressed in a wide variety of adult organs and tissues, with particular abundance in lung. However, their relative levels are different in many organs and thus their distribution is not completely coincident. Cx33 and Cx31.1 genes exhibit a much more restricted pattern of expression; mRNAs are detected only in testes and skin, respectively. Chromosomal mapping studies indicate that Cx26 and Cx46 are tightly linked on chromosome 14, and Cx37 and Cx31.1 are linked on chromosome 4, while the rest of the connexin genes are dispersed.     

Connexins Induce and Maintain Tight Junctions in Epithelial Cells

Connexins (Cx) are considered to play a crucial role in the differentiation of epithelial cells and to be associated with adherens and tight junctions. This review describes how connexins contribute to the induction and maintenance of tight junctions in epithelial cells, hepatic cells and airway epithelial cells. Endogenous Cx32 expression and mediated intercellular communication are associated with the expression of tight junction proteins of primary cultured rat hepatocytes. We introduced the human Cx32 gene into immortalized mouse hepatic cells derived from Cx32-deficient mice. Exogenous Cx32 expression and the mediated intercellular communication by transfection could induce the expression and function of tight junctions. Transfection also induced expression of MAGI-1, which localized at adherens and tight junction areas in a gap junctional intercellular communication (GJIC)–independent manner. Furthermore, expression of Cx32 was related to the formation of single epithelial cell polarity of the hepatic cells. On the other hand, Cx26 expression, but not mediated intercellular communication, contributed to the expression and function of tight junctions in human airway epithelial cells. We introduced the human Cx26 gene into the human airway epithelial cell line Calu-3 and used a model of tight junction disruption by the Na⁺/K⁺-ATPase inhibitor ouabain. Transfection with Cx26 prevented disruption of both tight junction functions, the fence and barrier, and the changes of tight junction proteins by treatment with ouabain in a GJIC–independent manner. These results suggest that connexins can induce and maintain tight junctions in both GJIC-dependent and –independent manners in epithelial cells.     

Altered permeability and modulatory character of connexin channels during mammary gland development

Abrupt developmental changes occur in structural form and function of connexin (Cx) channels in the mouse mammary gland. Microarray study shows that the principal connexin isoform in epithelial cells during pregnancy is Cx26, up-regulated and persisting from the virgin. After parturition, there is rapid induction of Cx32. In epithelial plasma membranes, size exclusion chromatography reveals that Cx32 organizes initially with Cx26 as heteromeric (Cx26-Cx32) hemichannels and later in heteromeric and homomeric Cx32 channels. Dramatic alterations of connexin channel function following these developmental changes in channel composition are characterized using native channels reconstituted into liposomes. Changes to channel stoichiometry increase the allowable physical size limits of permeant after parturition; the new Cx32 channels are wider than channels containing Cx26. Most remarkably, heteromeric Cx26-Cx32 channels are selectively permeability to adenosine 3',5' cyclic phosphate (cAMP), guanosine 3',5' cyclic phosphate (cGMP), and inositol 1,4,5-triphosphate (IP(3)), whereas homomeric channels are not. Homomeric Cx26 and heteromeric channels with high Cx26/Cx32 stoichiometry are also inhibited by taurine, an osmolyte playing a key role in milk protein synthesis. Taurine effect is reduced where heteromeric channels contain Cx32 > Cx26 and eliminated when channels contain only Cx32. Connexin channel stoichiometry, permeability, and chemical gating character change in precisely the desired fashion after parturition to maximize molecular and electrical coupling to support coordinated milk secretion.     

Identification of cells expressing Cx43, Cx30, Cx26, Cx32 and Cx36 in gap junctions of rat brain and spinal cord

We have identified cells expressing Cx26, Cx30, Cx32, Cx36 and Cx43 in gap junctions of rat central nervous system (CNS) using confocal light microscopic immunocytochemistry and freeze-fracture replica immunogold labeling (FRIL). Confocal microscopy was used to assess general distributions of connexins, whereas the 100-fold higher resolution of FRIL allowed co-localization of several different connexins within individual ultrastructurally-defined gap junction plaques in ultrastructurally and immunologically identified cell types. In >4000 labeled gap junctions found in >370 FRIL replicas of gray matter in adult rats, Cx26, Cx30 and Cx43 were found only in astrocyte gap junctions; Cx32 was only in oligodendrocytes, and Cx36 was only in neurons. Moreover, Cx26, Cx30 and Cx43 were co-localized in most astrocyte gap junctions. Oligodendrocytes shared intercellular gap junctions only with astrocytes, and these heterologous junctions had Cx32 on the oligodendrocyte side and Cx26, Cx30 and Cx43 on the astrocyte side. In 4 and 18 day postnatal rat spinal cord, neuronal gap junctions contained Cx36, whereas Cx26 was present in leptomenigeal gap junctions. Thus, in adult rat CNS, neurons and glia express different connexins, with "permissive" connexin pairing combinations apparently defining separate pathways for neuronal vs. glial gap junctional communication.     

Cx31.1 expression is modulated in HaCaT cells exposed to UV‐induced damage and scrape‐wounding

Connexin31.1 (Cx31.1) is a gap junction protein associated with apoptosis. In the skin, apoptosis is modulated by diabetes. A HaCaT skin model investigated whether normal (NGI) and high glucose and insulin (HGI; diabetic) conditions altered Cx31.1 expression, and if these were apoptosis linked. Cx31.1 was found in HaCaT and HeLa Ohio cells, with HaCaT Cx31.1 protein increased in HGI conditions, and around apoptotic cells. HeLa Cx31.1 channels were noncommunicative. Post scrape‐wounding, Cx31.1 increased at wound edges. Caspase 3/7 in scrape‐wounds media (containing cells) elevated in HGI. UV exposure raised Cx31.1, and caspase 3/7, in NGI and HGI. UV reduced cell viability in NGI cells, although not significantly in HGI. Cx31.1 is modulated during HaCaT cell wound closure, and associated with ‘diabetic’ conditions. Cx31.1 expression matched apoptosis levels, higher in HGI cultures. Cx31.1 is noncommunicating, modulated after wounding, linked to apoptosis, and may be associated with tissue turn‐over around diabetic wounds.     

Changes in Cellular Distribution of Connexins 32 and 26 during Formation of Gap Junctions in Primary Cultures of Rat Hepatocytes

In the adult rat hepatocyte, gap junction proteins consist of connexin 32 (Cx32) and connexin 26 (Cx26). Previously, we reported that both Cx32 and Cx26 were markedly induced and maintained in primary cultures of adult rat hepatocytes. The reappearing gap junctions were accompanied by increases in both the proteins and the mRNAs, and they were well maintained together with extensive gap junctional intercellular communication (GJIC) for more than 4 weeks. In the present study, we examined the cellular location of the gap junction proteins and the structures in the hepatocytes cultured in our system, using confocal laser microscopy and immunoelectron microscopy of cells processed for Cx32 and Cx26 immunocytochemistry and freeze-fracture analysis. In immunoelectron microscopy, the size of Cx32-immunoreactive gap junction structures on the plasma membrane increased with time of culture, and some of them were larger than those in liver sectionsin vivo.Freeze-fracture analysis also showed that the size of gap junction plaques increased and that the larger gap junction plaques were composed of densely packed particles. These results suggest that in this culture system, not only the synthesis of Cx proteins but also the size of the gap junction plaques was increased markedly. In the adluminal lateral membrane of the cells, Cx32-immunoreactive lines were observed and many small gap junction plaques were closely associated with a more developed tight junction network. In the basal region of the cells, small Cx32- and Cx26-immunoreactive dots were observed in the cytoplasm and several annular structures labeled with the antibody to Cx32 were observed in the cytoplasm. These results indicated the formation and degradation of gap junctions in the cultured hepatocytes.     

Functional expression of connexin30 and connexin31 in the polarized human airway epithelium

Gap junctions are documented in the human airway epithelium but the functional expression and molecular identity of their protein constituents (connexins, Cx) in the polarized epithelium is not known. To address this question, we documented the expression of a family of epithelial Cx (Cx26, Cx30, Cx30.3, Cx31, Cx31.1, Cx32, Cx37, Cx40, and Cx43) in primary human airway epithelial cells (AEC) grown on porous supports. Under submerged conditions, AEC formed a monolayer of airway cells whereas the air-liquid interface induced within 30-60 days AEC differentiation into a polarized epithelium for up to 6-9 months. Maturation of AEC was associated with the down-regulation of Cx26 and Cx43. The well-differentiated airway epithelium exhibited gap junctional communication between ciliated and between ciliated and basal cells. Interestingly, Cx30 was mostly present between ciliated cells whereas Cx31 was found between basal cells. These results are supportive of the establishment of signal-selective gap junctions with maturation of AEC, likely contributing to support airway epithelium function. These results lay the ground for studying the role of Cx-mediated cell-cell communication during repair following AEC injury and exploring Cx-targeted interventions to modulate the healing process.     

Connexins 26, 32 and 43 are expressed in virgin, pregnant and lactating mammary glands

Gap junctions (GJ) are formed by a number of homologous proteins termed connexins. Here expression of connexins Cx26, Cx32 and Cx43, was evaluated by immunofluorescence (IF) in mammary glands from virgin, pregnant and lactating rats. Cx26, Cx32 and Cx43 labeling was detected in epithelial parenchymal cells at all functional stages. Cx26 and Cx32 labeling was very low in glands from virgin animals, somewhat greater in glands from pregnant animals and significantly higher (in number and size) in lactating animals. In the last ones, Cx26 and Cx32 punctate labeling was localized to the basal and lateral membranes of alveolar epithelial cells and collecting ductules. Cx43 punctate labeling was restricted to the periphery of alveoli towards the basal pole of epithelial cells at all functional stages, and it enlarged slightly during lactation. At this localization, Cx43 may form GJ between myoepithelial cells and/or between epithelial and myoepithelial cells. Cx43 was also found to be steadily expressed in the connective tissue which surrounds and invades each parenchymal lobe, at all functional stages. At this localization, Cx43 may couple fibroblasts and/or adipose cells. IF studies in sections from lactating mice showed the same distribution of connexins. Immunoblots confirmed specificity of labeling and the presence of Cx32 and Cx43 in the mammary gland. The increase in connexin expression detected during pregnancy and lactation may be important for epithelial cell differentiation and secretion in the mammary gland.     

Expression of aquaporin 9 in the adult rat epididymal epithelium is modulated by androgens

Reabsorption of fluid and solutes across the epithelium lining the male excurrent duct is important for adequate sperm maturation, concentration, and storage. Water channels contribute to water movement across epithelia in many tissues. Aquaporin 9 (AQP9) is abundantly expressed in the apical membrane of principal cells that line the epididymis, and in reabsorptive and secretory epithelial cells of the male reproductive tract. In this study we show that the nonsteroidal antiandrogen flutamide, given to adult rats at a dose of 50 mg x kg(-1) x day(-1) for 2 wk via osmotic minipumps significantly decreased the amount of AQP9 in the epididymis. This down-regulation was observed by immunofluorescence of cryostat tissue sections and by Western blotting of epididymal brush border membrane preparations. In addition, castrated adult rats showed lower levels of epididymal AQP9 compared with adult controls, whereas systemic testosterone treatment of castrated adult rats induced a recovery of the expression of AQP9 to control levels. These data indicate that the expression of AQP9, a likely candidate for apical transepithelial fluid and solute transport in several regions of the male reproductive tract, is modulated by androgens in the adult rat epididymis.     

Chloral hydrate decreases gap junction communication in rat liver epithelial cells

Gap junction communication (GJC) is involved in controlling cell proliferation and differentiation. Alterations in GJC are associated with carcinogenesis, but the mechanisms involved are unknown. Chloral hydrate (CH), a by-product of chlorine disinfection of water, is carcinogenic in mice, and we demonstrated that CH reduced GJC in a rat liver epithelial cell line (Clone 9). To examine the mechanism(s) by which CH inhibits GJC, Clone 9 cells treated with CH were examined using Western blot, real-time polymerase chain reaction, immunocytochemical, and dye-communication techniques. Treatment with CH (0.1–5 mM for 24 h) resulted in a dose-dependent inhibition of GJC as measured by Lucifer yellow dye transfer. Western blot analysis demonstrated expression of connexin (Cx) 43 and 26 in control cells and reduced expression of Cx 43 but not Cx 26 protein from 0.1 to 1 mM CH. CH treatment from 2.5 to 5 mM caused an apparent increase in expression of both connexins that was concomitant with a reduction in mRNA expression for both connexins. Similarly, with immunocytochemistry, a dose-dependent decrease in Cx 43 staining at sites of cell–cell contact was apparent in CH (0.5–5 mM)-treated cultures, whereas no Cx 26 staining was observed. Thus, Clone 9 cells contain two types of connexins but only one type of plasma membrane channel. Understanding of the regulation of connexin may shed light on mechanisms responsible for inhibition of GJC by chemical carcinogens.     

Cx31.1 acts as a tumour suppressor in non-small cell lung cancer (NSCLC) cell lines through inhibition of cell proliferation and metastasis

Reduced connexin expression and loss of gap junction function is a characteristic of many cancers, including lung cancer. However, there are little reports about the relation between Cx31.1 and lung cancer. This study was conducted to investigate the effect of Cx31.1 on non-small cell lung cancer (NSCLC). We found that the Cx31.1 was down-regulated in NSCLC cell lines, and the expression levels were reversely related with their metastatic potential. We ectopically expressed Cx31.1 in H1299 NSCLC cell line to examine the influence of Cx31.1 overexpression. The results showed that overexpression of Cx31.1 in H1299 cells reduced cell proliferation, induced a delay in the G(1) phase, inhibited anchorage-independent growth and suppressed cell migration and invasion. The cell cycle delay and cell migration and invasion suppressive effects of Cx31.1 were partially reversed by siRNA targeting mRNA of Cx31.1. Moreover, xenografts of Cx31.1 overexpressing H1299 cells showed reduced tumourigenicity. These results suggested that Cx31.1 has tumour-suppressive properties. Further investigation indicated that cyclin D3 may be responsible for Cx31.1-induced G(1) phase delay. Importantly, Cx31.1 increased the expression of epithelial markers, such as cytokeratin 18, and decreased expression of mesenchymal markers, such as vimentin, indicating a Cx31.1-mediated partial shift from a mesenchymal towards an epithelial phenotype. We concluded that Cx31.1 inhibit the malignant properties of NSCLC cell lines, the mechanisms under this may include regulation of EMT.     

Connexin43 Is Another Gap Junction Protein in the Peripheral Nervous System

Abstract: That many cells express more than one connexin (Cx) led us to examine whether Cxs other than Cx32 are expressed in the PNS. In addition to Cx32 mRNA, Cx43 and Cx26 mRNAs were detected in rat sciatic nerve by northern blot analysis. Cx43 mRNA, but not Cx26 mRNA, was expressed in both the primary Schwann cell culture and immortalized Schwann cell line (T93). The steady-state levels of the Cx43 mRNA in the primary Schwann cell culture increased 2.0-fold with 100 µ M forskolin, whereas that of P₀ increased 7.0-fold. Immunoreactivity to Cx43 was detected on western blots of cultured Schwann cells, T93 cells, and sciatic nerves but not on blots of PNS myelin. Immunohistochemical study using human peripheral nerves revealed that anti-Cx43 antibody stained cytoplasm around nucleus of Schwann cells but not myelin, confirming western blot results. Although P₀ expression was markedly decreased by crush injury of the sciatic nerves, Cx43 expression showed no apparent change. Developmental profiles showed that Cx43 expression in the sciatic nerve increased rapidly after birth, peaked at about postnatal day 6, and then decreased gradually to a low level. In adult rats, the Cx43 mRNA value was much lower than that of Cx32. These findings suggest that Cx43 is localized in Schwann cell bodies and that, compared with P₀, its expression is less influenced by axonal contact and cyclic AMP levels. The high expression on postnatal day 6 indicates that Cx43 may be related to PNS myelination. Cx43 is another gap junction, but its function appears to differ from that of Cx32, as judged by the differences in their localization and developmental profiles.     

Gap-junctional communication between feeder cells and recipient normal epithelial cells correlates with growth stimulation

LA7 rat mammary tumor cells stimulate the proliferation, in culture, of three normal epithelial cell types, namely mouse mammary, rat mammary, and mouse thymic cells. Gap-junctional communication between LA7 feeders and mouse mammary cells was demonstrated by microinjection of lucifer yellow, which traveled from LA7 to the surrounding mouse mammary cells. The amount of 3H-uridine exchange between feeder and recipient mouse mammary, rat mammary, and mouse thymus cells correlated with the growth rate induced by the feeders. Cells of the Madin Darby canine kidney (MDCK) line, which do not appreciably stimulate mouse mammary cell growth when used as feeder cells, also exchange little 3H-uridine with them. Expression of connexins Cx43, 32, and 26 was studied in all these cell lines and strains by immunocytochemistry. Mouse mammary cells expressed Cx26, and a few mouse thymic cells expressed Cx32. LA7, mouse mammary, mouse thymic, and rat mammary cells all expressed easily detectable amounts of the gap-junction protein Cx43, in contrast to MDCK cells, which expressed only a hint of the protein. These results suggest that gap junctions composed of Cx43 are those by which the normal epithelial cells communicate with the LA feeders. Thus, the ability of feeder cells to stimulate proliferation in recipients correlates with the expression of Cx43 in both members of the feeder/recipient pair and the capacity to form functional gap junctions between these cells.     

Morphological and functional alterations in adult boar epididymis: Effects of prenatal and postnatal administration of flutamide

Background  

The dynamic cross-talk between epididymal cells is hormonally regulated and, in part, through direct cell-to-cell interactions. To date, no information is available regarding possible impact of anti-androgens on the proteins involved in the gap junctional communication within the boar epididymis. Thus, a question arised whether prenatal or postnatal exposure to an anti-androgen flutamide alters the expression of gap junction protein - connexin43 (Cx43) and androgen receptor (AR) expression in the caput, corpus and cauda epididymis and leads to delayed effects on morphology and function of adult pig epididymis.