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 sections in 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.     

Absorption and urinary excretion of the coffee diterpenes cafestol and kahweol in healthy ileostomy volunteers

Epidermal growth factor (EGF) has been found to induce enhanced gap junctional intercellular communication (GJIC) in the human kidney epithelial cell line K7. This is in contrast to what is reported for other cell types, which all show decreased GJIC in response to EGF. In the present study it is shown that 12-O-tetradecanoylphorbol-13-acetate (TPA) and EGF induce similar phosphorylation pattern of the gap junction protein connexin43 (Cx43) in K7 cells, although their effects on GJIC are opposite. Tyrosine phosphorylation of a 42 kD protein was observed to be induced concomitantly with phosphorylation of Cx43. EGF was however found to induce only serine phosphorylation of Cx43, indicating that the tyrosine kinase activity of the EGF receptor was not directly affecting the gap junction protein. The 42 kD protein phosphorylated on tyrosine was identified to be a mitogen activated protein (MAP) kinase. Both EGF and TPA was found to activate MAP kinase in these cells. Phosphorylation of Cx43 and enhancement of GJIC in response to EGF occurred with difference in time course. Phosphorylation of Cx43 was completed within 15 min, while the enhanced GJIC appeared 2-3 h later. It is therefore possible that regulation of synthesis or transport of Cx43 is responsible for the increase in GJIC, rather than direct involvement of Cx43 phosphorylation. This is in support of our previous finding that protein synthesis is necessary for EGF induced upregulation of GJIC in K7 cells.     

Inhibition of gap junctional intercellular communication by barbiturates in long-term primary cultured rat hepatocytes is correlated with liver tumour promoting activity

Rodent liver tumor formation can be promoted by certain barbiturates and this may involve their ability to inhibit hepatocyte gap junctional intercellular communication (GJIC). In order to address the mechanisms and specificity of action of barbiturates on hepatocyte gap junctions, we have compared the effects of liver tumor-promoting barbiturates (phenobarbital, sodium barbital and amobarbital: PB, SB and AB, respectively) and a non-liver tumor-promoting barbiturate (barbituric acid: BA) on primary cultured rat hepatocyte GJIC and connexin32 (Cx32) expression after short (1-24 h) and long (2-14 days) treatment. GJIC was evaluated by fluorescent dye microinjection (dye-coupling); Cx32 expression was monitored by Northern blot, Western blot and immunohistochemistry. Both parameters were maintained at high levels over 14 days by coculture of the cells with WB-F344 rat liver epithelial cells in the presence of dexamethasone. Treatment with PB (2 mM) for 1 h sharply reduced dye-coupling from approximately 90-30%, but the cells fully recovered by 24 h. No inhibition was seen with the other barbiturates over this 1-day treatment period. Longer treatments (2-14 days) with the promoters PB, SB and AB, however, gradually reduced hepatocyte dye-coupling to approximately 30-50%. The non-promoter, BA, did not affect hepatocyte GJIC. These decreases in hepatocyte dye-coupling occurred without changes in Cx32 or gap junction expression. Dye-coupling of WB-F344 cells and expression of their predominant gap junction protein, connexin43 (Cx43), were also not affected. Thus, the inhibition of GJIC was specific to liver tumor promoting barbiturates in hepatocytes, was time-dependent and was not due to altered Cx32 expression.     

Role of gap junctions in lung neoplasia

Reduced gap junctional intercellular communication (GJIC) has been noted in many types of neoplastic cells and may contribute to the neoplastic phenotype. This study assessed GJIC (by fluorescent dye-coupling) and gap junction protein (connexin) expression in mouse and human lung carcinoma cell lines and investigated whether reduced GJIC was involved in their neoplastic phenotype. Dye-coupling and connexin43 (Cx43) expression were much lower in most of the carcinoma lines (16 of 22) compared to nontransformed lung epithelial cells. Other connexins were not detected. A poorly communicating mouse lung carcinoma cell line (E9) was transfected with Cx43 or transduced with Cx32 and several stable clones were isolated that had 2- to 4-fold increased dye coupling. When evaluated for growth in vitro, the population doubling times were increased and the saturation densities were decreased in the clones. When assessed for tumorigenicity, the parental E9 cells formed tumors with a 100% incidence (6/6 mice), whereas the clones varied in tumorigenic response (0-88% incidence). The best communicating clone (E9-2) was not tumorigenic. The highly communicating Cx32 clone, E9/32-9, gave a tumor incidence of 88%. These results suggest that restoration of GJIC by forced connexin expression can reduce the growth and tumorigenicity of lung carcinoma cells in a connexin-specific manner.     

Perturbation in connexin 43 and connexin 26 gap-junction expression in mouse skin hyperplasia and neoplasia

To examine the possible role of gap junctions in mouse skin tumor progression, we generated a panel of mouse skin tissue samples exhibiting normal, hyperplastic, or neoplastic changes and characterized the expression of the gap-junction genes connexin 43 (Cx43) and connexin 26 (Cx26) by in situ hybridization and immunohistochemical analyses. In normal skin, these two gap junction genes were differentially expressed; Cx43 was found predominantly in the less differentiated lower spinous layers, whereas Cx26 was found in terminally differentiating upper spinous and granular layers. In hyperplastic epidermis exhibiting an expansion of the differentiated upper layer, i.e., epidermis with a thickened granular layer or in which the granular layer was replaced with keratinocytes exhibiting tricholemmal differentiation, expression of Cx43 and Cx26 remained segregated in the lower and upper spinous layers, respectively. However, in papillomas, Cx26 was localized in the lower but not upper spinous layer, an expression pattern identical to that of Cx43. In addition, the overall expression levels of both Cx43 and Cx26 appeared to be greatly elevated in the papillomas. It is interesting that such marked alteration in the pattern of Cx26 expression occurred within the context of hyperplastic changes histologically identical to those seen in the nonpapillomous hyperplasias. Interestingly, in neoplastic skin lesions containing a squamous cell carcinoma, Cx43 and Cx26 expression was extinguished. Moreover, expression of Cx43 was also significantly reduced in adjacent apparently nonneoplastic tissues. Overall, these observations show that perturbations in gap-junction gene expression are associated with skin hyperplasia and neoplasia. Such findings suggest a possible role for gap junctions in the malignant conversion of mouse epidermal cells.     

Connexin 32 gap junctions enhance stimulation of glucose output by glucagon and noradrenaline in mouse liver

Gap junctions connect neighboring cells via intercellular channels composed of connexins (Cx). Connexin 32 (Cx32) is the main connexin in hepatocytes. Gap junctions propagate a signal from periportal to perivenous hepatocytes generated by electrical stimulation of sympathetic liver nerves. Therefore, it was the aim of this study to examine the involvement of hepatocellular gap junctions in hormonal regulation. In perfused livers from wild-type mice and Cx32-deficient mice, the stimulation of glucose release by varying noradrenaline and glucagon concentrations was investigated. At saturating hormone concentrations, glucose release was the same in wild-type and Cx32-deficient livers. However, glucose output was significantly smaller in Cx32-deficient than wild-type livers at half-maximally effective hormone concentrations. Because the two hormones circulate at less than half-saturating concentrations and because they are degraded during passage of blood through the liver, they lose efficiency from the periportal to the perivenous zone. In wild-type livers, this decrease in efficiency can be partially compensated by intercellular signal propagation through gap junctions, resulting in higher hormone actions than in Cx32-deficient livers. It is concluded that gap junctions are not only involved in intercellular propagation of nervous, but also of hormonal signals from periportal to perivenous hepatocytes.     

Suppression of tumorigenicity of human lung carcinoma cells after transfection with connexin43

The human lung carcinoma cell line PG is defective in gap junctional intercellular communication (GJIC). Connexin43 (Cx43) mRNA, which is expressed in normal human lung cells, is undetectable in these tumor cells. To explore if up-regulation of Cx43 gene expression will suppress malignancy of PG cells, Cx43 cDNA was co-transfected with pSV2neo cDNA into PG cells. Control cells were transfected with the blank vector plus neo cDNA. Several stable Cx43 transfectant clones, which acquired high levels of Cx43 expression and the capacity of GJIC, were compared with control clones and the parental cell line, both of which lacked Cx43 expression and GJIC. The control clones resembled the parental cells in exhibiting high cell growth rate, weak attachment to the substratum and a high frequency of colony formation in soft agar. In contrast to the control cells, Cx43 transfected clones showed reduced growth rate, enhanced attachment to the substratum and inhibition of colony formation in soft agar. In vivo results from nude mice experiments showed high tumorigenicity with control clones and inhibition of tumorigenicity in Cx43 transfected clones. The consistency between in vitro and in vivo results strongly suggests a tumor suppressing effect of the Cx43 gene in human lung carcinoma cells.     

Gap junctional proteins, connexin 26, 32, and 43 in sheep ovaries throughout the estrous cycle

Ovarian follicles from days 13, 14, 15, and 16 and corpora lutea (CL) from days 2, 4, 8, 12, and 15 of the estrous cycle were evaluated for the presence of connexins by immunohistochemistry. In addition, CL from days 5, 10, and 15 of the estrous cycle were used for immunofluorescent detection of Cx43 followed by image analysis, and for Western immunoblot. In all tissues, staining for all connexins appeared punctate, indicating the presence of assembled gap junctions. Cx26 was present in the ovarian surface epithelium, stroma, and blood vessels within the stroma and hilus, and in the CL. In healthy antral follicles, Cx26 was present only in the theca layer, whereas Cx43 was present in granulosa and theca layers. In the majority of atretic follicles, connexins were not detected, but in 13% of the atretic follicles, Cx43 was present in the theca layer. Cx32 was detected in the blood vessels of ovarian stroma and in the CL, and Cx43 was detected in the CL. Localization and/or expression of connexins depended on stage of luteal development. Western analysis demonstrated that expression of Cx32 in luteal tissues was similar across the estrous cycle. The area of positive staining for Cx43 and expression of Cx43 in luteal tissues decreased (p < 0.05) as the estrous cycle progressed. The pattern of expression of connexins indicates that gap junctional proteins may be important in the regulation of folliculogenesis and follicular atresia, as well as growth, differentiation, and regression of the CL.     

Rapid turnover of connexin43 in the adult rat heart

Remodeling of the distribution of gap junctions is an important feature of anatomic substrates of arrhythmias in patients with healed myocardial infarcts. Mechanisms underlying this process are poorly understood but probably involve changes in gap junction protein (connexin) synthesis, assembly into channels, and degradation. The half-life of the principal cardiac gap junction protein, connexin43 (Cx43), is only 1.5 to 2 hours in primary cultures of neonatal myocytes, but it is unknown whether rapid turnover of Cx43 occurs in the adult heart or is unique to disaggregated neonatal myocytes that are actively reestablishing connections in vitro. To characterize connexin turnover dynamics in the adult heart and to elucidate its potential role in remodeling of gap junctions, we measured Cx43 turnover kinetics and characterized the proteolytic pathways involved in Cx43 degradation in isolated perfused adult rat hearts. Hearts were labeled for 40 minutes with Krebs-Henseleit buffer containing [35S]methionine, and then chase perfusions were performed with nonradioactive buffer for 0, 60, 120, and 240 minutes. Quantitative immunoprecipitation assays of Cx43 radioactivity in 4 hearts at each time point yielded a monoexponential decay curve indicating a Cx43 half-life of 1.3 hours. Proteolytic pathways responsible for Cx43 degradation were elucidated by perfusing isolated rat hearts for 4 hours with specific inhibitors of either lysosomal or proteasomal proteolysis. Immunoblot analysis demonstrated significant increases ( approximately 30%) in Cx43 content in hearts perfused with either lysosomal or proteasomal pathway inhibitors. Most of the Cx43 in hearts perfused with lysosomal inhibitors consisted of phosphorylated isoforms, whereas nonphosphorylated Cx43 accumulated selectively in hearts perfused with a specific proteasomal inhibitor. These results indicate that Cx43 turns over rapidly in the adult heart and is degraded by multiple proteolytic pathways. Regulation of Cx43 degradation could play an important role in gap junction remodeling in response to cardiac injury.     

Structural changes in lenses of mice lacking the gap junction protein connexin43

PURPOSE: To investigate the role of the gap junction protein connexin43 (Cx43), which is predominantly expressed in lens epithelial cells in the control of lens development and organization. METHODS: Newborn mice in which the Cx43 gene was disrupted by homologous recombination were used. Lenses from Cx43 (-/-) mice and wild-type littermates were processed by using 2% glutaraldehyde fixation for light and transmission electron microscopy and by freezing in liquid nitrogen for light and confocal microscopy of immunofluorescence in cryosections. RESULTS: In wild-type mice, Cx43 was immunolocalized to apical and lateral regions of lens epithelial cells and throughout the cornea, iris, ciliary body, and retina. In the bow, or equatorial, region of the lens, Cx43 disappeared gradually at the margins of the epithelial layer, whereas major intrinsic polypeptide, MP26, and alpha-crystallins were only detected in differentiated fiber cells. Ultrastructural studies revealed that epithelial cells and epithelial fiber cells were connected by large gap junctions. Lens fiber cells were closely apposed to apical boundaries of epithelial cells and apposed to one another along their entire lengths. In Cx43 (-/-) mice, epithelial cells were connected more loosely. The distribution of MP26 and alpha-crystallin in bow region fiber cells in Cx43 (-/-) lenses was not distinguishable from that in the lenses of wild-type mice. Cx46 and Cx50 were also expressed in superficial and cortical fiber cells, with similar distributions in Cx43 (-/-) and wild-type mice. However, organization of appositional membranes between lens fiber cells and between fiber and epithelial cells differed dramatically in the Cx43 (-/-) lens. In contrast to the close apposition of cells in lenses of normal mice, fiber cells in Cx43 (-/-) lenses were largely separated from apical surfaces of epithelial cells, and large vacuolar spaces were apparent between fiber cells, most prominently in deeper cortical regions. CONCLUSIONS: The normal differentiation of lens fiber cells in the bow region in lenses of Cx43 (-/-) mice, evidenced by similar distributions of Cx46, Cx50, MP26, and alpha-crystallin, suggests that the expression of Cx43 is not required for this process. However, these lenses exhibit grossly dilated extracellular spaces and intracellular vacuoles, indicative of early stages of cataract formation. These changes suggest that osmotic balance within the lens is markedly altered in Cx43 (-/-) animals, highlighting the importance of intercellular communication mediated by lens epithelial Cx43 gap junctions in the function of this tissue.     

Use of alternate promoters for tissue-specific expression of the gene coding for connexin32

The promoter of rat connexin32 (Cx32), the gap junction protein found in liver, was studied in transgenic mice. Cx32 transgenes, containing 2.5-kb of sequence upstream from the promoter, exon I, the entire 6.1-kb intron and the beginning of the coding sequence linked to the gene encoding luciferase (Luc), were found to be expressed in mouse in the same tissue-specific manner as previously reported for Cx32. Another construct lacking the promoter, but retaining 1.8 kb from the 3' end of the intron, was found to be expressed specifically in the nervous system. This result suggested that a second promoter, different from that used in liver, functions in nervous tissue. The use of this promoter in normal rats was corroborated by sequence analysis of reverse-transcribed PCR products obtained from rat nervous tissue RNA. The second promoter drives the synthesis of a second Cx32 mRNA species that is processed to remove a small 345-bp intron that shares its acceptor splice site with the large intron. This finding could have implications for the genetic basis of the X-linked form of Charcot-Marie-Tooth disease (CMT-X) in those patients that do not exhibit mutations in the Cx32-coding region.     

Effects of angiotensin II on expression of the gap junction channel protein connexin43 in neonatal rat ventricular myocytes

OBJECTIVES: To elucidate signal transduction pathways regulating expression of myocardial gap junction channel proteins (connexins) and to determine whether mediators of cardiac hypertrophy might promote remodeling of gap junctions, we characterized the effects of angiotensin II on expression of the major cardiac gap junction protein connexin43 (Cx43) in cultured neonatal rat ventricular myocytes. BACKGROUND: Remodeling of the distribution of myocardial gap junctions appears to be an important feature of anatomic substrates of ventricular arrhythmias in patients with heart disease. Remodeling of intercellular connections may be initiated by changes in connexin expression caused by chemical mediators of the hypertrophic response. METHODS: Cultures were exposed to 0.1 micromol/liter angiotensin II for 6 or 24 h, and Cx43 expression was characterized by immunoblotting, confocal microscopy and electron microscopy. RESULTS: Immunoblot analysis revealed a twofold increase in Cx43 content in cells treated for 24 h with angiotensin II (n=4, p < 0.05). This response was inhibited by the presence of 1.0 micromol/liter losartan, an AT1-receptor blocker. Confocal and electron microscopy demonstrated enhanced Cx43 immunoreactivity and increases in the number and size of gap junction profiles in cells exposed to angiotensin II for 24 h. These effects were also blocked by losartan. Immunoprecipitation of Cx43 from cells metabolically labeled with [35S]methionine demonstrated 2.4- and 2.9-fold increases in Cx43 radioactivity after 6 and 24 h exposure to angiotensin II, respectively (p < 0.03 at each time point). CONCLUSIONS: Angiotensin II up-regulates gap junctions in cultured neonatal rat ventricular myocytes by increasing Cx43 synthesis. Signal transduction pathways activated by angiotensin II under pathophysiologic conditions could initiate remodeling of conduction pathways, leading to the development of anatomic substrates of arrhythmias.     

Gap junction turnover, intracellular trafficking, and phosphorylation of connexin43 in brefeldin A-treated rat mammary tumor cells

Intercellular gap junction channels are thought to form when oligomers of connexins from one cell (connexons) register and pair with connexons from a neighboring cell en route to forming tightly packed arrays (plaques). In the current study we used the rat mammary BICR-M1Rk tumor cell line to examine the trafficking, maturation, and kinetics of connexin43 (Cx43). Cx43 was conclusively shown to reside in the Golgi apparatus in addition to sites of cell-cell apposition in these cells and in normal rat kidney cells. Brefeldin A (BFA) blocked Cx43 trafficking to the surface of the mammary cells and also prevented phosphorylation of the 42-kD form of Cx43 to 44- and 46-kD species. However, phosphorylation of Cx43 occurred in the presence of BFA while it was still a resident of the ER or Golgi apparatus yielding a 43-kD form of Cx43. Moreover, the 42- and 43-kD forms of Cx43 trapped in the ER/Golgi compartment were available for gap junction assembly upon the removal of BFA. Mammary cells treated with BFA for 6 h lost preexisting gap junction "plaques," as well as the 44- and 46-kD forms of Cx43 and functional coupling. These events were reversible 1 h after the removal of BFA and not dependent on protein synthesis. In summary, we provide strong evidence that in BICR-M1Rk tumor cells: (a) Cx43 is transiently phosphorylated in the ER/Golgi apparatus, (b) Cx43 trapped in the ER/Golgi compartment is not subject to rapid degradation and is available for the assembly of new gap junction channels upon the removal of BFA, (c) the rapid turnover of gap junction plaques is correlated with the loss of the 44- and 46-kD forms of Cx43.     

Ribavirin inhibits protein synthesis and cell proliferation induced by mitogenic factors in primary human and rat hepatocytes

Ribavirin, a guanosine analog, used in combination with interferon alpha (IFN-alpha) in the treatment of chronic hepatitis induced by hepatitis C virus (HCV) infection, has been shown to improve liver histology and to decrease transaminases even when administered alone. We analyzed the direct effects of ribavirin on the liver by using primary cultures of human and rat hepatocytes. Between 10 to 60 micromol/L, ribavirin was found to inhibit both the synthesis and secretion of whole proteins in a time- and dose-dependent fashion. Such an effect was confirmed by the measurement of albumin and haptoglobin secretion rates. [3H]-Thymidine incorporation was suppressed both in hepatocyte growth factor-stimulated human hepatocytes and in epidermal growth factor (EGF)-stimulated rat hepatocytes in the presence of ribavirin. The inhibitory effect on DNA synthesis was associated with a delayed progression to S phase of the cell cycle, as determined by flow cytometry and detection of cyclin A and cdc2 which are two proteins expressed during the S phase. The inhibition of DNA synthesis, caused by 50 micromol/L ribavirin, was completely restored by the addition of 80 micromol/L guanosine. These observations demonstrate that ribavirin at concentrations close to those found in plasma of treated patients can directly affect hepatic functions in vitro. Its effects could, however, be reduced in vivo by guanosine salvage supply.     

Gap junctions and growth control in liver regeneration and in isolated rat hepatocytes

The hepatocytes in the mature normal liver are tightly coupled through gap junctions, except during compensatory hyperplasia (regeneration) after partial hepatectomy when the gap junctions become down-regulated. The significance of this down-regulation has been a long-standing enigma. The present study of hepatocytes in primary culture and in the regenerating liver aimed at defining the relationship, if any, between hepatocyte gap junctional communication and proliferation. Gap junctional down-regulation in the regenerating liver appeared to be a specific phenomenon because desmosomes and the surface contact area between neighboring hepatocytes remained constant. All agents and conditions (dexamethasone in vivo; dexamethasone, cyclic adenosine monophosphate, serum, and high cell density in vitro) delaying gap junctional down-regulation also increased the lag before the cells reached competence to enter S phase. This raised the possibility that hepatocyte DNA replication was inhibited through preservation of gap junctions. However, we disproved this assumption by showing that the DNA replication (more specifically the G1/S transition rate constant) was inhibited even in hepatocytes completely devoid of gap junctional communication. The teleological advantage of linking gap junctional down-regulation to hepatocyte G1 progression therefore may not be to trigger DNA replication but to ensure that proliferating hepatocytes and hepatocytes responsible for liver-specific metabolic functions maintain separate pools of metabolites and signaling molecules.     

A characterization of permolybdate and its effect on cellular tyrosine phosphorylation, gap junctional intercellular communication and phosphorylation status of the gap junction protein, connexin43

Biological and analytical characterizations of permolybdate (a mixture of H2O2 and molybdate) were done. Molybdate (10 mM) and molybdenum(V) chloride (3 mM) did not affect gap junctional intercellular communication (GJIC), phosphorylation status of connexin43 (Cx43) or cellular tyrosine phosphorylation in early passage hamster embryonic cells (mainly fibroblast-like). High concentrations of H2O2 (3-10 mM) affected some of the parameters. Acidified permolybdate was clearly more stable than the unadjusted permolybdate. The maximum biological potency of acidified permolybdate was found at a molar ratio of 2:1 (H2O2:molybdate). The mixtures of molybdenum(V) chloride and H2O2 gave a maximum effect at 4:1 molar ratio (H2O2:molybdenum(V)). This can be explained by decomposition of H2O2 and by the generation of less biologically active compounds. Spectrophotometric analyses of the mixtures corroborated the biological results. The Mo(V) electron spin resonance spectrum disappeared upon addition of H2O2 to Mo(V) solutions, and no spectrum appeared when H2O2 was mixed with Mo(VI). Thus, permolybdate is probably diperoxomolybdate, a Mo(VI) compound. Regardless of the parent metal salt, the H2O2/metal salt mixtures showed concentration-dependent biphasic responses with an initial decrease in GJIC followed by an increase. A dissociation between alteration in Cx43 phosphorylation status and GJIC was obtained under certain conditions. The biological activities of permolybdate were only partially mimicked by phenylarsine oxide, an alternative protein tyrosine phosphatase inhibitor.     

Assembly of hepatic gap junctions. Topography and distribution of connexin 32 in intracellular and plasma membranes determined using sequence-specific antibodies

The subcellular distribution in rat liver and the topography in intracellular and plasma membranes of connexin 32, a major protein component of gap junctions, was studied using sequence-specific anti-peptide antibodies generated to extracellular and intracellular domains of the protein. The distribution of connexin 32 in liver analyzed using SDS-polyacrylamide gel electrophoresis and Western blotting showed the relative protein levels in the subcellular fractions to be: lateral plasma membranes > Golgi membranes > sinusoidal plasma membranes > lysosomes. Low amounts of connexin 32 were detected in microsomes, endosomes, and bile canalicular plasma membranes. Six highly conserved cysteine residues are located in the amino acid sequences comprising the two extracellular loops of all connexins thus far isolated, and these loops are positioned to extend the channel in the lipid bilayers across the intercellular region of the gap junction. In the present work, the intramolecular disulfide bonds linking the extracellular loops in gap junctions were shown to be present in connexins located in plasma membranes, Golgi, and a microsomal fraction, and it was concluded that the disulfide linkages were formed in the endoplasmic reticulum. In addition, immature configurations of connexin 32, probably occurring during membrane insertion, were detected in liver microsomal fractions. The results contribute to charting of the biogenetic routes followed by connexins in hepatocytes and the general mechanisms of gap junction assembly.