Comparative analysis of the major polypeptides from liver gap junctions and lens fiber junctions.

Gap junctions from rat liver and fiber junctions from bovine lens have similar septilaminar profiles when examined by thin-section electron microscopy and differ only slightly with respect to the packing of intramembrane particles in freeze-fracture images. These similarities have often led to lens fiber junctions being referred to as gap junctions. Junctions from both sources were isolated as enriched subcellular fractions and their major polypeptide components compared biochemically and immunochemically. The major liver gap junction polypeptide has an apparent molecular weight of 27,000, while a 25,000-dalton polypeptide is the major component of lens fiber junctions. The two polypeptides are not homologous when compared by partial peptide mapping in SDS. In addition, there is not detectable antigenic similarity between the two polypeptides by immunochemical criteria using antibodies to the 25,000-dalton lens fiber junction polypeptide. Thus, in spite of the ultrastructural similarities, the gap junction and the lens fiber junction are comprised of distinctly different polypeptides, suggesting that the lens fiber junction contains a unique gene product and potentially different physiological properties.

Proteoglycans and glycosaminoglycans induce gap junction synthesis and function in primary liver cultures.

Intercellular communication via gap junctions, as measured by dye and electrical coupling, disappears within 12 h in primary rat hepatocytes cultured in serum-supplemented media or within 24 h in cells in a serum-free, hormonally defined medium (HDM) designed for hepatocytes. Glucagon and linoleic acid/BSA were the primary factors in the HDM responsible for the extended life span of the electrical coupling. After 24 h of culture, no hormone or growth factor tested could restore the expression of gap junctions. After 4-5 d of culture, the incidence of coupling was undetectable in a serum-supplemented medium and was only 4-5% in HDM alone. However, treatment with glycosaminoglycans or proteoglycans of 24-h cultures, having no detectable gap junction protein, resulted in synthesis of gap junction protein and of reexpression of electrical and dye coupling within 48 h. Most glycosaminoglycans were inactive (heparan sulfates, chondroitin-6 sulfates) or only weakly active (dermatan sulfates, chondroitin 4-sulfates, hyaluronates), the weakly active group increasing the incidence of coupling to 10-30% with the addition of 50-100 micrograms/ml of the factor. Treatment of the cells with 50-100 micrograms/ml of heparins derived from lung or intestine resulted in cells with intermediate levels of coupling (30-50%). By contrast, 10-20 micrograms/ml of chondroitin sulfate proteoglycan, dermatan sulfate proteoglycan, or liver-derived heparin resulted in dye coupling in 80-100% of the cells, with numerous cells showing dye spread from a single injected cell. Sulfated polysaccharides of glucose (dextran sulfates) or of galactose (carrageenans) were inactive or only weakly active except for lambda-carrageenan, which induced up to 70% coupling (albeit no multiple coupling in the cultures). The abundance of mRNA (Northern blots) encoding gap junction protein and the amounts of the 27-kD gap junction polypeptide (Western blots) correlated with the degree of electrical and dye coupling indicating that the active glycosaminoglycans and proteoglycans are inducing synthesis and expression of gap junctions. Thus, proteoglycans and glycosaminoglycans, especially those found in abundance in the extracellular matrix of liver cells, are important in the regulation of expression of gap junctions and, thereby, in the regulation of intercellular communication in the liver. The relative potencies of heparins from different tissue sources at inducing gap junction expression are suggestive of functional tissue specificity for these glycosaminoglycans.

CLIP-115, a novel brain-specific cytoplasmic linker protein, mediates the localization of dendritic lamellar bodies.

Intracellular localization of organelles may depend in part on specific cytoplasmic linker proteins (CLIPs) that link membranous organelles to microtubules. Here, we characterize rat cDNAs encoding a novel, brain-specific CLIP of 115 kDa. This protein contains two N-terminal microtubule-binding domains and a long coiled-coil region; it binds to microtubules and is homologous to CLIP-170, a protein mediating the binding of endosomes to microtubules. CLIP-115 is enriched in the dendritic lamellar body (DLB), a recently discovered organelle predominantly present in bulbous dendritic appendages of neurons linked by dendrodendritic gap junctions. Local microtubule depolymerization leads to a temporary reduction of DLBs. These results suggest that CLIP-115 operates in the control of brain-specific organelle translocations.

The ion-optical prototype of the low energy neutral atom sensor of the Interstellar Boundary Explorer Mission (IBEX).

The direct measurement of the energetic neutral atoms originating from the heliospheric termination shock and beyond as well as neutral interstellar gas penetrating into the heliosphere requires a very sensitive neutral particle imaging instrument in the energy range of 10-1000 eV. We present the development of the prototype of the low energy sensor for the Interstellar Boundary Explorer (IBEX) mission: IBEX-Lo is a neutral particle mass spectrometer dedicated to the measurement of energetic neutral atoms in this energy range. The response of the sensor to incident neutral hydrogen, helium, and oxygen atoms is discussed as well as the properties of the sensor's ion optics, the neutral-to-negative conversion surfaces, and other instrumental parameters.

Colocalized alterations in connexin32 and cytochrome P450IIB1/2 by phenobarbital and related liver tumor promoters.

Direct intercellular signal transduction is achieved by the passage of small molecules through gap junctions (GJ). Previous studies in our laboratory showed that the liver tumor promoter phenobarbital (PB) reversibly decreases the abundance of the GJ protein connexin32 (Cx32) in both preneoplastic-altered hepatic foci and centrolobular hepatocytes (M. J. Neveu et al., Cancer Commun., 2: 21-31, 1990). Because the inhibitory effects of PB on GJ intercellular communication are prevented by the nonspecific cytochrome P-450 inhibitor SKF-525A (J. E. Klauning, et al., Toxicol. Appl. Pharmacol., 102: 533-563, 1990), we investigated whether alterations in Cx32 are coincident with changes in the major PB-inducible cytochrome P-450, termed b/e or IIB1/2. Immunostaining of liver cryosections from rats fed dietary PB demonstrated that centrolobular hepatocytes that exhibit reduced Cx32 express enhanced cytochrome P450IIB1/2 protein. In contrast, no change in the periportal distribution of connexin26 immunoreactivity was found in PB-treated rats. In addition, rats were treated with the structurally related barbiturates pentobarbital, amobarbital, barbital, and barbituric acid. We found that the extent of the hepatic lobule occupied by coincident centrolobular alterations in Cx32 and P-450 staining correlates with the ability of the compounds to promote liver oncogenesis. To determine the molecular mechanisms responsible for the modification in Cx32 staining, we examined the mRNA and protein levels of Cx32 and P450IIB1/2 in total-tissue homogenates from PB-treated rats. Northern blotting demonstrated thatdietary PB dramatically induced P-450IIB1 mRNA, but the same RNA samples failed to show alterations in Cx32 steady-state transcripts. Consistent with these findings, the level of Cx32 protein in total liver homogenates did not change in rats chronically fed PB. Examination of Cx32 solubility in 20 mM NaOH demonstrated that PB treatment results in the generation of a NaOH-soluble form of Cx32 (i.e., 47 kDa). In addition, trypsinized paraffin-embedded liver sections from PB-treated rats exhibited diffuse cytoplasmic Cx32 staining that was restricted to centrolobular cells. Our results show that PB and related barbiturate tumor promoters reversibly down-regulate punctate Cx32 staining in centrolobular hepatocytes posttranslationally, possibly through modification(s) in the transport, assembly, and/or turnover of GJs.

Cell-to-cell communication in osteoblastic networks: cell line-dependent hormonal regulation of gap junction function.

We have characterized the distribution, expression, and hormonal regulation of gap junctions in primary cultures of rat osteoblast-like cells (ROBs), and three osteosarcoma cell lines, ROS 17/2.8, UMR-106, and SAOS-2, and a continuous osteoblastic cell line, MC3T3-E1. All cell lines we examined were functionally coupled. ROS 17/2.8 were the more strongly coupled, while ROB and MC3T3-E1 were moderately coupled and UMR-106 and SAOS-2 were weakly coupled. Exposure to parathyroid hormone (PTH) for 1 h increased functional coupling in ROB cells in a concentration-dependent manner. Furthermore, PTH(3-34), an analog of PTH with binds to the PTH receptor and thus attenuates PTH-stimulated cAMP accumulation, also attenuated PTH-stimulated functional coupling in ROB. This suggests that PTH increases functional coupling partly through a cAMP-dependent mechanism. A 1 h exposure to PTH did not affect coupling in ROS 17/2.8, UMR-106, MC3T3-E1, or SAOS-2. To examine whether connexin43 (Cx43), a specific gap junction protein, is present in functionally coupled osteoblastic cells, we characterized Cx43 distribution and expression. Indirect immunofluorescence with antibodies to Cx43 revealed that ROS 17/2.8, ROB, and to a lesser extent MC3T3-E1 and UMR-106, expressed Cx43 immunoreactivity. SAOS-2 showed little if any Cx43 immunoreactivity. Cx43 mRNA and Cx43 protein were detected by Northern blot analysis and immunoblot analysis, respectively, in all cell lines examined, including SAOS-2. Our findings suggest that acute exposure to PTH regulates gap junction coupling, in a cell-line dependent manner, in osteoblastic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Connexin43 in MDCK cells: regulation by a tumor-promoting phorbol ester and Ca2+.

Prior to confluence, cultures of Madin Darby canine kidney (MDCK) cells expressed gap junctional communication, as assessed by fluorescent dye transfer, as well as relatively high levels of an anti-connexin43 immunoreactive component referred to as connexin43 (Cx43). After confluence, dye coupling and levels of Cx43 were dramatically reduced. Immunofluorescence analysis of the distribution of Cx43 in subconfluent cultures showed punctate labeling on the plasma membrane at regions of cell apposition and a more diffuse labeling in perinuclear regions. Western blots of total cell homogenates showed that the dephosphorylated form of Cx43 was more abundant than the phosphorylated forms. Phosphorylation of Cx43 was not significantly affected by 8-Bromo-cAMP or 8-Bromo-cGMP. However, 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited dye coupling and induced an increase in the amount of phosphorylated forms of Cx43 at the expense of the dephosphorylated form. This effect occurred as rapidly as 5 min after TPA treatment without apparent changes in distribution of Cx43 or cell morphology. These results suggest that second messenger pathways involving protein kinase C, but not cAMP- or cGMP-dependent protein kinase, led to changes in electrophoretic mobility of Cx43, revealed by Western blot, consistent with an alteration in the state of phosphorylation of the gap junction protein. Treatments with staurosporine, a protein kinase inhibitor, or okadaic acid, a protein phosphatase inhibitor, either alone or in combination with TPA, indicated that the abundance of the dephosphorylated form of Cx43 in MDCK cells was due to low kinase activity. It was also found that lowering the concentration of extracellular Ca2+, which reduced cell contact, did not affect the abundance, the state of phosphorylation, or the TPA-induced phosphorylation of Cx43. These results suggest that neither extracellular Ca2+ nor cell contact is required for basal or TPA-induced phosphorylation of Cx43.

On the mechanisms of cell uncoupling induced by a tumor promoter phorbol ester in clone 9 cells, a rat liver epithelial cell line.

It is known that in Clone 9 (C9) cells, intercellular gap junctional communication (IGJC) is rapidly blocked by the tumor promoter phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), but it recovers spontaneously a few hours later and becomes refractory to TPA (Yada et al., J. Membr. Biol. 88, 217-232 (1985)). We now report that gap junctions between C9 cells contain at least two junctional proteins, connexin26 (Cx26) and connexin43 (Cx43), and that the TPA-induced changes in IGJC correlate temporally to changes in the state of phosphorylation of Cx43. The latter changes were prevented by inhibition of protein kinase C. Phosphoamino acid analysis and two-dimensional tryptic peptide maps of 32P-labeled Cx43 showed that during the TPA-induced phosphorylation at least two of the phosphorylated forms of Cx43 were differentially phosphorylated in seryl residues as compared to control. TPA induced a drastic reduction in junctional conductance as well as a redistribution of unitary gap junction channel event sizes seen in control cells. These changes were associated with retrieval of Cxs from the plasma membrane. Reappearance of gap junctions formed by Cx43 but not by Cx26 accounted for the spontaneous recovery in IGJC. It is proposed that gap junctions between C9 cells contain two types of channels each formed by Cx43 or Cx26 and that they are differentially affected during the action of TPA.

Connexin32 in oligodendrocytes and association with myelinated fibers in mouse and rat brain.

The distribution and cellular localization of connexin32 (Cx32) in the brain and spinal cord of the mouse and rat was investigated by light microscope (LM) and electron microscope (EM) immunohistochemistry by using several different antibodies against Cx32. By double immunofluorescence staining for Cx32 and either the oligodendrocyte markers cyclic nucleotide phosphodiesterase (CNPase) or Rip, Cx32 was consistently found in oligodendrocyte cell bodies and proximal processes. Cx32 immunoreactivity was also clearly visualized along CNPase- and Rip-positive myelinated fibers. Both immunopositive cells and fibers were heterogeneously distributed and were often more intensely labeled when dispersed in or associated with regions of gray matter than when concentrated in major white matter tracts. Labeling of myelin sheaths along fibers was restricted to subpopulations of myelinated axons. In the cerebellar cortex, for example, it was selectively localized to sheaths around Purkinje cell axons. Punctate staining, distinct from that corresponding to cells or fibers, was evident in the olfactory bulb and hippocampus. By EM, oligodendrocytes exhibited cytoplasmic labeling associated with rough endoplasmic reticulum and Golgi apparatus. Their processes were intermittently stained, most intensely when surrounding myelinated fibers and occasionally in paranodal loops. Cx32-immunoreactive gap junctions with symmetric labeling (staining on both junctional membranes) were observed between oligodendrocytic somata and processes as well as between presumptive oligodendrocytic processes. Unidentifiable elements forming asymmetrically labeled gap junctions (staining only one side of junctional membranes) were less frequently encountered. Western blot analysis confirmed anti-Cx32 antibody detection of Cx32 in whole brain homogenates and an enrichment of the protein in isolated myelin fractions. These results are consistent with earlier ultrastructural studies showing the occurrence of inter-oligodendrocytic gap junctions, but indicate that these may be more prevalent than previously thought. Furthermore, the results suggest a specialized role of gap junctions composed of Cx32 along myelinated fibers belonging to subpopulations of neurons.

Gap junction protein in weakly electric fish (Gymnotide): immunohistochemical localization with emphasis on structures of the electrosensory system.

Electrotonic transmission via gap junctions appears to be essential for both the relay and integration of information in nuclear groups involved in the electrolocation and electrocommunication systems of weakly electric fish. An affinity-purified antibody against the 27 kD gap-junctional polypeptide (GJP) from rat liver was used to determine immunohistochemically the distribution of GJP-immunoreactivity (GJP-IR) in electrosensory structures and some other brain regions of the gymnotiform fish, Apteronotus leptorhynchus. At the ultrastructural level, immunolabelling with this antibody was localized, in part, to neuronal and glial gap junctions where it was assumed to recognize a junctional polypeptide. By light microscopy, the vast majority of immunoreactive elements appeared either as fine puncta or as varicosities along fibers that exhibited immunostained intervaricose segments. Diffuse immunoreactivity within cell bodies was rare, being most evident in giant relay neurons and presumptive glial cells within the pacemaker nucleus and in neurons within the posterior raphe nucleus. The distribution of punctate and fibrous GJP-IR was remarkably heterogeneous with respect to density; large areas of the forebrain and most major fiber tracts were nearly devoid of immunoreactivity, whereas concentrations of puncta delineating patches within the inferior lobe of the hypothalamus and the vagal sensory nucleus were so dense as to appear as uniform deposition of immunoperoxidase reaction product at low magnification. Some structures known to be associated with the electrosensory system, including the nucleus electrosensorius and nucleus praeeminentialis, were among the brain regions containing the highest concentrations of immunoreactivity. At the cellular level, expected patterns of GJP-IR were observed in the pacemaker nucleus, torus semicircularis, and electrosensory lateral line lobe. In each of these structures punctate immunoreactivity was seen in apposition to cell bodies or dendrites of neurons known to receive gap junction contacts. In addition, the dendrites of neurons within the prepacemaker nucleus were laden with a striking array of puncta, suggesting that interactions via gap junctions may be a significant feature of these neurons. These immunohistochemical results are consistent with previous electrophysiological and ultrastructural observations pointing to the importance of electrotonic communication in the electrosensory system of weakly electric fish, and suggest that gap junctions may also contribute to neural transmission in central nervous system related to other functions in these teleosts.(ABSTRACT TRUNCATED AT 400 WORDS)

Gap junction protein in rat hippocampus: correlative light and electron microscope immunohistochemical localization.

Immunohistochemical techniques and an affinity-purified antibody directed against the 27-kD gap-junctional protein (GJP) from rat liver were used to determine the ultrastructural localization of GJP in the rat hippocampus. At the light microscope level, dense GJP immunoreactivity having a stringlike appearance was seen in a very small percentage of medium-sized neuronal somata located in the stratum pyramidale, and diffuse immunostaining was seen in many small cell bodies in the stratum pyramidale, stratum oriens, and the alveus. Abundant GJP-immunoreactive (GJP-IR) varicose fibers were observed in the strata pyramidale, radiatum, and oriens but were less concentrated in the alveus. Numerous punctate GJP-IR elements were observed in all hippocampal layers. Upon EM analysis, GJP-IR neuronal somata in the stratum pyramidale were found to be, without exception, nonpyramidal neurons as judged by such distinguishing features as their fusiform perikarya, indented nucleus, and well-developed rough endoplasmic reticulum (RER). Immunostaining within these cells was largely localized to the Golgi apparatus and associated vesicular components. Small, diffusely GJP-IR cells were identified ultrastructurally as protoplasmic and fibrous astrocytes. Immunostaining within these cells was localized to the Golgi apparatus, RER, and small, ribosomelike bodies 15-25 nm in diameter. Among neuronal processes GJP immunoreactivity was found within dendrites, axons, and axonal terminals. The latter structures contained numerous GJP-IR vesicles having an average diameter of about 40 nm. A frequent observation indicating some degree of specificity of the anti-GJP antibody employed here was immunostaining of typical gap junctions between dendrites and, more commonly, between processes of glial cells. Occasionally, however, GJP-IR dendrodendritic, axodendritic, and axoaxonic contacts were found that could be considered, at best, as being gap-junction-like (gj-L). In these cases, asymmetric immunostaining of adjacent plasma membranes forming gj-L structures was not uncommon. These results confirm the existence of gap junctions between dendrites in the rat hippocampus and demonstrate that GJP immunoreactivity on cytoplasmic membranes is restricted either to typical neuronal and glial gap junctions or to gj-L structures at circumscribed sites of contact between various types of neuronal elements where GJP may contribute to a novel mechanism of neural communication.

Connexin43 and astrocytic gap junctions in the rat spinal cord after acute compression injury.

To examine the possible role of interastrocytic gap junctions in the maintenance of tissue homeostasis after spinal cord damage, we initiated studies of the astrocytic gap junctional protein connexin43 (Cx43) in relation to temporal and spatial parameters of neuronal loss, reactive gliosis, and white matter survival in a rat model of traumatic spinal cord injury (SCI). Cx43 immunolocalization in normal and compression-injured spinal cord was compared by using two different sequence-specific anti-Cx43 antibodies that have previously exhibited different immunorecognition properties at lesion sites in brain. At 1- and 3-day survival times, gray matter areas with mild to moderate neuronal depletion exhibited a loss of immunolabeling with one of the two antibodies. At the lesion epicenter, these areas consisted of a zone that separated normal staining distal to the lesion from intensified labeling seen with both antibodies immediately adjacent to the lesion. Loss of immunoreactivity with only one of the two antibodies suggested masking of the corresponding Cx43 epitope. By 7 days post-SCI, Cx43 labeling was absent with both antibodies in all regions extending up to 1 mm from the lesion site. Reactive astrocytes displaying glial fibrillary acidic protein (GFAP) appeared by 1 day and were prominent by 3 days post-SCI. Their distribution in white and gray matter corresponded closely to that of Cx43 staining at 1 day, but less so at 3 days when GFAP-positive profiles were present at sites where Cx43 labeling was absent. By 7 days post-SCI, Cx43 again co-localized with GFAP-positive cells in the surviving subpial rim, and with astrocytic processes on radially oriented vascular profiles investing the central borders of the lesion. The results indicate that alterations in Cx43 cellular localization and Cx43 molecular modifications reflected by epitope masking, which were previously correlated with gap junction remodeling following excitotoxin-induced lesions in brain, are not responses limited to exogenously applied excitotoxins; they also occur in damaged spinal cord and are evoked by endogenous mechanisms after traumatic SCI. The GFAP/Cx43 co-localization results suggest that during their transformation to a reactive state, spinal cord astrocytes undergo a transitional phase marked by altered Cx43 localization or expression.

On the organization of astrocytic gap junctions in rat brain as suggested by LM and EM immunohistochemistry of connexin43 expression.

Gap junctions and the intercellular communication syncytium they form between glial cells are thought to play a critical role in glial maintenance of appropriate metabolic environments in neural tissues. We have previously suggested (Yamamoto et al., Brain Res. 508:313-319, '90) that the vast majority of astrocytes in rat brain express connexin43, one of several recently identified gap junction proteins. Here, we confirm ultrastructurally that astrocytes in a number of brain regions of rat are immunolabelled with an antibody against connexin43 and that neurons and oligodendrocytes are devoid of labelling. The distribution of connexin43 immunoreactivity throughout the brain is presented at the light microscope (LM) level. By LM, immunoreactive structures consisted primarily of round or elongated puncta ranging from 0.3 microns to 4 microns in length and of annular profiles ranging from 1 to 10 microns in diameter. Immunolabelled fibrous processes were only occasionally seen and no labelling was observed in astrocytic cell bodies. Long, linear arrays of puncta were rare in gray matter but were common in white matter where they were arranged parallel to myelinated fibers. Puncta organized in a honeycomb pattern were seen near the cerebral cortical surface and frequently around blood vessels. Regional immunoreaction density, which was a reflection of either the concentration or staining intensity of immunoreactive elements, was remarkably heterogeneous; dramatic differences in labelling intensity frequently delineated anatomical boundaries between adjacent nuclei. Abrupt as well as graded fluctuations of immunoreaction intensity were also observed within nuclear structures. By electron microscopy (EM), gap junctions of fibrous and protoplasmic astrocytes were intensely stained and labelled organelles were often observed intracellularly in areas near gap junctions. These junctions and the spread of immunoreaction product to perijunctional organelles in their vicinity were considered to correspond to puncta seen by LM. Labelling within astrocytic cell bodies was seen in only a few instances. In some brain areas, astrocytic processes commonly gave rise to immunoreactive lamellae that partially ensheathed neuronal cell bodies, axon terminals, dendrites, and synaptic glomeruli. Such lamellae were considered to correspond to immunoreactive annular profiles seen by LM. Perivascular endfoot processes of astrocytes displayed intense staining of their gap junctions and portions of their apposing membranes.(ABSTRACT TRUNCATED AT 400 WORDS)

Gap junction protein in rat hippocampus: light microscope immunohistochemical localization.

An affinity-purified antibody against a 27-kD rat liver gap-junctional protein (GJP) was used to determine the distribution of GJP immunoreactivity in sections of rat hippocampus. Four heterogeneously distributed GJP-immunostaining patterns were observed. The two most common were punctate immunoreactive elements ranging in size from 0.3 to 0.7 microns and networks of immunoreactive varicose fibers coursing in a variety of directions within the various hippocampal layers and ranging in length from a few microns up to 200 microns. The density of punctate immunostaining was highest within a portion of the stratum pyramidale, at the border between the stratum pyramidale and stratum oriens, and at the border between the molecular and granule cell layers of the dentate gyrus. Moderate to low densities were observed in other hippocampal areas. Immunoreactive fibers were most concentrated within the border portions of the stratum pyramidale and oriens, moderately distributed in the stratum radiatum and the remaining part of the stratum oriens, and sparse in the alveus. In the dentate gyrus, fiber networks were most evident at the border between the granule cell and molecular layers and very unevenly distributed in the molecular layer. The two other patterns observed included intense filamentous immunostaining within a small number of neuronal perikarya located mainly in the stratum pyramidale of areas CA2 and CA3, but rarely in area CA1 or the dentate gyrus, and diffuse immunostaining of small cell bodies dispersed throughout the hippocampus but most numerous in the vicinity of the stratum pyramidale and in the alveus. All of these immunostaining patterns were seen at all rostrocaudal hippocampal levels. These results suggest that if GJP-immunoreactive fibers and neurons observed in the hippocampus have the capacity to form gap junctions, then electrotonic transmission may constitute an important means of information processing within this structure.

Expression of connexins in the developing olfactory system of the mouse.

To gain insight into the function of gap junctions' connexin43, connexin32 and connexin26 in a neural structure that retains neuronal turnover capacities throughout adulthood, the expression of these molecules has been investigated in the developing and adult olfactory system by immunocytochemical and biochemical methods. Connexin43 was detectable from the olfactory placode stage. During early embryonic development, the levels of connexin43 expression remained low. An increase in the expression of this connexin occurred perinatally. Expression of connexin43 became very high during the postnatal stages and adulthood. Electron microscopy (EM) immunocytochemistry of the olfactory system showed connexin43 expression in non-neuronal cells. Strong regional differences in the expression of connexin43 in the olfactory epithelium were observed. No apparent relationship between connexin43 expression and turnover activity of olfactory neurons was detected. Western blots of olfactory tissues revealed the presence of three different isoforms of connexin43. Connexin32 was detected in the olfactory bulb at late postnatal stages including adulthood. Connexin32 was observed on some cells tentatively identified as oligodendrocytes. Connexin26 was localized onto leptomeninges. Some immunofluorescence was also obtained in the periglomerular region and in the subependymal layer of the bulb. Northern blot analysis revealed the presence of mRNA of connexin32 and connexin26 in the adult olfactory system. Our results substantiate the cell specific expression of these three types of connexins and they document the primary of connexin43 in olfactory tissues. Moreover, our findings indicate that although expression of connexin43 in the olfactory system is developmentally regulated, it is not directly associated with the neuronal cell turnover of the olfactory epithelium.

Evidence for the co-localization of another connexin with connexin-43 at astrocytic gap junctions in rat brain.

Gap junctions between astrocytes as well as between astrocytes and oligodendrocytes in rat brain were immunohistochemically labelled with a monoclonal and an affinity-purified polyclonal antibody generated against connexin-26. By light microscopy, the immunolabelling patterns obtained were, with a few exceptions, remarkably similar to previously described distribution patterns of the gap junctional protein connexin-43, which is expressed by astrocytes and is localized at astrocytic gap junctions. By electron microscopy, immunoreactivity with these two anti-connexin-26 antibodies was restricted to astrocytes; inter-astrocytic gap junctional membranes were symmetrically labelled, heterologous oligo-astrocytic junctional membranes were asymmetrically labelled only on the astrocyte side and oligo-oligodendrocyte junctions were unlabelled. Two additional anti-connexin-26 antibodies that were found to produce punctate labelling in leptomeninges and liver failed to do so in brain parenchyma, consistent with reports indicating the absence of authentic connexin-26 in this tissue. Antibodies that labelled astrocytic gap junctions exhibited no cross-reaction with connexin-43 or connexin-32, as demonstrated by western blotting, but recognized liver connexin-26 as well as several brain proteins, including an approximately 32000 mol. wt protein that did not correspond to connexin-32 and a 26000 mol. wt protein that co-migrated with liver connexin-26. These results suggest that connexin-26, or more likely a protein having sequence homology with connexin-26, is targeted to astrocytic gap junctions and raise the possibility of the existence of connexins that may be co-expressed with connexin-43 in most, but perhaps not all, astrocytes.

Connexin-43 in rat spinal cord: localization in astrocytes and identification of heterotypic astro-oligodendrocytic gap junctions.

Connexin-43 in relation to gap junctions between astrocytes and between other cell types in rat spinal cord was investigated immunohistochemically. In gray matter, connexin-43 was distributed thoughout all laminae, but was more concentrated in the substantia gelatinosa and around the central canal. Ultrastructurally, immunostaining was present in the cytoplasm of, and at gap junctions between, fine astrocytic processes, most of which ensheathed neuronal elements. In white matter, connexin-43 was localized to somata of fibrous astrocytes, their glial fibrillary acidic protein-positive processes running parallel to myelinated axons, and at gap junctions between these processes. Labelling was also evident in thick radially-directed astrocytic processes displaying pockets of staining near immunopositive gap junctions. Near the cord surface, staining was present in cell bodies of subpial astrocytes and at gap junctions between their tangential processes which formed most of the glia limitans. Radially-directed processes of subpial astrocytes formed symmetrically- and asymmetrically-labelled gap junctions with each other and extended fine branches into surrounding white matter where they made contact and often formed gap junctions with oligodendrocytic processes at the outer surface of myelinated fibres. Immunopositive astrocyte processes also made heterologous gap junctions with unstained oligodendrocyte cell bodies. Ependymal cells lining the central canal exhibited apical cytoplasmic labelling, as well as symmetrically-labelled gap junctions at their apices. Ependymal cells also formed asymmetrically-labelled gap junctions at which the junctional membranes of unlabelled cells, presumed to be tanycytes, were unstained. The results indicate the expression of connexins in addition to connexin-43 at asymmetrically-labelled gap junctions between some astrocytic processes, between astrocytes and oligodendrocytes and between some ependymal cells. The presence of gap junctions between astrocyte and oligodendrocyte processes at the outer surface of myelin suggests incorporation of the latter into the extensive gap junctionally-coupled astrocytic syncytium.

Functional demonstration of connexin-protein binding using surface plasmon resonance.

Surface plasmon resonance (SPR) allows examination of protein-protein interactions in real time, from which both binding affinities and kinetics can be directly determined. We have used the SPR technique to search for proteins in heart tissue that would be candidate binding partners for the cardiac gap junction protein, connexin43 (Cx43). Heart lysate showed a strong, pH-dependent binding to the carboxyl terminus (CT) of Cx43 (amino acids 254-382) covalently linked to an SPR cuvette. Binding was inhibited by the presence of v-src transfected 3T3 cell lysate, suggesting that binding partners in these two lysates may compete for overlapping epitopes on Cx43CT. The combined application of proteomic and functional studies is expected to identify which proteins within heart tissue interact with Cx43 and what roles they may play in gap junction function.

Epitopes of gap junctional proteins localized to neuronal subsurface cisterns.

Several lines of evidence indicate the existence of channels that mediate the movement of calcium from the extracellular space directly into some intracellular calcium storage compartment and from one intracellular membrane-bounded compartment to another. The possibility that such channels resemble intercellular communication pathways formed by gap junction proteins (connexins) was investigated in rat brain. Antibodies against a rat liver gap junction protein (connexin32) were found to recognize several distinct proteins on Western blots of brain homogenates. In motoneurons these antibodies immunohistochemically labelled portions of neuronal endoplasmic reticulum membranes that form subsurface cisterns (SSCs) adjacent to the plasma membrane. These results suggest that SSCs and connexin-like proteins may be involved in the process of calcium mobilization in neurons.

Elevated connexin43 immunoreactivity at sites of amyloid plaques in Alzheimer's disease.

The distribution of the astrocytic gap junctional protein, connexin43 (Cx43) was compared immunohistochemically with that of amyloid plaques in Alzheimer's Disease (AD) brain. By light microscopy, cortical areas containing numerous beta/A4 amyloid plaques exhibited increased immunostaining density for Cx43 and some plaques corresponded exactly to sites of intensified Cx43 immunoreactivity. By electron microscopy, Cx43 was localized to astrocytic gap junctions in AD brain. Increased Cx43 expression in AD may represent an attempt to maintain tissue homeostasis by augmented intercellular communication via gap junction formation between astrocytic processes that invest senile plaques, or alternatively, an aberrant induction of astrocytic Cx43 expression which may further compromise homeostasis and exacerbate pathological conditions in the microenvironment of amyloid plaques.