Rolf Dermietzel

Angiogenesis of the blood–brain barrier in vitro and the function of cerebral pericytes

Cerebral pericytes constitute an essential component of the blood‐brain barrier (BBB) and are involved in blood vessel assembly. Recently, we reported on the induction of a BBB‐specific enzyme expressed by cerebral pericytes (pericytic aminopeptidase N/pAPN) in coculture with cerebral endothelial cells. We completed this in vitro BBB system by adding astrocytes to these mixed cultures of endothelial cells and pericytes. Under these triculture conditions, endothelial cells and pericytes reorganize into capillary‐like structures (CLSs). Capillary formation can also be achieved by the application of transforming growth factor beta 1 (TGF‐β1) in the culture medium of endothelial‐pericyte cultures lacking astrocytes. In contrast to the effect achieved by astrocytes, pericytes did not assemble with endothelial cells. In both cases (application of astrocytes or TGF‐β1), endothelial cells underwent apoptosis. However, endothelial cells that form CLSs in the presence of pericytes appeared to be resistant to induction of apoptosis. On the basis of these observations, we concluded that astrocytes have a profound influence on the morphogenetic events underlying the organization of the vessel wall; that the effect of TGF‐β1 is different from the astrocytic effect because it lacks induction of endothelial‐pericyte association; and that pericytes stabilize CLSs formed by endothelial cells in coculture with astrocytes.

Protein Kinase A-mediated Phosphorylation of Connexin36 in Mouse Retina Results in Decreased Gap Junctional Communication between AII Amacrine Cells

Gap junctions in AII amacrine cells of mammalian retina participate in the coordination of the rod and cone signaling pathway involved in visual adaptation. Upon stimulation by light, released dopamine binds to D1 receptors on AII amacrine cells leading to increased intracellular cAMP (cyclic adenosine monophosphate) levels. AII amacrine cells express the gap junctional protein connexin36 (Cx36). Phosphorylation of Cx36 has been hypothesized to regulate gap junctional activity of AII amacrine cells. However, until now in vivo phosphorylation of Cx36 has not been reported. Indeed, it had been concluded that Cx36 in bovine retina is not phosphorylated, but in vitro phosphorylation for Cx35, the bass ortholog of Cx36, had been shown. To clarify this experimental discrepancy, we examined protein kinase A (PKA)-induced phosphorylation of Cx36 in mouse retina as a possible mechanism to modulate the extent of gap junctional coupling. The cytoplasmic domains of Cx36 and the total Cx36 protein were phosphorylated in vitro by PKA. Mass spectroscopy revealed that all four possible PKA consensus motifs were phosphorylated; however, domains point mutated at the sites in question showed a prevalent usage of Ser-110 and Ser-293. Additionally, we demonstrated that Cx36 was phosphorylated in cultured mouse retina. Furthermore, activation of PKA increased the level of phosphorylation of Cx36. cAMP-stimulated, PKA-mediated phosphorylation of Cx36 protein was accompanied by a decrease of tracer coupling between AII amacrine cells. Our results link increased phosphorylation of Cx36 to down-regulation of permeability through gap junction channels mediating light adaptation in the retina.

COEXPRESSION OF CONNEXIN45 AND -32 IN OLIGODENDROCYTES OF RAT BRAIN

Connexin proteins are the subunits of gap junction channels, and are encoded by a gene family. Although several connexin mRNAs were detected in brain, only a few connexin-proteins have been localized to specific cell types in this tissue. Here we describe expression of connexin45 protein in oligodendrocytes in rat hippocampus. Double immunofluorescent staining using specific antibodies to connexin45 and connexin32 paired with cell-type specific marker proteins revealed that connexin45 and connexin32 were co-expressed and colocalized in oligodendrocytes. Each of the connexin antibodies gave rise to the same pattern of punctate fluorescence in the plasma membrane of cell bodies and proximal processes of oligodendrocytes. Connexins in the plasma membrane of oligodendrocytes may form gap junctions between oligodendrocytes, or between oligodendrocytes and astrocytes. Expression of connexin45 in oligodendrocytes may prevent dysmyelinating effects of connexin32 mutations in the central nervous system of Charcot-Marie-Tooth (X-type) patients.

Gap junction formation in rabbit uterine epithelium in response to embryo recognition

Gap junction formation was studied in the uterine epithelium of nonpregnant, pregnant, and pseudopregnant rabbits in the periimplantation phase (6, 7, 8 days post coitum/post human gonadotropin injection) using freeze-fracture and immunocytochemistry as well as intracellular Lucifer yellow injection. At implantation (7 days post coitum) the uterine epithelial cells of the implantation chamber become junctionally coupled as evidenced by all three methods used. Gap junction protein (26K) becomes detectable immunocytochemically with a monoclonal antibody at 6 days post coitum in the epithelium surrounding the blastocyst, i.e., in the forming implantation chamber. The same sequence of events, starting with the presence of the gap junction protein before cell-to-cell coupling becomes evident, was observed in the blastocyst-free segments 1 day later. In contrast, uterine epithelium of nonpregnant and pseudopregnant animals in comparable phases shows an extremely low degree of coupling. The presence of the blastocyst is a necessary condition for the induction of gap junctions as demonstrated by unilateral pregnancy produced by tubal ligation. Thus, gap junction formation is one of the first maternal responses to a locally acting signal of the blastocyst.

Expression of the gap junction protein connexin43 in the subependymal layer and the rostral migratory stream of the mouse: evidence for an inverse correlation between intensity of connexin43 expression and cell proliferation activity

Abstract.Connexins constitute the channel-forming proteins of gap junctions. Gap junctions are considered to be involved in the regulation of cell proliferation. To verify this hypothesis for connexin43, the most abundant connexin in brain tissue, we have analyzed the expression of this gap junction protein in the subependymal layer and the rostral migratory stream of the murine telencephalon. These regions reveal high proliferative activity, even during postnatal stages and in adulthood. Proliferating cells were labeled in vivo by means of the bromodeoxyuridine method and were later processed for double immunocytochemistry by using an antibody to connexin43. The relationship between connexin43 expression and cell proliferation was also determined in primary cell cultures of olfactory bulbs from newborn mice. The intercellular coupling efficiency of cultured bulbar cells was also analyzed by dye-transfer experiments in combination with the bromodeoxyuridine technique. In the rostral migratory stream, connexin43 was upregulated during postnatal development, coinciding with a decrease of BrdU incorporation. Comparative quantification of the intensity of connexin43 immunoreactivity by confocal laser microscopy and of BrdU-labeled cells showed a clear reverse correlation between connexin43 expression and cell proliferation in the rostral migratory stream during postnatal development. A marked reverse correlation of both parameters was also observed in primary cell cultures from olfactory bulbs at day 6 after seeding.

Pannexin 1 Constitutes the Large Conductance Cation Channel of Cardiac Myocytes

A large conductance (∼300 picosiemens) channel (LCC) of unknown molecular identity, activated by Ca2+ release from the sarcoplasmic reticulum, particularly when augmented by caffeine, has been described previously in isolated cardiac myocytes. A potential candidate for this channel is pannexin 1 (Panx1), which has been shown to form large ion channels when expressed in Xenopus oocytes and mammalian cells. Panx1 function is implicated in ATP-mediated auto-/paracrine signaling, and a crucial role in several cell death pathways has been suggested. Here, we demonstrate that after culturing for 4 days LCC activity is no longer detected in myocytes but can be rescued by adenoviral gene transfer of Panx1. Endogenous LCCs and those related to expression of Panx1 share key pharmacological properties previously used for identifying and characterizing Panx1 channels. These data demonstrate that Panx1 constitutes the LCC of cardiac myocytes. Sporadic openings of single Panx1 channels in the absence of Ca2+ release can trigger action potentials, suggesting that Panx1 channels potentially promote arrhythmogenic activities.

Mitral and tufted cells of the mouse olfactory bulb possess gap junctions and express connexin43 mRNA.

Analyses of freeze-fracture replicas of mouse olfactory bulb reveal the presence of gap junctions in the plasma membranes of the cell bodies of mitral cells. Due to their localization and morphology we presume that they interconnect mitral and granule cells. Since the quality of electrical transmission between neurons is considered to be determined by the biochemical nature of the gap junction channel forming proteins (connexins) we performed immunohistochemistry and in situ hybridization using probes for connexin43 (Cx43), the most abundant connexin in brain tissue. Attribution of Cx43 immunolabel to specific neurons could not definitely be assessed by means of immunohistochemistry. In situ hybridization, however, using a specific cRNA probe for Cx43 revealed a label confined to cell bodies of mitral and tufted cells of the olfactory bulb. These data indicate that Cx43 is expressed by bulbar neurons and suggest that Cx43 is a molecular constituent of gap junction channels in neurons.

Intracellular Cysteine 346 Is Essentially Involved in Regulating Panx1 Channel Activity

Pannexins constitute a family of proteins exhibiting predominantly hemichannel activity. Pannexin channels have been suggested to participate in a wide spectrum of biological functions such as propagation of calcium waves, release of IL-1β, and responses to ischemic conditions. At present, the molecular mechanisms regulating pannexin hemichannel activity are essentially unknown. Because cysteines have been shown to constitute key elements in regulating hemichannel properties of the connexin-type we performed site-directed mutagenesis of intracellular cysteine residues of Panx1. Cysteine to serine exchange (Cys → Ser) at the C-terminal position amino acid 346 led to a constitutively leaky hemichannel and subsequently to cell death. Increased channel activity was demonstrated by dye uptake and electrophysiological profiling in injected Xenopus laevis oocytes and transfected N2A cells. Mutations of the remaining intracellular cysteines did not result in major changes of Panx1 channel properties. From these data we conclude that the Cys-346 residue is important for proper functioning of the Panx1 channel.

Gap Junctions in the Nervous System: An Introduction

Acentury ago, it was generally believed that consciousness and movement resulted from the flow of substances freely throughout the interconnected neural network. This Reticular Theory eventually gave way to the Neuronal Doctrine in which the nervous system was envisioned as a composite of discrete cells, where direct transfer of information among neurons is a rare event, occurring only in specialized nuclei and under specific circumstances. Nevertheless, as chapters in this volume testify, gap junctions, the structural elements responsible for direct intercellular communication, are increasingly detected between cells in both the central and peripheral nervous systems of mammals, including man.

Isolation and characterization of Chinese hamster cells defective in cell-cell coupling via gap junctions☆

Chinese hamster Wg3-h-o cells which were descended from DON cells have been mutagenized and selected for derivatives defective in metabolic cooperation via gap junctions (i.e., mec-). The selection protocol included four consecutive cycles of cocultivating mutagenized cells, deficient in hypoxanthine phosphoribosyltransferase (HPRT) and wild-type cells in the presence of thioguanine (cf Slack, C, Morgan, R H M & Hooper, M L, Exp cell res 117 (1978) 195-205) [8]. We carried out the last two selection cycles in the presence of 1 mM dibutyryl cyclic adenosine monophosphate (db-cAMP). The isolated Chinese hamster CI-4 cells which expressed the mec- phenotype most stringently showed the following characteristics: 1. In standard culture medium no cell-cell coupling was detected among CI-4 cells when assayed by injections of the fluorescent dye Lucifer yellow or by electrical measurements. Between 73 and 100% of the mec+ parental cells were coupled under these conditions. Up to 14% positive contacts were found between CI-4 cells and Chinese hamster Don cells (mec+). Confluent CI-4 cells grown in the presence of 1 mM db-cAMP showed 9% coupled cells. 2. No gap junction plaques were found on electron micrographs of freeze-fractured, confluent CI-4 cells. The mec+ parental cells showed small gap junction plaques (0.013% of the total cell surface analyzed). 3. CI-4 cells exhibited 16% positive contacts and the parental Wg3-h-o cells showed 92% positive contacts in autoradiographic measurements of metabolic cooperation with DON cells. On an extracellular matrix, prepared from normal embryonic fibroblasts, metabolic cooperation between CI-4 and DON cells was autoradiographically measured to be 68%. Other cells of spontaneous mec- phenotype (for example mouse L cells or human fibrosarcoma HT1080 cells) also appeared to exhibit increased metabolic cooperation when grown on an extracellular matrix and assayed by autoradiographic measurements. When tested by Lucifer yellow injections, however, only very few positive contacts were found for CI-4/DON cell pairs and no positive contacts were found among mouse L cells grown on an extracellular matrix. 4. The mec- defect in the genome of CI-4 cells was cured in somatic cell hybrids with mouse embryonic fibroblasts or with mouse embryonal carcinoma cells. The results of isozyme and karyotype studies of mec-, as well as mec+ somatic cell hybrids suggest that mouse chromosome 16 may be involved in complementation of the mec- defect.