Cellular mechanisms of connexin32 mutations associated with CNS manifestations

Both oligodendrocytes and myelinating Schwann cells express the gap junction protein connexin32 (Cx32). Mutations in the gene encoding Cx32 (GJB1) cause the X-linked form of Charcot-Marie-Tooth disease (CMTX). Although most CMTX patients do not have clinical central nervous system (CNS) manifestations, subclinical evidence of CNS dysfunction is common. We investigated the cellular effects of a subgroup of GJB1/Cx32 mutations that have been reported to cause clinical CNS dysfunction. We hypothesized that these mutants have dominant-negative effects on other connexins expressed by oligodendrocytes, specifically Cx45. We expressed these and other Cx32 mutants in communication-incompetent as well as Cx45-expressing HeLa cells, and analyzed the transfected cells by immunocytochemistry and immunoblotting. In communication-incompetent cells, the mutants associated with CNS phenotypes failed to reach the cell membrane and were instead retained in the endoplasmic reticulum (A39V, T55I) or Golgi apparatus (M93V, R164Q, R183H), although rare gap junction plaques were found in cells expressing M93V or R183H. In HeLa cells stably expressing Cx45, these Cx32 mutants showed a similar expression pattern, and did not alter the pattern of Cx45 expression. These results indicate that Cx32 mutants that are associated with a CNS phenotype do not interact with Cx45, but may instead have other toxic effects in oligodendrocytes.     

Connexin29 expression,immunocytochemistry and freeze-fracture replica immunogold labelling (FRIL) in sciatic nerve

The recently discovered connexin29 (Cx29) was reported to be present in the central and peripheral nervous systems (CNS and PNS), and its mRNA was found in particular abundance in peripheral nerve. The expression and localization of Cx29 protein in sciatic nerve were investigated using an antibody against Cx29. The antibody recognized Cx29 in HeLa cells transfected with Cx29 cDNA, while nontransfected HeLa cells were devoid of Cx29. Immunoblotting of sciatic nerve homogenate revealed monomeric and possibly higher molecular weight forms of Cx29. These were distinguished from connexin32 (Cx32), which also is expressed in peripheral nerve. Double immunofluorescence labelling for Cx29 and Cx32 revealed only partial colocalization of the two connexins, with codistribution at intermittent, conical-shaped striations along nerve fibers. By freeze-fracture replica immunogold labelling (FRIL), Cx32 was found in gap junctions in the outermost layers of myelin, whereas Cx29-immunogold labelling was found only in the innermost layer of myelin in close association with hexagonally arranged intramembrane particle (IMP) 'rosettes' and gap junction-like clusters of IMPs. Although both Cx32 and Cx29 were detected in myelin of normal mice, only Cx29 was present in Schwann cell membranes in Cx32 knockout mice. The results confirm that Cx29 is a second connexin expressed in Schwann cells of sciatic nerve. In addition, Cx29 is present in distinctive IMP arrays in the inner most layer of myelin, adjacent to internodal axonal plasma membranes, where this connexin may have previously unrecognized functions.     

Differential Dose-dependent Effects of α-, β-Carotenes and Lycopene on Gap-junctional Intercellular Communication in Rat Liver in vivo

In order to examine the relevance of alteration of gap-junctional intercellular communication (GJIC) to chemopreventive activity against carcinogenesis, the effects of α and β -carotene as well as lycopene, typical chemopreventive carotenoids, on cell coupling via gap junctions in rat liver in vivo were studied using a direct functional dye-transfer technique. We found that all three test compounds given at a dose of 50 mg/kg-body weight (b.w.) daily, 5 times by gavage, inhibited GJIC, while similar treatment with 5 mg/kg b.w. caused enhancement, especially in the β-carotene and lycopene-treated groups. At the dose level of 0.5 mg/kg b.w., the three compounds had no effect. The findings show that all three agents differentially modulate GJIC depending on the dose, with beneficial effects on cell communication only detected at the one dose. The result suggests that determination of the dose of chemicals to be used is crucial for human intervention studies.     

血管平滑肌细胞的内皮依赖性超极化(英文)

In response to various neurohumoral substances en-dothelial cells release nitric oxide (NO) and prostacy-clin, and produce hyperpolarization of the underlying vascular smooth muscle cells, possibly by releasing another factor termed endothelium-derived hyperpolarizing factor (EDHF). NO and prostacyclin stimulate smooth muscle soluble guanylate and adenylate cyclase respectively and can activate, depending on the vascular tissue studied, ATP-sensitive potassium ( KATP) and large conductance calcium-activated potassium channels (BKca). Furthermore, NO directly activates BKca. In contrast to NO and prostacyclin, EDHF-mediated responses are sensitive to the combination of charybdotox-in plus apamin but do not involve KATP or BKca. As hyperpolarization of the endothelial cells is required to observe endothelium-dependent hyperpolarization, an electric coupling through myoendothelial gap junctions may explain the phenomenon. An alternative explanation is that the hyperpolarization of the endothelial cells cau     

Human fetal neurons in culture: Intercellular communication and voltage- and ligand-gated responses

We established high density primary cultures of neural cells from dissociated second trimester human fetal brains using a novel spin seeding method. Under our culture conditions, the majority of the cells exhibited neuronal phenotypes as evidenced by morphological criteria, immunoreactivity to the 66 kDa neurofilament protein and expression of TTX-sensitive Na⁺ channels and cell excitability. These cultures were enriched in glutamic acid decarboxylase (GAD), the synthetic enzyme of the neurotransmitter GABA, and responded with Ca²⁺ influx to acute application of glutamate. Interestingly, the human fetal neurons in culture did not express either dopaminergic or cholinergic phenotypes. In addition, the population of neurons obtained express a high incidence of gap junction-mediated intercellular communication. These studies provide evidence that functional neuronal properties arise early during prenatal development in humans and offer the potential to evaluate pharmacological agents on primary human neurons. © 1994 Wiley-Liss, Inc.     

Characterization of gap junctions between osteoblast-like cells in culture

Summary The structure of gap junctions in osteoblast-like cells (OBs) and the connexins (cx) that build up these structures were characterized by ultrastructural, immunocytochemical, and molecular techniques. Ultrastructural studies revealed numerous gap junctions which were mostly located on processes of neighboring cells. Immunofluorescence labeling using two different antibodies (specific to mouse live cx26 and cx32 and to a peptide-specific rat heart gap junction protein cx43) gave evidence that in OBs, gap junctions consist mainly of cx43. The presence of cx43 in cultured OB was also confirmed by Western blot analysis. Dye-coupling with Lucifer yellow led to a staining of up to 30 neighboring cells. Parallel intracellular recordings showed that membrane potential amplitude changes (4–5 mV) are typically related to those in the coupled cells. Thus, there is morphological and functional evidence for intercellular communication between OB in culture. OBs in culture express the same connexins as observed in vivo and may serve as a model to investigate electrophysiological events in response to different stimulation signals.     

A Comparison of Flow Field Structures of Two Tri-Leaflet Polymeric Heart Valves

Polymeric heart valves have the potential to reduce thrombogenic complications associated with current mechanical valves and overcome fatigue-related problems experienced by bioprosthetic valves. In this in vitro study, the velocity fields inside and downstream of two different prototype tri-lealfet polymeric heart valves were studied. Experiments were conducted on two 23 mm prototype polymeric valves, provided by AorTech Europe, having open or closed commissure designs and leaflet thickness of 120 and 80 m, respectively. A two-dimensional LDV system was used to measure the velocity fields in the vicinity of the two valves under simulated physiological conditions. Both commissural design and leaflet thickness were found to affect the flow characteristics. In particular, very high levels of Reynolds shear stress of 13,000 dynes/cm² were found in the leakage flow of the open commisure design. Maximum leakage velocities in the open and closed designs were 3.6 m/s and 0.5 m/s respectively; the peak forward flow velocities were 2.0 m/s and 2.6 m/s, respectively. In both valve designs, shear stress levels exceeding 4,000 dyne/cm² were observed at the trailing edge of the leaflets and in the leakage and central orifice jets during peak systole. Additionally, regions of low velocity flow conducive to thrombus formation were observed in diastole. The flow structures measured in these experiments are consistent with the location of thrombus formation observed in preliminary animal experiments.     

Chronic endothelin exposure inhibits connexin43 expression in cultured cortical astroglia

Severe brain lesions are accompanied by sustained increases in endothelin (ET) levels, which in turn profoundly affect brain microcirculation and neural cell function. A known response of astrocytes to acute increases in ET levels is the rapid and transient closure of gap junctions and the subsequent decrease of gap junction-mediated intercellular communication (GJIC). Because evidence exists that the loss of GJIC alters astrocytic gene expression, we analyzed the effects of chronic ET exposure on astrocytic gap junction coupling. We found that within 24 hr, cultured cortical astrocytes respond to low nanomolar concentrations (2-10 nM) of either ET-1 or ET-3 with a robust inhibition of connexin (Cx)43 expression, the major junctional protein in astrocytes, and a subsequent decline of GIJC. We further observed that in the continuous presence of ETs, Cx43 expression remained inhibited for at least 7 days. In addition, a similar decrease of Cx43 expression occurred in cultured spinal cord astrocytes maintained with ET-1 for 3 days. Applying ETs in combination with the highly selective ETA and ETB receptor antagonists, BQ123 and BQ788, respectively, revealed that the inhibitory influences on astrocytic Cx43 expression depend on activation of ETB receptors. We suggest that the observed ET-dependent inhibition of Cx43 expression and the resulting decline of GJIC might represent a major pathway by which ETs regulate astrocytic gene expression in the injured brain.     

Quinine, a blocker of neuronal cx36 channels, suppresses seizure activity in rat neocortex in vivo

PURPOSE: The selective contribution of neuronal gap junction (GJ) communication via connexin 36 (Cx36) channels to epileptogenesis and to the maintenance and propagation of seizures was investigated in both the primary focus and the mirror focus by using pharmacologic approaches with the 4-aminopyridine in vivo epilepsy model. METHODS: ECoG recording was performed on anesthetized adult rats, in which either quinine, a selective blocker of Cx36, or the broad-spectrum GJ blockers carbenoxolone and octanol were applied locally, before the induction or at already active epileptic foci. RESULTS: The blockade of Cx36 channels by quinine before the induction of epileptiform activity slightly reduced the epileptogenesis. When quinine was applied after 25-30 repetitions of seizures, a new discharge pattern appeared with frequencies >15 Hz at the initiation of seizures. In spite of the increased number of seizures, the summated ictal activity decreased, because of the significant reduction in the duration of the seizures. The amplitudes of the seizure discharges of all the patterns decreased, with the exception of those with frequencies of 11-12 Hz. The blockade of Cx36 channels and the global blockade of the GJ channels resulted in qualitatively different modifications in ictogenesis. CONCLUSIONS: The blockade of Cx36 channels at the already active epileptic focus has an anticonvulsive effect and modifies the manifestation of the 1- to 18-Hz seizure discharges. Our findings indicate that the GJ communication via Cx36 channels is differently involved in the synchronization of the activities of the networks generating seizure discharges with different frequencies. Additionally, we conclude that both neuronal and glial GJ communication contribute to the manifestation and propagation of seizures in the adult rat neocortex.