Biomechanical analysis of anterior bone graft augmentation with reversed shoulder arthroplasty in large combined glenoid defects compared with total bony joint line reconstruction (modified bony-increased-offset reversed shoulder arthroplasty).

BACKGROUND: The aim of this biomechanical study was to compare 2 surgical techniques for the reconstruction of large, combined, uncontained glenoid defects with reversed shoulder arthroplasty (RSA). METHODS: Three groups of scapulae with RSA were tested by the application of a physiological combination of compressive/shear loads in Sawbones (Pacific Research Laboratories, Inc., Vashon Island, WA, USA) and cadavers. Two of the groups (both Sawbones and cadaveric specimens) consisted of anterior combined defects (14 mm in depth), and the third group served as a control group (only Sawbones specimens). The first group with an anterior combined defect was reconstructed with anterior bone grafts to contain the defect and cancellous bone to fill the central defect before RSA with partial bony joint line reconstruction (p-BJR). In the second group with an anterior combined defect, the dorsal rim was reamed and the joint line was reconstructed with a bone disc fully covering the peg. This total BJR (t-BJR) corresponds to the technique of bony-increased-offset-RSA (BIO-RSA). RESULTS: At 150 µm of displacement, the loadings in the inferior-superior (IS) direction were significantly more stable than those in the anterior-to-posterior (AP) direction within both reconstructed defect groups (P ≤ .002). In contrast, no significant differences were found between the partial BJR and t-BJR group in either direction (Sawbones: AP: P = .29; IS: P = .44; cadavers: AP: P = .67; IS: P = .99). The control group revealed significantly higher values in all loadings of the IS direction and significantly higher loadings at 40 µm and 150 µm in the AP direction. CONCLUSION: Both techniques could be applied for such complex defects provided that there is sufficient medial bone stock for a t-BJR. Significantly greater stability was found in the IS direction than in the AP direction within each group, which could be explained by the longer screw anchoring within the superior and inferior columns. Both defect groups were less stable than the group of intact glenoids.

Effects on channel properties and induction of cell death induced by c-terminal truncations of pannexin1 depend on domain length.

Pannexin1 (Panx1) is an integral membrane protein and known to form multifunctional hexameric channels. Recently, Panx1 was identified to be responsible for the release of ATP and UTP from apoptotic cells after site-specific proteolysis by caspases 3/7. Cleavage at the carboxy-terminal (CT) position aa 376-379 irreversibly opens human Panx1 channels and leads to the release of the respective nucleotides resulting in recruitment of macrophages and in subsequent activation of the immunologic response. The fact that cleavage of the CT at this particular residues terminates in a permanently open channel raised the issue of functional relevance of the CT of Panx1 for regulating channel properties. To analyze the impact of the CT on channel gating, we generated 14 truncated versions of rat Panx1 cleaved at different positions in the C-terminus. This allowed elaboration of the influence of defined residues on channel formation, voltage-dependent gating, execution of cell mortality, and susceptibility to the Panx1 inhibitor carbenoxolone. We demonstrate that expression of Panx1 proteins, which were truncated to lengths between 370 and 393 residues, induces differential effects after expression in Xenopus laevis oocytes as well as in Neuro2A cells with strongest impact downstream the caspase 3/7 cleavage site.

Internal ribosomal entry site (IRES) activity generates endogenous carboxyl-terminal domains of Cx43 and is responsive to hypoxic conditions.

Connexin43 (Cx43) is the most abundant gap junction protein in higher vertebrate organisms and has been shown to be involved in junctional and non-junctional functions. In addition to the expression of full-length Cx43, endogenously produced carboxyl-terminal segments of Cx43 have been described and have been suggested to be involved in manifold biological functions, such as hypoxic preconditioning and neuronal migration. Molecular aspects, however, behind the separate generation of carboxyl-terminal segments of Cx43 have remained elusive. Here we report on a mechanism that may play a key role in the separate production of these domains. First, stringent evidence derived from siRNA treatment and specific knockouts revealed significant loss of the low molecular weight fragments of Cx43. By applying a dicistronic vector strategy on transfected cell lines, we were able to identify putative IRES activity (nucleotides 442­637) in the coding region of Cx43, which resides upstream from the nucleotide sequence encoding the carboxyl terminus (nucleotides 637­1149). Functional responsiveness of the endogenous expression of Cx43 fragments to hypoxic/ischemic treatment was evaluated in in vitro and in vivo models, which led to a significant increase of the fastest migrating form (20 kDa) under conditions of metabolic deprivation. By nano-MS spectrometry, we achieved stringent evidence of the identity of the 20-kDa segment as part of the carboxyl-terminal domain of full-length Cx43. Our data prove the existence of endogenously expressed carboxyl-terminal domains, which may serve as valuable tools for further translational application in ischemic disorders.

Pannexin1 channel proteins in the zebrafish retina have shared and unique properties.

In mammals, a single pannexin1 gene (Panx1) is widely expressed in the CNS including the inner and outer retinae, forming large-pore voltage-gated membrane channels, which are involved in calcium and ATP signaling. Previously, we discovered that zebrafish lack Panx1 expression in the inner retina, with drPanx1a exclusively expressed in horizontal cells of the outer retina. Here, we characterize a second drPanx1 protein, drPanx1b, generated by whole-genome duplications during teleost evolution. Homology searches strongly support the presence of pannexin sequences in cartilaginous fish and provide evidence that pannexins evolved when urochordata and chordata evolution split. Further, we confirm Panx1 ohnologs being solely present in teleosts. A hallmark of differential expression of drPanx1a and drPanx1b in various zebrafish brain areas is the non-overlapping protein localization of drPanx1a in the outer and drPanx1b in the inner fish retina. A functional comparison of the evolutionary distant fish and mouse Panx1s revealed both, preserved and unique properties. Preserved functions are the capability to form channels opening at resting potential, which are sensitive to known gap junction and hemichannel blockers, intracellular calcium, extracellular ATP and pH changes. However, drPanx1b is unique due to its highly complex glycosylation pattern and distinct electrophysiological gating kinetics. The existence of two Panx1 proteins in zebrafish displaying distinct tissue distribution, protein modification and electrophysiological properties, suggests that both proteins fulfill different functions in vivo.

Connexins and Cap-independent translation: role of internal ribosome entry sites.

Cap-independent translation using an internal ribosome entry site instead of the 5'-Cap structure has been discovered in positive-sense RNA viruses and eukaryotic genomes including a subset of gap junction forming connexins genes. With a growing number of mutations found in human connexin genes and studies on genetically modified mouse models mechanisms highlighting the important role of gap junctional communication in multicellular organism it is obvious that mechanism need to be in place to preserve this critical property even under conditions when Cap-mediated translation is scrutinized. To ensure sustained gap junctional communication, rapid initiation of translation of preexisting connexin mRNAs is one possibility, and the presence of internal ribosome entry sites in gap junction genes comply with such a requirement. In this review, we will summarize past and recent findings to build a case for IRES mediated translation as an alternative regulatory pathway facilitating gap junctional communication. This article is part of a Special Issue entitled Electrical Synapses.

Neuroglial activation and Cx43 expression are reduced upon transplantation of human umbilical cord blood cells after perinatal hypoxic-ischemic injury.

Glial cells play a crucial role in the pathomechanism of perinatal hypoxic-ischemic brain injury (HI) and are involved in the maintenance of a chronic state of inflammation that causes delayed neuronal damage. Activation of astrocytes is one factor prolonging brain damage and contributing to the formation of a glial scar that limits neuronal plasticity. In this context, the major astrocytic gap junction protein Connexin 43 (Cx43) has been ascribed various functions including regulation of astrocytic migration and proliferation. Here, we investigate glial responses like microglia/macrophages and astrocytic activation in a rat model of neonatal HI and characterize changes of these parameters upon transplantation of human umbilical cord blood cells (hUCB). As an alleviation of motor function in lesioned rats has previously been described in transplanted animals, we analyze the putative correlation between motor function and glial activation over time. The lesion-induced impairment of motor function, assessed by forelimb use bias, muscle strength and distal spasticity, was alleviated upon transplantation of hUCB short and long term. HI induced an acute inflammatory reaction with activation of microglia/macrophages and reactive astrogliosis associated with perilesional upregulation of Cx43 that slowly declined during the chronic post-ischemic phase. hUCB transplantation accelerated the regression of inflammatory events, narrowed the perilesional astrocytic wall and led to a downregulation of the investigated astrocytic proteins. Thus, in the immature brain, hUCB may indirectly reduce secondary cell death upon hypoxia-ischemia and facilitate post-ischemic plasticity through the attenuation of reactive gliosis. This article is part of a Special Issue entitled Electrical Synapses.

Calmodulin dependent protein kinase increases conductance at gap junctions formed by the neuronal gap junction protein connexin36.

The major neuronal gap junction protein connexin36 (Cx36) exhibits the remarkable property of "run-up", in which junctional conductance typically increases by 10-fold or more within 5-10min following cell break-in with patch pipettes. Such conductance "run-up" is a unique property of Cx36, as it has not been seen in cell pairs expressing other connexins. Because of the recent observation describing CaMKII binding and phosphorylation sites in Cx36 and evidence that calmodulin dependent protein kinase II (CaMKII) may potentiate electrical coupling in neurons of teleosts, we have explored whether CaMKII activates mammalian Cx36. Consistent with this hypothesis, certain Cx36 mutants lacking the CaMKII binding and phosphorylation sites or wild type Cx36 treated with certain cognate peptides corresponding to binding or phosphorylation sites blocked or strongly attenuated run-up of junctional conductance. Likewise, KN-93, an inhibitor of CaMKII, blocked run-up, as did a membrane permeable peptide corresponding to the CaMKII autoinhibitory domain. Furthermore, run-up was blocked by phosphatase delivered within the pipette and not affected by treatment with the phosphatase inhibitor okadaic acid. These results imply that phosphorylation by CaMKII strengthens junctional currents of Cx36 channels, thereby conferring functional plasticity on electrical synapses formed of this protein.

Transplantation of human umbilical cord blood cells mediated beneficial effects on apoptosis, angiogenesis and neuronal survival after hypoxic-ischemic brain injury in rats.

Transplantation of human umbilical cord blood (hucb) cells in a model of hypoxic-ischemic brain injury led to the amelioration of lesion-impaired neurological and motor functions. However, the mechanisms by which transplanted cells mediate functional recovery after brain injury are largely unknown. In this study, the effects of hucb cell transplantation were investigated in this experimental paradigm at the cellular and molecular level. As the pathological cascade in hypoxic-ischemic brain injury includes inflammation, reduced blood flow, and neuronal cell death, we analyzed the effects of peripherally administered hucb cells on these detrimental processes, investigating the expression of characteristic marker proteins. Application of hucb cells after perinatal hypoxic-ischemic brain injury correlated with an increased expression of the proteins Tie-2 and occludin, which are associated with angiogenesis. Lesion-induced apoptosis, determined by expression of cleaved caspase-3, decreased, whereas the number of vital neurons, identified by counting of NeuN-positive cells, increased. In addition, we observed an increase in the expression of neurotrophic and pro-angiogenic growth factors, namely BDNF and VEGF, in the lesioned brain upon hucb cell transplantation. The release of neurotrophic factors mediated by transplanted hucb cells might cause a lower number of neurons to undergo apoptosis and result in a higher number of living neurons. In parallel, the increase of VEGF might cause growth of blood vessels. Thus, hucb transplantation might contribute to functional recovery after brain injury mediated by systemic or local effects.

Pannexin1 stabilizes synaptic plasticity and is needed for learning.

Pannexin 1 (Panx1) represents a class of vertebrate membrane channels, bearing significant sequence homology with the invertebrate gap junction proteins, the innexins and more distant similarities in the membrane topologies and pharmacological sensitivities with gap junction proteins of the connexin family. In the nervous system, cooperation among pannexin channels, adenosine receptors, and K(ATP) channels modulating neuronal excitability via ATP and adenosine has been recognized, but little is known about the significance in vivo. However, the localization of Panx1 at postsynaptic sites in hippocampal neurons and astrocytes in close proximity together with the fundamental role of ATP and adenosine for CNS metabolism and cell signaling underscore the potential relevance of this channel to synaptic plasticity and higher brain functions. Here, we report increased excitability and potently enhanced early and persistent LTP responses in the CA1 region of acute slice preparations from adult Panx1(-/-) mice. Adenosine application and N-methyl-D-aspartate receptor (NMDAR)-blocking normalized this phenotype, suggesting that absence of Panx1 causes chronic extracellular ATP/adenosine depletion, thus facilitating postsynaptic NMDAR activation. Compensatory transcriptional up-regulation of metabotropic glutamate receptor 4 (grm4) accompanies these adaptive changes. The physiological modification, promoted by loss of Panx1, led to distinct behavioral alterations, enhancing anxiety and impairing object recognition and spatial learning in Panx1(-/-) mice. We conclude that ATP release through Panx1 channels plays a critical role in maintaining synaptic strength and plasticity in CA1 neurons of the adult hippocampus. This result provides the rationale for in-depth analysis of Panx1 function and adenosine based therapies in CNS disorders.

Single cysteines in the extracellular and transmembrane regions modulate pannexin 1 channel function.

Pannexins form high-conductance ion channels in the membranes of many vertebrate cells. Functionally, they have been associated with multiple functional pathways like the propagation of calcium waves, ATP release, responses to ischemic conditions and apoptosis. In contrast to accumulating details which uncovered their functions, the molecular mechanisms for pannexin channel regulation and activation are hardly understood. To further elucidate regulatory mechanisms, we substituted cysteine residues, expected key elements for channel function, in extracellular and transmembrane regions of Pannexin 1 (Panx1). Most apparently, substitution of the transmembrane cysteine C40 resulted in constitutively open channels with profoundly increased activity. Hence, Xenopus laevis oocytes injected with corresponding cRNA showed strongly impaired viability, anomalous dye uptake and greatly increased whole-cell conductivity. All changes induced by C40 substitution were significantly reduced by the Panx1 channel blocker carbenoxolone, indicating that channel activity of the mutated Panx1 had been affected. In contrast, no changes occurred after substitution of the two other transmembrane cysteines, C215 and C227, in terms of channel conductivity. Finally, substitution of any of the four extracellular cysteines resulted in complete loss of channel function in both X. laevis oocytes and transfected N2A cells. From this, we conclude that cysteine residues of Panx1 reveal differential functional profiles for channel activation and drug sensitivity.

Neuroanatomical correlates of suicide in psychosis: the possible role of von Economo neurons.

Suicide is the most important incident in psychiatric disorders. Psychological pain and empathy to pain involves a neural network that involves the anterior cingulate cortex (ACC) and the anterior insula (AI). At the neuronal level, little is known about how complex emotions such as shame, guilt, self-derogation and social isolation, all of which feature suicidal behavior, are represented in the brain. Based on the observation that the ACC and the AI contain a large spindle-shaped cell type, referred to as von Economo neuron (VEN), which has dramatically increased in density during human evolution, and on growing evidence that VENs play a role in the pathophysiology of various neuropsychiatric disorders, including autism, psychosis and dementia, we examined the density of VENs in the ACC of suicide victims. The density of VENs was determined using cresyl violet-stained sections of the ACC of 39 individuals with psychosis (20 cases with schizophrenia, 19 with bipolar disorder). Nine subjects had died from suicide. Twenty specimen were available from the right, 19 from the left ACC. The density of VENs was significantly greater in the ACC of suicide victims with psychotic disorders compared with psychotic individuals who died from other causes. This effect was restricted to the right ACC. VEN density in the ACC seems to be increased in suicide victims with psychosis. This finding may support the assumption that VEN have a special role in emotion processing and self-evaluation, including negative self-appraisal.

Olfactory neuron-specific expression of A30P α-synuclein exacerbates dopamine deficiency and hyperactivity in a novel conditional model of early Parkinson's disease stages.

Mutations in the N-terminus of the gene encoding α-synuclein (α-syn) are linked to autosomal dominantly inherited Parkinson's disease (PD). The vast majority of PD patients develop neuropsychiatric symptoms preceding motor impairments. During this premotor stage, synucleinopathy is first detectable in the olfactory bulb (OB) and brain stem nuclei; however its impact on interconnected brain regions and related symptoms is still less far understood. Using a novel conditional transgenic mouse model, displaying region-specific expression of human mutant α-syn, we evaluated effect and reversibility of olfactory synucleinopathy. Our data showed that induction of mutant A30P α-syn expression increased transgenic deposition into somatodendritic compartment of dopaminergic neurons, without generating fibrillar inclusions. We found reversibly reduced levels of dopamine and metabolites in the OB, suggesting an impact of A30P α-syn on olfactory neurotransmitter content. We further showed that mutant A30P expression led to neurodegenerative changes on an ultrastructural level and a behaviorally hyperactive response correlated with novelty, odor processing and stress associated with an increased dopaminergic tone in midbrain regions. Our present data indicate that mutant (A30P) α-syn is directly implicated in reduction of dopamine signaling in OB interneurons, which mediates further alterations in brain regions without transgenic expression leading functionally to a hyperactive response. These modulations of neurotransmission may underlie in part some of the early neuropsychiatric symptoms in PD preceding dysfunction of the nigrostriatal dopaminergic system.

Ccdc66 null mutation causes retinal degeneration and dysfunction.

Retinitis pigmentosa (RP) is a group of human retinal disorders, with more than 100 genes involved in retinal degeneration. Canine and murine models are useful for investigating human RP based on known, naturally occurring mutations. In Schapendoes dogs, for example, a mutation in the CCDC66 gene has been shown to cause autosomal recessively inherited, generalized progressive retinal atrophy (gPRA), the canine counterpart to RP. Here, a novel mouse model with a disrupted Ccdc66 gene was investigated to reveal the function of protein CCDC66 and the pathogenesis of this form of gPRA. Homozygous Ccdc66 mutant mice lack retinal Ccdc66 RNA and protein expression. Light and electron microscopy reveal an initial degeneration of photoreceptors already at 13 days of age, followed by a slow, progressive retinal degeneration over months. Retinal dysfunction causes reduced scotopic a-wave amplitudes, declining from 1 to 7 months of age as well as an early reduction of the photopic b-wave at 1 month, improving slightly at 7 months, as evidenced by electroretinography. In the retina of the wild-type (WT) mouse, protein CCDC66 is present at highest levels after birth, followed by a decline until adulthood, suggesting a crucial role in early development. Protein CCDC66 is expressed predominantly in the developing rod outer segments as confirmed by subcellular analyses. These findings illustrate that the lack of protein CCDC66 causes early, slow progressive rod-cone dysplasia in the novel Ccdc66 mutant mouse model, thus providing a sound foundation for the development of therapeutic strategies.

Electromagnetic field effect or simply stress? Effects of UMTS exposure on hippocampal longterm plasticity in the context of procedure related hormone release.

Harmful effects of electromagnetic fields (EMF) on cognitive and behavioural features of humans and rodents have been controversially discussed and raised persistent concern about adverse effects of EMF on general brain functions. In the present study we applied radio-frequency (RF) signals of the Universal Mobile Telecommunications System (UMTS) to full brain exposed male Wistar rats in order to elaborate putative influences on stress hormone release (corticosteron; CORT and adrenocorticotropic hormone; ACTH) and on hippocampal derived synaptic long-term plasticity (LTP) and depression (LTD) as electrophysiological hallmarks for memory storage and memory consolidation. Exposure was computer controlled providing blind conditions. Nominal brain-averaged specific absorption rates (SAR) as a measure of applied mass-related dissipated RF power were 0, 2, and 10 W/kg over a period of 120 min. Comparison of cage exposed animals revealed, regardless of EMF exposure, significantly increased CORT and ACTH levels which corresponded with generally decreased field potential slopes and amplitudes in hippocampal LTP and LTD. Animals following SAR exposure of 2 W/kg (averaged over the whole brain of 2.3 g tissue mass) did not differ from the sham-exposed group in LTP and LTD experiments. In contrast, a significant reduction in LTP and LTD was observed at the high power rate of SAR (10 W/kg). The results demonstrate that a rate of 2 W/kg displays no adverse impact on LTP and LTD, while 10 W/kg leads to significant effects on the electrophysiological parameters, which can be clearly distinguished from the stress derived background. Our findings suggest that UMTS exposure with SAR in the range of 2 W/kg is not harmful to critical markers for memory storage and memory consolidation, however, an influence of UMTS at high energy absorption rates (10 W/kg) cannot be excluded.

Unified patch clamp protocol for the characterization of Pannexin 1 channels in isolated cells and acute brain slices.

In the central nervous system, Pannexin 1 (Panx1) channels are implicated in a variety of physiological and pathological conditions. One of the prerequisites to enlighten the role of Panx1 is the development and standardization of reliable methods. Here, we address the applicability of voltage clamp protocols to identify Panx1 channel mediated currents in neurons of acutely dissected brain slices. We improved an established protocol and report on a modified paradigm that robustly evokes Panx1 channel currents. Crucial advances are the use of physiologic ion gradient conditions and a preconditioning step of depolarizing membrane potential ramps of long duration. This new paradigm provides significant impact on membrane current generation at hypo- and depolarized holding potential steps post voltage ramp preconditioning in heterologous expression systems and primary hippocampal CA1 neurons of mouse brain slices in vitro. Finally, we demonstrate that under these conditions the analysis of tail currents elicited by repolarization of the cells from preconditioning holding potential depolarization permits an independent method to isolate Panx1 mediated channel activity. In summary, this study provides a comprehensive methodological improvement in the biophysical analysis of Panx1 channels with a particular focus on investigations under physiological conditions in complex tissues.

In vitro motility of liver connexin vesicles along microtubules utilizes kinesin motors.

Trafficking of the proteins that form gap junctions (connexins) from the site of synthesis to the junctional domain appears to require cytoskeletal delivery mechanisms. Although many cell types exhibit specific delivery of connexins to polarized cell sites, such as connexin32 (Cx32) gap junctions specifically localized to basolateral membrane domains of hepatocytes, the precise roles of actin- and tubulin-based systems remain unclear. We have observed fluorescently tagged Cx32 trafficking linearly at speeds averaging 0.25 µm/s in a polarized hepatocyte cell line (WIF-B9), which is abolished by 50 µM of the microtubule-disrupting agent nocodazole. To explore the involvement of cytoskeletal components in the delivery of connexins, we have used a preparation of isolated Cx32-containing vesicles from rat hepatocytes and assayed their ATP-driven motility along stabilized rhodamine-labeled microtubules in vitro. These assays revealed the presence of Cx32 and kinesin motor proteins in the same vesicles. The addition of 50 µM ATP stimulated vesicle motility along linear microtubule tracks with velocities of 0.4-0.5 µm/s, which was inhibited with 1 mM of the kinesin inhibitor AMP-PNP (adenylyl-imidodiphosphate) and by anti-kinesin antibody but only minimally affected by 5 µM vanadate, a dynein inhibitor, or by anti-dynein antibody. These studies provide evidence that Cx32 can be transported intracellularly along microtubules and presumably to junctional domains in cells and highlight an important role of kinesin motor proteins in microtubule-dependent motility of Cx32.

Pannexin channels are not gap junction hemichannels.

Pannexins, a class of membrane channels, bear significant sequence homology with the invertebrate gap junction proteins, innexins and more distant similarities in their membrane topologies and pharmacological sensitivities with the gap junction proteins, connexins. However, the functional role for the pannexin oligomers, or pannexons, is different from connexin oligomers, the connexons. Many pannexin publications have used the term "hemichannels" to describe pannexin oligomers while others use the term "channels" instead. This has led to confusion within the literature about the function of pannexins that promotes the idea that pannexons serve as gap junction hemichannels and thus have an assembly and functional state as gap junctional intercellular channels. Here we present the case that unlike the connexin gap junction intercellular channels, so far, pannexin oligomers have repeatedly been shown to be channels that are functional in single membranes, but not as intercellular channel in appositional membranes. Hence, they should be referred to as channels and not hemichannels. Thus, we advocate that in the absence of firm evidence that pannexins form gap junctions, the use of the term "hemichannel" be discontinued within the pannexin literature.

Anti-inflammatory effects of the anticonvulsant drug levetiracetam on electrophysiological properties of astroglia are mediated via TGFß1 regulation.

BACKGROUND AND PURPOSE: The involvement of astrocytes as immune-competent players in inflammation and the pathogenesis of epilepsy and seizure-induced brain damage has recently been recognized. In clinical trials and practice, levetiracetam (LEV) has proven to be an effective antiepileptic drug (AED) in various forms of epileptic seizures, when applied as mono- or added therapy. Little is known about the mechanism(s) of action of LEV. Evidence so far suggests a mode of action different from that of classical AEDs. We have shown that LEV restored functional gap junction coupling and basic membrane properties in an astrocytic inflammatory model in vitro. EXPERIMENTAL APPROACH: Here, we used neonatal rat astrocytes co-cultured with high proportions (30%) of activated microglia or treated with the pro-inflammatory cytokine interleukin-1ß to provoke inflammatory responses. Effects of LEV (50 µg·mL⁻¹) on electrophysiological properties of astrocytes (by whole cell patch clamp) and on secretion of TGFß1 (by (ELISA)) were studied in these co-cultures. KEY RESULTS: LEV restored impaired astrocyte membrane resting potentials via modification of inward and outward rectifier currents, and promoted TGFß1 expression in inflammatory and control co-cultures. Furthermore, LEV and TGFß1 exhibited similar facilitating effects on the generation of astrocyte voltage-gated currents in inflammatory co-cultures and the effects of LEV were prevented by antibody to TGFß1. CONCLUSIONS AND IMPLICATIONS: Our data suggest that LEV is likely to reduce the harmful spread of excitation elicited by seizure events within the astro-glial functional syncytium, with stabilizing consequences for neuronal-glial interactions.

Pannexin 1 constitutes the large conductance cation channel of cardiac myocytes.

A large conductance (∼300 picosiemens) channel (LCC) of unknown molecular identity, activated by Ca(2+) 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 Ca(2+) release can trigger action potentials, suggesting that Panx1 channels potentially promote arrhythmogenic activities.