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Connexin and pannexin hemichannels are regulated by redox potentials. Cxs and Panx- hemichannels in peripheral and central chemosensing in mammals. Opening of pannexin and connexin based-channels increases the excitability of nodose ganglion sensory neurons. Hemichannels; from the molecule to the function. Carbon Monoxide CO a novel inhibitor of connexin hemichannels. Petrosal ganglion: a more complex role than originally imagined.

Neural reflex regulation of systemic inflammation: potential new targets for sepsis therapy. ATP is required and advances cytokine-induced gap junction formation in microglia in vitro. Gap junction channels and hemichannels in the CNS: Regulation by signaling molecules. Impact of microglial activation on astroglial connexin expression and function in brain inflammation. ISBN: Nitric oxide signaling in the retina: What have we learned in two decades?

Release of gliotransmitters through astroglial connexin 43 hemichannels is necessary for fear memory consolidation in the basolateral amygdala. Shifting from hypoxia to hyperoxia to assess the peripheral chemosensory drive of ventilation. Adv Exp Med Biol Biphasic effect of linoleic acid on connexin 46 hemichannels. Connexin in lens physiology and cataract formation. Chronic hypoxia upregulates adenosine 2a receptor expression in chromaffin cells via hypoxia inducible factor-2a: Role in modulating secretion.

Immunosensory signaling by carotid body chemoreceptors. Respir Physiol Neurobiol Voltage-dependent facilitation of Cx46 hemichannels. Cell membrane permeabilization via connexin hemichannels in living and dying cells. Fifty years of progress in carotid body physiology. Cardio-vascular responses to hyperoxic withdrawal of arterial chemosensory drive. Study Undergraduate Why study here? Research Publications and Research Outputs A role for connexins in inflammatory dis Advanced search.

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Ovideo-Orta ; B. Kwak; W. CRC Press, CRC Press. Here we show that a low concentration of tonabersat can attenuate ATP release from connexin43 hemichannels following injury and reperfusion conditions in vitro , indicating direct hemichannel block. High concentrations of tonabersat, however, reduced GJ coupling in a scrape-load dye-spread model down to about half , with sustained long-term exposure causing internalization and degradation of membrane connexin43 plaques via the lysosomal pathway.

This study demonstrates that tonabersat is not only acting on connexin26 GJ formation or glia neurons as previously reported, but that it can also inhibit connexin43 hemichannels in other cell types and can partially regulate connexin43 GJ coupling at higher concentrations. Carbenoxolone CBX; Sigma , a nonspecific inhibitor of connexin and pannexin channels, and a broad-spectrum inhibitor of GJ channels [ 64 ], was dissolved in ultrapure H 2 O at a stock concentration of 10 mM.

Briefly, 2. Cells were incubated in culture medium overnight. Hypoxic, acidic, ion-shifted Ringer injury HAIR solution that mimics ionic concentrations and acid—base shifts of the interstitial space in hypoxic—ischemic brains [ 65 ] was used to trigger hemichannel opening. In ischemia or ischemia—reperfusion models, hCMVEC were incubated in standard Ringer solution only as a negative control. Injury solution or injury solution followed by standard Ringer solution was used as a positive control for ischemia injury and ischemia—reperfusion models, respectively. At the end of each experiment incubating solutions were removed and immediately placed on ice.

Standard curves were generated in each experiment from an ATP standard 0— nM to quantify the concentration of ATP in the test samples. Treatment groups had a sample size of 2 wells per experiment, and the concentration of ATP in each sample was measured in triplicates over 10 repeated readings. The internal pipette solution contained mM KCl, 10 mM sodium aspartate, 0. A voltage clamp was used to elicit hemichannel currents by holding the membrane potential Vm at 0 mV for ms, followed by positive voltage steps of 10 mV to 80 mV in 10 mV increments at 15 s each.

Voltage-clamp recording and drug perfusion were conducted simultaneously. Tonabersat was washed out for 5 min before a 1-min recording of the washout period. The data were low-pass filtered at 1 kHz and digitized at sampling intervals of 0. Once confluency was reached, the cells were preincubated in tonabersat 0. The cells were then incubated in 0. Three images within each well from 3 independent experiments were taken for analysis. Cells were rinsed 3 times with PBS containing 0.

Real-time reverse transcription polymerase chain reaction PCR was used to determine the relative levels of connexin43 mRNA. Cells without treatment were also used as a further control. Primer sequences for human connexin43, GenBank Accession No. The following day, cells were washed twice with PBS and incubated in culture medium with tonabersat 0.

To induce light damage rats were exposed to bright light for 24 h. Light exposure started consistently around a. The light source covered broadband fluorescence, from to nm, with no extra heat generated. This broadband fluorescence light has been used in previous studies on albino rats [ 54 , 70 ]. Animals were able to move freely in the cage and had access to food and water at libitum. The light-damage process was continuous otherwise, except for a brief period during tonabersat injection. Light-damaged SD rats with vehicle only injection were used as sham control animals.

There were 6 animals per group with 12 eyes per group analyzed. Tonabersat was administered as a double injection in the rat peritoneum, the first injection 2 h into the h light-damage period and the second injection immediately after the h light-damage procedure. Electroretinogram recording ERG for assessment of retinal function was performed as described previously [ 54 ].

SD rats were dark-adapted overnight for 12 to 14 h before the ERG recording. The ERG baseline was recorded before light damage and after 24 h, and 7 and 14 days of intense light damage. The active electrode was U-shaped and was kept in contact with the eyelid—cornea interface. The inactive electrode was V shaped and hooked around the front teeth and was in contact with the wet tongue. Full-field ERG responses were elicited by a twin-flash 0.

Recordings were performed in a Faraday cage to reduce electrical noise. The flash unit triggered paired flashes that had identical luminous energy.

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The rod and cone mixed response were recorded after the initial flash, and the response was subtracted from the second flash to represent function from the cone photoreceptors only. Published algorithms were used in the analysis of the amplitudes of a-wave and b-wave ERG for each eye [ 50 , 54 ]. A spectral domain optical coherence tomography Micron IV; Phoenix Research Labs, Pleasanton, CA, USA approach was used to investigate the thickness of the retinal layers in tonabersat or sham-treated light-damaged rats. This procedure was executed immediately after ERG recordings under the same anesthesia and pupil dilation [ 54 ].

StreamPix 6 software, version 7. Ten B-scans, acquired 2 mm from the optic nerve in the dorsal retina, were taken and averaged.

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Images were analyzed using InSight software, version 1. The total thickness of the retina was measured from the retinal pigment epithelium to the edge of the nerve-fiber layer. The outer nuclear layer was measured from the border of the retinal pigment epithelium to the outer plexiform layer interface. Tonabersat inhibits hemichannels and inhibits adenosine triphosphate ATP release from human cerebral microvascular endothelial cells hCMVEC in injury and reperfusion.

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  • Quantification of total extracellular ATP release is presented as a percentage of the injury control for each treatment group. C In the absence of probenecid there was no significant reduction in extracellular ATP in the presence of 0. ATP is quantified as a percentage of the injury—reperfusion IR control. C black dotted lines and O red dotted lines indicate closed and open state, respectively.

    The values next to the lines indicate starting current. When compared with the injury group As has been reported previously [ 71 ], Peptide5 significantly reduced ATP release to Peptide5 in combination with probenecid is synergistic and reduced ATP release almost to the same level as CBX [ 71 ]. We compared these results with the effect of tonabersat in combination with probenecid Fig.

    ATP release was significantly reduced when probenecid 1 mM was combined with tonabersat at concentrations of 0. This effect was concentration dependent—tonabersat at the lowest concentration, 0. However, during the reperfusion period, there was no evidence for pannexin channel opening and tonabersat was as effective as Peptide5 whether probenecid was present or not Fig.

    Baseline ATP release was slightly higher in this experimental group where the medium had been replaced after 2 h of ischemia in order to mimic reperfusion. Peptide5 significantly reduced ATP release to A combined treatment of Peptide5 and probenecid also significantly reduced ATP compared with reperfusion injury As for Peptide5, addition of probenecid with tonabersat had no further effect under reperfusion conditions.

    To confirm that ATP release was from healthy cells, the effect of tonabersat 0. Furthermore, there was no significant reduction in cell viability in response to vehicle 0. A high level of activity was observed at positive membrane potentials in aCSF Fig. The single channel unitary currents were partially recoverable following washout of Tonabersat and return to normal aCSF Fig. Conductances for 70 mV before, during, and after tonabersat were Functional effect of tonabersat on intercellular communication in human cerebral microvascular endothelial cells hCMVEC in vitro.

    A Examples of Lucifer Yellow dye spread to neighboring cells via coupled gap junction as seen after scrape loading. Left-hand panels are phase contrast imaging showing cells and the scrape line; the right-hand panels show the dye uptake and spread. Tonabersat treatment for 1 h has minimal effect on coupling, but dye spread is reduced after tonabersat treatment for 2 h. Treatment up to 1 h before scrape loading did not significantly affect dye spread but the amount of uncoupling statistically significant increased with Tonabersat treatment times from 2 to 24 h.

    Tonabersat internalizes and degrades connexin43 gap junction plaques via the lysosomal pathway in ARPE cells. B, C Normalized to untreated control and expressed as a percentage. NH 4 Cl prevents junctional degradation, indicating that it would otherwise occur via the lysosomal pathway. No significant difference in connexin43 labeling was present between untreated control and 10 mM NH4Cl or vehicle control [0.

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    H Tonabersat has no effect on connexin43 transcription. We then proposed that tonabersat at higher doses may facilitate turnover of connexin43 plaques from the plasma membrane. To test this, we immobilized the lysosomal degradation pathway in ARPE cells using ammonium chloride NH 4 Cl , a weak base lysosome inhibitor [ 68 ]. In addition, there was now dense labeling visible around the perinucleus in what appears to be the Golgi body Fig. Treatment with NH 4 Cl for 1 h, or the vehicle control 0. However, following treatment with tonabersat in the presence of 10 mM NH 4 Cl, total connexin43 labeling was significantly increased by Following 6 h of treatment in tonabersat and 10 mM NH 4 Cl, punctate connexin43 labeling was distributed throughout the cell Fig.

    In contrast to the 1-h treatment period, there was no visible connexin43 labeling between cell-to-cell contacts Fig.

    Connexin Cell Communication Channels Roles In The Immune System And Immunopathology

    Treatment with NH 4 Cl for 6 h, or 0. However, 6-h treatment with Tonabersat in the presence of 10 mM NH 4 Cl significantly increased the total amount of connexin43 labeling by To confirm that tonabersat-mediated reduction in connexin43 plaques seen in Figure 3 A was caused by internalization, and not a downregulation in connexin43 mRNA transcription, we used real-time reverse transcriptase PCR to compare the relative differences in connexin43 mRNA following a 1-h treatment with tonabersat.

    Tonabersat treatment reduces the loss of retinal function in the bright-light damage model 6 animals per group, 12 eyes analyzed. Representative electroretinogram ERG waveforms for light intensities ranging from —3. LD and tonabersat-treated animals in a light-damaged rat retina model. Tonabersat leads to significantly improved a-wave amplitude compared with A sham-treated and B b-wave amplitude compared with sham-treated assessed at 2 weeks. Further analysis showed that the mixed b-wave amplitude of the ERG was also better preserved as a function of time post-treatment.

    The mixed b-wave that reflects the inner retina function was preserved in tonabersat-treated rats as a function of time compared with sham rats at intensities —3. There was a significant difference in b-wave amplitude 24 h post-treatment at —2. Interestingly, the cone PII amplitude observed at the highest flash intensity, 2. Tonabersat treatment in the light-damaged rat model prevents the loss of both retinal and choroidal thickness. Representative fundus and optical coherence tomography OCT images of sham-treated animal retina are seen in A and tonabersat-treated animal retina in B 2 weeks postinjury and treatment.

    Both the outer nucleus layer and choroid thickness is reduced in the injured but sham-treated animals, but retinal thickness was preserved with tonabersat treatment. A major finding of this study is that tonabersat significantly inhibits connexin hemichannel opening. While immunohistochemistry indicated internalization of connexin43 plaques with tonabersat treatment, based on data from the scrape-loading assay it appears that a number of functional GJ channels remain dispersed within the membrane.

    This was consistent with reduced damage seen in an established model of in vivo injury, the retinal bright light damage model, previously used to show the effects of Peptide5 connexin hemichannel block on reducing inflammation and preserving retinal function and integrity [ 50 ].

    Extracellular calcium levels fall following brain injury [ 76 ] and at the same time the injured brain is subject to acidosis [ 77 ]. The injured site is also subject to glucose and oxygen deprivation that leads to energy failure and oxidative stress. Therefore, in our in vitro hemichannel assay, we used hypoxic acidic ion shifted Ringer solution [ 78 ] as it most closely resembles the complex changes of an acute insult that extends to reperfusion injury in vivo [ 40 , 71 ].

    We used the hypoxic acidic ion-shifted Ringer solution to stimulate hemichannels to open in hCMVEC, mimicking injury conditions and quantified the amount of ATP released into the extracellular space as a function of channel activity. During ischemia, ATP release from connexin hemichannels was seen to be 1. Using the in vitro injury model described above then, we have shown a concentration-dependent inhibition of connexin hemichannels by tonabersat.

    As higher tonabersat concentrations were not linked to cell toxicity, it is possible that tonabersat may have dual effects during ischemic injury whereby connexin hemichannels are targeted for inhibition, but pannexin channels may be triggered to open in an off-target effect. While further research is required to confirm any action of tonabersat on pannexin channels, our present data suggest that a low concentration of tonabersat combined with a pannexin channel blocker, such as probenecid, may be most effective during ischemic injury in vitro.

    Ischemia is not a single event, and cellular injury to peritraumatic regions is attributed to lesion spread postischemia [ 81 ]. Connexin43 hemichannels have been shown to be upregulated 4 to 8 h after injury in several in vivo models [ 37 , 43 , 46 , 53 , 54 ], and this has been linked to cell swelling [ 82 , 83 ], vessel leak, inflammation, and cell death [ 16 , 37 , 84 ].

    As discussed above, connexin hemichannels open in response to conditions that are typical of ischemia, but whether hemichannels open both during and after the initial ischemic injury has been debated. Therefore, we also created post-ischaemia—reperfusion conditions in vitro by returning to normal physiologic conditions after 2 h in HAIR solution. Postischemia, connexin hemichannels accounted for almost all ATP release Fig. These data suggest that a low concentration of tonabersat alone can inhibit connexin hemichannel-mediated ATP release postischemia.

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    • Tonabersat-mediated inhibition of ATP release through connexin43 hemichannels could have implications for attenuating inflammation and inflammasome activation postinjury for review see [ 40 , 84 ]. Intracellular ATP is crucial for energy transfer [ 85 ], but a rapid increase of extracellular ATP upon ischemia [ 86 ], spinal cord injury [ 42 ], or epilepsy-initiated seizures [ 87 ] can activate ATP-gated receptors i. This event is associated, in particular, with the activation of the NLR pyrin domain containing 3 inflammasome protein complex, a major component of the innate immune system [ 91 ].

      These proinflammatory cytokines can induce secondary damage at the systemic level sepsis [ 94 ], as well as local lesion spread in the peritraumatic region, including proliferation of astrocytes astrogliosis [ 95 ]. In an in vivo bright light-induced retina damage model, the inflammatory response starts in the choroid before leading to oxidative stress in the retina, and those changes are associated with increased connexin43 expression [ 54 ].

      Loss of retinal function can be attributed to open connexin43 hemichannels as a low dose of connexin43 mimetic Peptide5 that targets connexin43 hemichannels but not GJ [ 56 ], significantly improved functional outcomes [ 50 ]. Better functional outcomes for neurons in both the rod and cone phototransduction pathways was likely owing to reduced choroid inflammation and a suppressed glial-mediated inflammatory response bought about by hemichannel block.

      We have applied the same in vivo model used by [ 50 ] to show that tonabersat may also be protective against functional loss following retinal bright-light damage, reported to be a model for the dry form of age-related macular degeneration [ 96 ]. In most phase II clinical trials that involve tonabersat for the treatment of migraine, it has been used at a daily dosage of 20 to 80 mg [ 7 ].