Connexin 43 (Cx43) is thought to be present largely in the plasma membrane and its function solely to provide low resistance electrical connection between myocytes. A recent report suggested the presence of Cx43 in the mitochondria as well. We confirmed the presence of Cx43 in the mitochondria isolated from adult rat ventricles with the Cx43 immunoreactivity fractionating to the outer mitochondrial membrane. Mitochondrial Cx43 is mostly phosphorylated only detected by a phosphospecific antibody. Using a Ca ++ -sensitive electrode and Western blot, we showed that the gap junction inhibitors 18-β-glycyrrhetinic acid (β-GA), oleamide, and heptanol all induced concomitant release of Ca ++ and cytochrome C in isolated mitochondria whereas the inactive analog 18-β-glycyrrhizic acid failed to do so. In low density neonatal myocyte culture with no appreciable cell-cell contacts, β-GA induced apoptosis as assessed by TUNEL staining. Our results suggest a novel role of Cx43 as a regulator of mitochondrial physiology and myocyte apoptosis.

GABAergic interneurons of the spinal cord substantia gelatinosa regulate the transmission of nociceptive information. Hydrogen peroxide (H2O2) is likely a diffusible messenger contributing to the development of long-lasting pathological pain states after nerve injury. In this study, we examined the presynaptic effects of H2O2 on the inhibitory interneurons of mouse substantia gelatinosa (SG) using whole-cell patch-clamp recordings from spinal cord slices. H2O2 increased the frequency of GABAergic miniature inhibitory postsynaptic current (mIPSC) in a concentration-dependent (10-1000 microM) manner. The profound increase in mIPSC frequency was diminished by thapsigargin or cyclopiazonic acid suggesting that the intracellular stored pool was the source of presynaptic calcium. Further examination revealed the 2-aminoethoxydiphenil borate blockable inositol-(1,4,5) trisphosphate receptor (IP3R) regulated pool of stored calcium as the likely source. The phospholipase C (PLC) blocker, 1-(6-[([17beta]-3-methoxyestra-1,3,5[10]-trien-17-yl)-amino]hexyl)-1H-pyrrole-2,5-dione (U73122), did not block the frequency increase, which suggested that the site of action of H2O2 lies downstream in the IP3 signalling pathway, and nifedipine-sensitivity of the frequency increase indicated a possible role of calcium-induced calcium-release. However, a direct examination of L-type voltage-gated calcium channels (VGCC) demonstrated that H2O2 did not increase the calcium influx through these channels. The H2O2 effect on mIPSC frequency was markedly reduced in the opisthotonus (Opt) mutant mice with a known deletion in the IP3R1 gene. We demonstrated that H2O2 increased presynaptic activity in the GABAergic interneurons by the release of calcium from the IP3R-regulated intracellular pool. The presynaptic IP3R could emerge as a novel target for preventing H2O2-induced synaptic plasticity in substantia gelatinosa leading to pathological pain states.

The current study aimed to understand the anti-apoptotic effect of overexpressed gap junction forming protein connexin (Cx) 43 in C6 glioma cells. C6 cells exposed to hydrogen peroxide (H2O2) or staurosporine demonstrated morphological and biochemical changes consistent with apoptosis, whereas C6 cells expressing Cx43 demonstrated relative resistance to H2O2, but not to staurosporine. This selective protection against H2O2 was due to inhibition of caspase-3 activation in Cx43 expressing cells. siRNA knockdown experiments in rat primary astrocytes confirmed the presence of endogenous Cx43-mediated anti-apoptotic effect. Cx43 interacts with the upstream apoptosis signal-regulating kinase 1 known to mediate H2O2-induced apoptosis providing a possible mechanism for protection. These findings provided new evidence for regulation of the mitogen activated protein kinase pathway and apoptosis by Cx43 implicating this protein in intracellular signaling beyond its role as a gap junction forming protein on the plasma membrane.

BackgroundAirway instrumentation can evoke upper airway reflexes including bronchoconstriction and cough which can cause serious complications including airway trauma, laryngospasm or bronchospasm which may in turn lead to difficulty with ventilation and hypoxemia. These airway events are mediated in part by irritant-induced neuronal modulation of airway tone and cough responses. We investigated whether the commonly used anesthetic agents dexmedetomidine, lidocaine or remifentanil attenuated neuronal and airway smooth muscle responses in the upper airways of guinea pigs.MethodsThe ability of dexmedetomidine, lidocaine or remifentanil to attenuate direct cholinergic nerve stimulation, C-fiber stimulation or direct smooth muscle contraction were studied using isolated tracheal rings from male guinea pigs under four paradigms; (1) the magnitude of contractile force elicited by cholinergic electrical field stimulation (EFS); (2) the amount of acetylcholine released during cholinergic EFS; (3) the direct airway smooth muscle relaxation of a sustained acetylcholine-induced contraction and (4) the magnitude of C-fiber mediated contraction.ResultsDexmedetomidine (1–100 μM) and lidocaine (1 mM) attenuated cholinergic 30Hz EFS-induced tracheal ring contraction while remifentanil (10 μM) had no effect. Dexmedetomidine at 10 μM (p = 0.0047) and 100 μM (p = 0.01) reduced cholinergic EFS-induced acetylcholine release while lidocaine (10 μM-1 mM) and remifentanil (0.1–10 μM) did not. Tracheal ring muscle force induced by the exogenous addition of the contractile agonist acetylcholine or by a prototypical C-fiber analogue of capsaicin were also attenuated by 100 μM dexmedetomidine (p = 0.0061 and p = 0.01, respectively). The actual tracheal tissue concentrations of dexmedetomidine achieved (0.54–26 nM) following buffer application of 1–100 μM of dexmedetomidine were within the range of clinically achieved plasma concentrations (12 nM).ConclusionsThe α2 adrenoceptor agonist dexmedetomidine reduced cholinergic EFS-induced contractions and acetylcholine release consistent with the presence of inhibitory α2 adrenoceptors on the prejunctional side of the postganglionic cholinergic nerve-smooth muscle junction. Dexmedetomidine also attenuated both exogenous acetylcholine-induced contraction and C-fiber mediated contraction, suggesting a direct airway smooth muscle effect and an underlying mechanism for cough suppression, respectively.

Asthma, a common disorder associated with airway inflammation and hyperresponsiveness, remains a significant clinical burden in need of novel therapeutic strategies. Patients are increasingly seeking complementary and alternative medicine approaches to control their symptoms, including the use of natural products. Ginger, a natural product that we previously demonstrated acutely relaxes airway smooth muscle (ASM), has long been reported to possess anti-inflammatory properties, although a precise mechanistic understanding is lacking. In these studies, we demonstrate that chronic administration of whole ginger extract or 6-shogaol, a bioactive component of ginger, mitigates in vivo house dust mite antigen-mediated lung inflammation in mice. We further show that this decrease in inflammation is associated with reduced in vivo airway responsiveness. Utilizing in vitro studies, we demonstrate that 6-shogaol augments cAMP concentrations in CD4 cells, consistent with phosphodiesterase inhibition, and limits the induction of nuclear factor-κB signaling and the production of proinflammatory cytokines in activated CD4 cells. Sustained elevations in cAMP concentration are well known to inhibit effector T cell function. Interestingly, regulatory T cells (Tregs) utilize cAMP as a mediator of their immunosuppressive effects, and we demonstrate here that 6-shogaol augments the Treg polarization of naïve CD4 cells in vitro. Taken together with previous reports, these studies suggest that ginger and 6-shogaol have the potential to combat asthma via two mechanisms: acute ASM relaxation and chronic inhibition of inflammation.