Gap Junctions and Hemichannels Composed of Connexins and Pannexins Mediate the Secondary Brain Injury following Intracerebral Hemorrhage

Intracerebral hemorrhage (ICH) is a devastating disease with high mortality and morbidity; the mortality rate ranges from 40% at 1 month to 54% at 1 year; only 12%−39% achieve good outcomes and functional independence. ICH affects nearly 2 million patients worldwide annually. In ICH development, the blood leakage from ruptured vessels generates sequelae of secondary brain injury (SBI). This mechanism involves activated astrocytes and microglia, generation of reactive oxygen species (ROS), the release of reactive nitrogen species (RNS), and disrupted blood brain barrier (BBB). In addition, inflammatory cytokines and chemokines, heme compounds, and products of hematoma are accumulated in the extracellular spaces, thereby resulting in the death of brain cells. Recent evidence indicates that connexins regulate microglial activation and their phenotypic transformation. Moreover, communications between neurons and glia via gap junctions have crucial roles in neuroinflammation and cell death. A growing body of evidence suggests that, in addition to gap junctions, hemichannels (composed of connexins and pannexins) play a key role in ICH pathogenesis. However, the precise connection between connexin and pannexin channels and ICH remains to be resolved. This review discusses the pathological roles of gap junctions and hemichannels in SBI following ICH, with the intent of discovering effective therapeutic options of strategies to treat ICH.

Osteocytic Connexin Hemichannels Modulate Oxidative Bone Microenvironment and Breast Cancer Growth

Osteocytes, the most abundant bone cell types embedded in the mineral matrix, express connexin 43 (Cx43) hemichannels that play important roles in bone remodeling and osteocyte survival. Estrogen deficiency decreases osteocytic Cx43 hemichannel activity and causes a loss in osteocytes’ resistance to oxidative stress (OS). In this study, we showed that OS reduced the growth of both human (MDA-MB-231) and murine (Py8119) breast cancer cells. However, co-culturing these cells with osteocytes reduced the inhibitory effect of OS on breast cancer cells, and this effect was ablated by the inhibition of Cx43 hemichannels. Py8119 cells were intratibially implanted in the bone marrow of ovariectomized (OVX) mice to determine the role of osteocytic Cx43 hemichannels in breast cancer bone metastasis in response to OS. Two transgenic mice overexpressing dominant-negative Cx43 mutants, R76W and Δ130-136, were adopted for this study; the former inhibits gap junctions while the latter inhibits gap junctions and hemichannels. Under normal conditions, Δ130-136 mice had significantly more tumor growth in bone than that in WT and R76W mice. OVX increased tumor growth in R76W but had no significant effect on WT mice. In contrast, OVX reduced tumor growth in Δ130-136 mice. To confirm the role of OS, WT and Δ130-136 mice were administered the antioxidant N-acetyl cysteine (NAC). NAC increased tumor burden and growth in Δ130-136 mice but not in WT mice. Together, the data suggest that osteocytes and Cx43 hemichannels play pivotal roles in modulating the oxidative microenvironment and breast cancer growth in the bone.

Efferent neurons control hearing sensitivity and protect hearing from noise through the regulation of gap junctions between cochlear supporting cells

It is critical for hearing that the descending cochlear efferent system provide a negative feedback to hair cells to regulate hearing sensitivity and provide the protection of hearing from noise. Here, we report that the medial olivocochlear (MOC) efferent nerves, which project to outer hair cells (OHCs), also could innervate OHC surrounding supporting cells (SCs) to regulate hearing sensitivity. MOC nerve fibers are cholinergic and acetylcholine (ACh) is a primary neurotransmitter. MOC nerve endings, presynaptic vesicular acetylcholine transporters (VAChT), and postsynaptic ACh receptors were visible in SCs and the SC area. Application of ACh in the SC could evoke a typical inward current, which reduced gap junctions (GJs) between SCs and consequently declined OHC electromotility, which is an active cochlear amplification and can increase hearing sensitivity. This indirect, GJ-mediated inhibition enhanced the direct inhibition of ACh on OHC electromotility but had long-lasting influence. In vivo experiments further demonstrated that deficiency of this GJ-mediated efferent pathway declined the regulation of active cochlear amplification and compromised the protection against noise. In particular, distortion production otoacoustic emission (DPOAE) showed a delayed reduction after noise exposure. Our findings reveal a new pathway for the MOC efferent system via innervating SCs to control active cochlear amplification and hearing sensitivity. These data also suggest that this GJ-mediated efferent pathway may play a critical role in the long-term efferent inhibition and is required for protecting hearing from noise trauma.

First Responders to Hyperosmotic Stress in Murine Astrocytes: Connexin 43 Gap Junctions Are Subject to an Immediate Ultrastructural Reorganization

In a short-term model of hyperosmotic stress, primary murine astrocytes were stimulated with a hyperosmolar sucrose solution for five minutes. Astrocytic gap junctions, which are mainly composed of Connexin (Cx) 43, displayed immediate ultrastructural changes, demonstrated by freeze–fracture replica immunogold labeling: their area, perimeter, and distance of intramembrane particles increased, whereas particle numbers per area decreased. Ultrastructural changes were, however, not accompanied by changes in Cx43 mRNA expression. In contrast, transcription of the gap junction regulator zonula occludens (ZO) protein 1 significantly increased, whereas its protein expression was unaffected. Phosphorylation of Serine (S) 368 of the Cx43 C–terminus has previously been associated with gap junction disassembly and reduction in gap junction communication. Hyperosmolar sucrose treatment led to enhanced phosphorylation of Cx43S368 and was accompanied by inhibition of gap junctional intercellular communication, demonstrated by a scrape loading-dye transfer assay. Taken together, Cx43 gap junctions are fast reacting elements in response to hyperosmolar challenges and can therefore be considered as one of the first responders to hyperosmolarity. In this process, phosphorylation of Cx43S368 was associated with disassembly of gap junctions and inhibition of their function. Thus, modulation of the gap junction assembly might represent a target in the treatment of brain edema or trauma.

Advances of Traditional Chinese Medicine Regulating Connexin43 in the Prevention and Treatment of Myocardial Infarction

Gap junctions are the main form of interaction between cardiomyocytes, through which the electrochemical activities between cardiomyocytes can be synchronized to maintain the normal function of the heart. Connexins are the basis of gap junctions. Changes in the expression, structural changes (e.g., phosphorylation and dephosphorylation), and distribution of connexins can affect the normal electrophysiological activities of the heart. Myocardial infarction (MI) and concurrent arrhythmia, shock, or heart failure can endanger life. The structural and functional damage of connexin (Cx) 43 in cardiomyocytes is a central part of the pathological progression of MI and is one of the main pathological mechanisms of arrhythmia after MI. Therefore, increasing Cx43 expression has become one of the main measures to prevent MI. Also, intervention in Cx43 expression can improve the structural and electrical remodeling of the myocardium to improve MI prognosis. Here, research progress of Cx43 in MI and its prevention and treatment using Traditional Chinese Medicine formulations is reviewed.

New Insights on the Role of Connexins and Gap Junctions Channels in Adipose Tissue and Obesity

Due to the inability to curb the excessive increase in the prevalence of obesity and overweight, it is necessary to comprehend in more detail the factors involved in the pathophysiology and to appreciate more clearly the biochemical and molecular mechanisms of obesity. Thus, understanding the biological regulation of adipose tissue is of fundamental relevance. Connexin, a protein that forms intercellular membrane channels of gap junctions and unopposed hemichannels, plays a key role in adipogenesis and in the maintenance of adipose tissue homeostasis. The expression and function of Connexin 43 (Cx43) during the different stages of the adipogenesis are differentially regulated. Moreover, it has been shown that cell–cell communication decreases dramatically upon differentiation into adipocytes. Furthermore, inhibition of Cx43 degradation or constitutive overexpression of Cx43 blocks adipocyte differentiation. In the first events of adipogenesis, the connexin is highly phosphorylated, which is likely associated with enhanced Gap Junction (GJ) communication. In an intermediate state of adipocyte differentiation, Cx43 phosphorylation decreases, as it is displaced from the membrane and degraded through the proteasome; thus, Cx43 total protein is reduced. Cx is involved in cardiac disease as well as in obesity-related cardiovascular diseases. Different studies suggest that obesity together with a high-fat diet are related to the production of remodeling factors associated with expression and distribution of Cx43 in the atrium.

Development of an in Vitro Assay to Assess Gap Junction Activity in Cumulus-Oocyte Complexes (COC) in the Rat

<p>The capacity of an oocyte to mature during ovarian follicular development is a key process in reproductive biology. Bidirectional communication between mammalian oocytes and their associated follicular somatic cells (cumulus-cells) is essential for oocyte maturation. Historically, studies examining the control of ovarian follicular development focused mainly on the endocrine (external) signalling but recently intraovarian (paracrine) regulation has also been shown to be important. In addition, signalling via gap junctions between follicular cells had also been crucial for oocyte maturation and follicular development. In antral follicles, gap junction activity between the oocyte and adjacent cumulus cells first increase during follicular growth and shortly before ovulation they decrease as the oocyte resumes meiosis once more before ovulation. The range of factors that modulate gap junction activity of oocyte-cumulus cell complexes (COC) is largely unknown. The aims of these studies were to develop an assay to assess the rate of transfer of low molecular weight materials from cumulus cells to the oocyte via gap junctions. The first objective was to validate a bioassay by which to test the effects of hormones, second messengers, and growth factors on gap junction activity in rat cumulus-oocyte complexes. In this study, COCs were collected from antral follicles of untreated post-pubertal Sprague Dawley rats. Gap junction activity was measured in the presence or absence of different treatments using the fluorescence dye, Calcein-AM and in the presence of a phosphodiesterase type 3 inhibitor (PDE3) milrinone. Transfer of the calcein dye from cumulus cells into the oocyte was measured at various times using CRAIC fluorescence system. The results showed that removal of the COCs from their follicular environments disrupted the gap junction activity which recovered over time in culture media. COC were sensitive to changes in pH concentration and gap junction activity could be blocked with 8 ocatnol-1 but not carbenoxolone. Treating rat COCs with dibutyryl cAMP or agents that maintained or increased intracellular cAMP levels like milrinone or forskolin were unable to modulate gap junction activity. Further, the combined effect of the oocyte-derived growth factors: growth differentiating factor 9 (GDF9) with bone morphogenetic protein 15 (BMP15) was also unable to modulate the rate of calcein dye transfer from cumulus cells to the oocyte. Ovarian steroids such as oestradiol and testosterone by themselves were unable to modulate the gap junction activity of rat COC but the combined treatment of testosterone plus forskolin or testosterone plus forskolin plus insulin-like growth factor 1 (IGF-1) increased the rate of dye transfer from cumulus cells to the oocyte. In conclusion, a fluorescence dye transfer assay was developed to measure the effects of different treatments on gap junction activity in rat COC. Under in vitro conditions, it was established that the combination of steroid and cAMP stimulators or a steroid, cAMP stimulator with IGF1 but not these reagents individually could enhance the recovery of gap junction function in rat COC. The outcomes of these experiments may help to provide new insights into developing suitable in vitro conditions, for the in vitro maturation of mammalian oocytes. Also, the newly developed assay may serve as a useful in vitro model to evaluate the effects of hormones, nutritional supplements and other factors on COC functions.</p>

Development of an in Vitro Assay to Assess Gap Junction Activity in Cumulus-Oocyte Complexes (COC) in the Rat

<p>The capacity of an oocyte to mature during ovarian follicular development is a key process in reproductive biology. Bidirectional communication between mammalian oocytes and their associated follicular somatic cells (cumulus-cells) is essential for oocyte maturation. Historically, studies examining the control of ovarian follicular development focused mainly on the endocrine (external) signalling but recently intraovarian (paracrine) regulation has also been shown to be important. In addition, signalling via gap junctions between follicular cells had also been crucial for oocyte maturation and follicular development. In antral follicles, gap junction activity between the oocyte and adjacent cumulus cells first increase during follicular growth and shortly before ovulation they decrease as the oocyte resumes meiosis once more before ovulation. The range of factors that modulate gap junction activity of oocyte-cumulus cell complexes (COC) is largely unknown. The aims of these studies were to develop an assay to assess the rate of transfer of low molecular weight materials from cumulus cells to the oocyte via gap junctions. The first objective was to validate a bioassay by which to test the effects of hormones, second messengers, and growth factors on gap junction activity in rat cumulus-oocyte complexes. In this study, COCs were collected from antral follicles of untreated post-pubertal Sprague Dawley rats. Gap junction activity was measured in the presence or absence of different treatments using the fluorescence dye, Calcein-AM and in the presence of a phosphodiesterase type 3 inhibitor (PDE3) milrinone. Transfer of the calcein dye from cumulus cells into the oocyte was measured at various times using CRAIC fluorescence system. The results showed that removal of the COCs from their follicular environments disrupted the gap junction activity which recovered over time in culture media. COC were sensitive to changes in pH concentration and gap junction activity could be blocked with 8 ocatnol-1 but not carbenoxolone. Treating rat COCs with dibutyryl cAMP or agents that maintained or increased intracellular cAMP levels like milrinone or forskolin were unable to modulate gap junction activity. Further, the combined effect of the oocyte-derived growth factors: growth differentiating factor 9 (GDF9) with bone morphogenetic protein 15 (BMP15) was also unable to modulate the rate of calcein dye transfer from cumulus cells to the oocyte. Ovarian steroids such as oestradiol and testosterone by themselves were unable to modulate the gap junction activity of rat COC but the combined treatment of testosterone plus forskolin or testosterone plus forskolin plus insulin-like growth factor 1 (IGF-1) increased the rate of dye transfer from cumulus cells to the oocyte. In conclusion, a fluorescence dye transfer assay was developed to measure the effects of different treatments on gap junction activity in rat COC. Under in vitro conditions, it was established that the combination of steroid and cAMP stimulators or a steroid, cAMP stimulator with IGF1 but not these reagents individually could enhance the recovery of gap junction function in rat COC. The outcomes of these experiments may help to provide new insights into developing suitable in vitro conditions, for the in vitro maturation of mammalian oocytes. Also, the newly developed assay may serve as a useful in vitro model to evaluate the effects of hormones, nutritional supplements and other factors on COC functions.</p>