Gap Junction Inhibitor Research Articles

Loading history changes the morphology and compressive force-induced expression of receptor activator of nuclear factor kappa B ligand/osteoprotegerin in MLO-Y4 osteocytes.

BackgroundIn this study, we investigated the effect of the mechanical loading history on the expression of receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) in MLO-Y4 osteocyte-like cells.MethodsThree hours after MLO-Y4 osteocytes were seeded, a continuous compressive force (CCF) of 31 dynes/cm2 with or without additional CCF (32 dynes/cm2) was loaded onto the osteocytes. After 36 h, the additional CCF (loading history) was removed for a recovery period of 10 h. The expression of RANKL, OPG, RANKL/OPG ratio, cell numbers, viability and morphology were time-dependently examined at 0, 3, 6 and 10 h. Then, the same additional CCF was applied again for 1 h to all osteocytes with or without the gap junction inhibitor to examine the expression of RANKL, OPG, the RANKL/OPG ratio and other genes that essential to characterize the phenotype of MLO-Y4 cells. Fluorescence recovery after photobleaching technique was also applied to test the differences of gap-junctional intercellular communications (GJIC) among MLO-Y4 cells.ResultsThe expression of RANKL and OPG by MLO-Y4 osteocytes without a loading history was dramatically decreased and increased, respectively, in response to the 1-h loading of additional weight. However, the expression of RANKL, OPG and the RANKL/OPG ratio were maintained at the same level as in the control group in the MLO-Y4 osteocytes with a loading history but without gap junction inhibitor treatment. Treatment of loading history significantly changed the capacity of GJIC and protein expression of connexin 43 (Cx43) but not the mRNA expression of Cx43. No significant difference was observed in the cell number or viability between the MLO-Y4 osteocyte-like cells with and without a loading history or among different time checkpoints during the recovery period. The cell morphology showed significant changes and was correlated with the expression of OPG, Gja1 and Dmp1 during the recovery period.ConclusionOur findings indicated that the compressive force-induced changes in the RANKL/OPG expression could be habituated within at least 11 h by 36-h CCF exposure. GJIC and cell morphology may play roles in response to loading history in MLO-Y4 osteocyte-like cells.

Unique cellular protrusions mediate breast cancer cell migration by tethering to osteogenic cells

Migration and invasion are key properties of metastatic cancer cells. These properties can be acquired through intrinsic reprogramming processes such as epithelial-mesenchymal transition. In this study, we discovered an alternative “migration-by-tethering” mechanism through which cancer cells gain the momentum to migrate by adhering to mesenchymal stem cells or osteoblasts. This tethering is mediated by both heterotypic adherens junctions and gap junctions, and leads to a unique cellular protrusion supported by cofilin-coated actin filaments. Inhibition of gap junctions or depletion of cofilin reduces migration-by-tethering. We observed evidence of these protrusions in bone segments harboring experimental and spontaneous bone metastasis in animal models. These data exemplify how cancer cells may acquire migratory ability without intrinsic reprogramming. Furthermore, given the important roles of osteogenic cells in early-stage bone colonization, our observations raise the possibility that migration-by-tethering may drive the relocation of disseminated tumor cells between different niches in the bone microenvironment.

Spreading depolarization evoked by endothelin-1 is inhibited by octanol but not by carbenoxolone

Spreading depolarization (SD) has been implicated in the pathogenesis of delayed cerebral ischemia (DCI) after subarachnoid hemorrhage. Endothelin-1 (ET-1) is a powerful trigger of SD and may be involved in DCI. The SD-causing mechanism is assumed to result from ET-1-induced microarterial spasm and ischemia. However, ET-1 is also a potent, astrocyte-specific gap junction (GJ) inhibitor. There are two competing hypotheses on the role of astrocytic GJs in SD. One postulates that they mediate SDs, since long-chain alcohols such as octanol inhibit GJs and inhibit SD at high concentrations. The other postulates that astrocytic GJs protect against SD and that their inhibition increases susceptibility to SD and SD velocity. Here, we found in rats that brain topical application of carbenoxolone, a more specific GJ inhibitor, failed to inhibit ET-1-induced SDs in vivo, whereas octanol, a less specific GJ inhibitor, partially blocked them at high concentrations. These results suggest that GJs are not required for initiation or propagation of ET-1-induced SDs, and that octanol inhibits SDs by effects unrelated to GJs. The results do not exclude that the specific inhibition of astrocytic GJs by ET-1 contributes to the generation of SDs, which should be further investigated in future studies.

Propofol protects against oxygen/glucose deprivation‑induced cell injury via gap junction inhibition in astrocytes.

Stroke is one of the leading causes of mortality and disability worldwide with limited clinical therapies available. The present study isolated primary astrocytes from the brains of rats and treated them with oxygen-glucose deprivation and re-oxygenation (OGD/R) to mimic hypoxia/reperfusion (H/R) injury in vitro to investigate stroke. It was revealed that propofol (2,6-diisopropylphenol), an intravenous sedative and anesthetic agent, protected against oxygen/glucose-deprivation (OGD) and induced cell injury. Furthermore, propofol exerted a protective effect by inhibiting gap junction function, which was also revealed to promote cell death in astrocytes. The present study further identified that propofol suppressed gap junction function by downregulating the protein expression levels of connexin43 (Cx43), which is one of the most essential components of gap junctions in astrocytes. In addition, when the expression levels of Cx43 were downregulated using small interfering RNA, OGD/R-induced cell death was decreased. Conversely, cell death was enhanced when Cx43 was overexpressed, which was reversed following propofol treatment. In summary, propofol protects against OGD-induced injury in astrocytes by decreasing the protein expression levels of Cx43 and suppressing gap junction function. The present study improved our understanding of how propofol protects astrocytes from OGD/R-induced injury.

Zoxazolamine-induced stimulation of cardiomyogenesis from embryonic stem cells is mediated by Ca2+, nitric oxide and ATP release

Ca2+-activated potassium (KCa) channels of small and intermediate conductance influence proliferation, apoptosis, and cell metabolism. We analysed whether prolonged activation of KCa channels by zoxazolamine (ZOX) induces differentiation of mouse embryonic stem (ES) cells towards cardiomyocytes. ZOX treatment of ES cells dose-dependent increased the number and diameter of cardiac foci, the frequency of contractions as well as mRNA expression of the cardiac transcription factor Nkx-2.5, the cardiac markers cardiac troponin I (cTnI), α-myosin heavy chain (α-MHC), ventricular myosin light chain-2 (MLC2v), and the pacemaker hyperpolarization-activated, cyclic nucleotide-gated 4 channel (HCN4). ZOX induced hyperpolarization of membrane potential due to activation of IKCa, raised intracellular Ca2+ concentration ([Ca2+]i) and nitric oxide (NO) in a Ca2+-dependent manner. The Ca2+ response to ZOX was inhibited by chelation of Ca2+ with BAPTA-AM, release of Ca2+ from intracellular stores by thapsigargin and the phospholipase C (PLC) antagonist U73,122. Moreover, the ZOX-induced Ca2+ response was blunted by the purinergic receptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) as well as the specific P2Y1 antagonist MRS 2,179, suggesting purinergic receptor-stimulated signal transduction. Consequently, ZOX initiated ATP release from differentiating ES cells, which was inhibited by the chloride channel inhibitor NPPB and the gap junction inhibitor carbenoxolone (CBX). The stimulation of cardiomyogenesis by ZOX was blunted by the nitric oxide synthase (NOS) inhibitor l-NAME, as well as CBX and NPPB. In summary, our data suggest that ZOX enhances cardiomyogenesis of ES cells by ATP release presumably through gap junctional hemichannels, purinergic receptor activation and intracellular Ca2+ response, thus promoting NO generation.

Abstract B19: Role of cell-cell communication in endocrine therapy-resistant breast cancer

Abstract Endocrine therapy is the mainstay for the treatment of estrogen-receptor positive (ER+) breast cancer. However, frequent emergence of endocrine resistance is a major clinical challenge. Cell-cell cooperation in the tumor microenvironment plays a vital role in acquiring resistance, but its role is poorly understood. We studied the interactions among endocrine-resistant (R) and -sensitive (S) breast cancer cells in the presence and absence of ER-targeted endocrine therapy. We measured changes in admixed cell population remodeling by dual fluorescent labeling of LCC1 (S) with eGFP (green fluorescent protein) and their derived resistant variant (selected against the antiestrogen fulvestrant) LCC9 (R) with mCherry (red fluorescent protein), respectively. Thus, unlike most prior studies, these are genetically related cells, rather than ER+ and ER- cell lines obtained from different patients. Our results revealed that “S”cells (LCC1) acquired the phenotype of “R” cells when cultured in a mixed population. A ratio of 5:1 of S:R cells in mixed population was sufficient to confer resistance to fulvestrant to the S cells within a six-day period. Furthermore, we show that acquirement of this resistant phenotype for the S cells was partially blocked by inhibition of either gap junctions or exosome secretion, suggesting a role of cell-cell contact as well as extracellular vesicles. Global proteomic analysis of pure and admixed S and R cell populations showed that R and S can alter key molecular features of each other when present in mixed cell populations. We used a Bayesian approach to fit single culture cell populations to infer density-dependent growth parameters (growth rate, carrying capacity) and a generalized Lotka-Volterra model to understand how S and R coculture populations may be depressing or supporting growth of the other. Our results identify a net mutualistic interaction between the susceptible and resistant cancer strains, demonstrating that there are ecological dynamics to cancer resistance. In some tumors, ecological dynamics may be more mechanistically relevant than simple Darwinian-based clonal selection in driving tumor heterogeneity and determining response to treatment. Our study strongly indicates that different tumor cell types that may exist in tumors are able to influence each other, which leads to alteration of phenotype. Note: This abstract was not presented at the conference. Citation Format: Surojeet Sengupta, Rong Hu, Zachary Susswein, Adeola Fagunloye, Shweta Bansal, Robert Clarke. Role of cell-cell communication in endocrine therapy-resistant breast cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B19.

The Amplitude-Normalized Area of a Bipolar Electrogram as a Measure of Local Conduction Delay in the Heart.

BackgroundRe-entrant ventricular tachycardia may be non-inducible or haemodynamically compromising, requiring assessment of the electrophysiological properties of the myocardium during sinus rhythm (i.e., substrate mapping). Areas of heart tissue with slow conduction can act as a critical isthmus for re-entrant electrical excitation and are a potential target for ablation therapy.AimTo develop and validate a novel metric of local conduction delay in the heart, the amplitude-normalized electrogram area (norm_EA).MethodsA computational model of a propagating mouse action potential was used to establish the impact of altering sodium channel conductance, intracellular conductivity, fibrosis density, and electrode size/orientation on bipolar electrogram morphology. Findings were then validated in experimental studies in mouse and guinea pig hearts instrumented for the recording of bipolar electrograms from a multipolar linear mapping catheter. norm_EA was calculated by integrating the absolute area of a bipolar electrogram divided by the electrogram amplitude. Electrogram metrics were correlated with the local conduction delay during sodium channel block, gap junction inhibition, and acute ischemia.ResultsIn computational simulations, reducing sodium channel conductance and intracellular conductivity resulted in a decrease in signal amplitude and increase in norm_EA (reflecting a broadening of electrogram morphology). For larger electrodes (3 mm diameter/7.1 mm2 area), the change in norm_EA was essentially linear with the change in local conduction delay. Experimental studies supported this finding, showing that the magnitude of change in norm_EA induced by flecainide (1–4 μM), carbenoxolone (10–50 μM), and low-flow ischemia (25% of initial flow rate) was linearly correlated with the local conduction delay in each condition (r2 = 0.92). Qualitatively similar effects were observed in guinea pig hearts perfused with flecainide. Increasing fibrosis density in the computational model also resulted in a decrease in signal amplitude and increase in norm_EA. However, this remains to be validated using experimental/clinical data of chronic infarct.Conclusionnorm_EA is a quantitative measure of local conduction delay between the electrode pair that generates a bipolar electrogram, which may have utility in electrophysiological substrate mapping of non-inducible or haemodynamically compromising tachyarrhythmia.

Modafinil potentiates cocaine self-administration by a dopamine-independent mechanism: possible involvement of gap junctions.

Modafinil and methylphenidate are medications that inhibit the neuronal reuptake of dopamine, a mechanism shared with cocaine. Their use as "smart drugs" by healthy subjects poses health concerns and requires investigation. We show that methylphenidate, but not modafinil, maintained intravenous self-administration in Sprague-Dawley rats similar to cocaine. Both modafinil and methylphenidate pretreatments potentiated cocaine self-administration. Cocaine, at self-administered doses, stimulated mesolimbic dopamine levels. This effect was potentiated by methylphenidate, but not by modafinil pretreatments, indicating dopamine-dependent actions for methylphenidate, but not modafinil. Modafinil is known to facilitate electrotonic neuronal coupling by actions on gap junctions. Carbenoxolone, a gap junction inhibitor, antagonized modafinil, but not methylphenidate potentiation of cocaine self-administration. Our results indicate that modafinil shares mechanisms with cocaine and methylphenidate but has a unique pharmacological profile that includes facilitation of electrotonic coupling and lower abuse liability, which may be exploited in future therapeutic drug design for cocaine use disorder.

Vascular smooth muscle cell phenotypic transition regulates gap junctions of cardiomyocyte

Atrial fibrillation (AF) is one of the most prevalent arrhythmias. Myocardial sleeves of the pulmonary vein are critical in the occurrence of AF. Our study aims to investigate the effect of synthetic vascular smooth muscle cells (SMCs) on gap junction proteins in cardiomyocytes. (1) Extraction of vascular SMCs from the pulmonary veins of Norway rats. TGF-β1 was used to induce the vascular SMCs switching to the synthetic phenotype and 18-α-GA was used to inhibit gap junctions of SMCs. The contractile and synthetic phenotype vascular SMCs were cocultured with HL-1 cells; (2) Western blotting was used to detect the expression of Cx43, Cx40 and Cx45 in HL-1 cells, and RT-PCR to test microRNA 27b in vascular SMCs or in HL-1 cells; (3) Lucifer yellow dye transfer experiment was used to detect the function of gap junctions. (1) TGF- β1 induced the vascular SMCs switching to synthetic phenotype; (2) Cx43 was significantly increased, and Cx40 and Cx45 were decreased in HL-1 cocultured with synthetic SMCs; (3) The fluorescence intensity of Lucifer yellow was higher in HL-1 cocultured with synthetic SMCs than that in the cells cocultured with contractile SMCs, which was inhibited by18-α-GA; (4) the expression of microRNA 27b was increased in HL-1 cocultured with synthetic SMCs, which was attenuated markedly by 18-α-GA. (5) the expression of ZFHX3 was decreased in HL-1 cocultured with synthetic SMCs, which was reversed by 18-α-GA. The gap junction proteins of HL-1 were regulated by pulmonary venous SMCs undergoing phenotypic transition in this study, accompanied with the up-regulation of microRNA 27b and the down-regulation of ZFHX3 in HL-1 cells, which was associated with heterocellular gap junctions between HL-1 and pulmonary venous SMCs.

Control of Vasoconstriction by Myoendothelial Ca2+ Signaling Through Gap Junctions

In resistance arteries, smooth muscle cells (SMCs) and endothelial cells (ECs) are communicated through gap junctions (i.e. myoendothelial gap junctions), which provide a signaling pathway for control of vasomotor tone. In this context, the Ca2+ signaling associated to smooth muscle contraction might also be transmitted via myoendothelial gap junctions and activate a regulatory feedback mechanism (myoendothelial feedback) by the production of endothelium‐dependent vasodilators. It is thought that IP3 is the main Ca2+‐related signaling transmitted from smooth muscle cells to endothelial cells, but the direct contribution of Ca2+ to the myoendothelial feedback is not clear. We investigated the temporal pattern of vasoconstriction in mesenteric resistance arteries, and their temporal relationship with the increase of Ca2+ in SMC and EC. We used intact mesenteric resistance arteries of Sprague dawley male rats (≤ 10 mm in length) to measure the vasoconstriction and changes in [Ca2+]i of smooth muscle cells and endothelial cells observed in response to phenylephrine (PE) or KCl using the fluorescent indicators of Ca2+, Fluo‐4 and X‐Rhod‐1.Changes in [Ca2+]i were also evaluated in primary cultures of mesenteric endothelial cells. All procedures were approved by the local Bioethical Committee (ID:180806005). The vasoconstriction induced by PE and KCl was not only associated with an increase of [Ca2+]i in smooth muscle cells, but also in endothelial cells, which was prevented by the blockade of L‐type voltage‐dependent Ca2+ channels with 10 μM nifedipine or the inhibition of gap junctions with 50 μM 18‐b‐Glycyrrhetenic acid. Although the stimulation with PE or KCl did not evoke an increment of [Ca2+]i in primary cultures of endothelial cells, the response induced by these vasoconstrictors was larger after blocking nitric oxide (NO) production with NG‐nitro‐L‐arginine (100 μM). These results suggest that the direct diffusion of Ca2+ contributes to the NO‐mediated component of the myoendothelial feedback.Support or Funding InformationConicyt 21171840

Cell‐specific heterogeneity of Cx43‐positive gap junctions in the mammalian kidney

ObjectiveConnexin 43 (Cx43) is a gap junction‐forming protein which mediates the intercellular passage of molecules < 1000 Da. In the kidney, Cx43 was reported in glomerular vessels and mesangium. Own results prompted us to reevaluate the distribution of Cx43 and its phosphorylated form, phospho‐S368‐Cx43 (pCx43), within the different zones of the rat kidney and in cultured fibroblasts in more detail.MethodsCx43 and pCx43 were localized by immunofluorescence in rat kidney sections and cultured human fibroblasts. High‐resolution immunostaining of Cx43 was achieved by ultrastructural immunogold labelling on electron microscopy sections. Double‐labelings with anti‐aquaporin 1 (AQP1), aquaporin 2 (AQP2), zonula occludens‐1 (ZO‐1), and cyclooxygenase‐2 (COX‐2) were conducted to clarify Cx43 distribution in the rat renal medulla. Cultured human fibroblasts served to study intercellular transfer of fluorescent lucifer yellow.ResultsCx43 was distributed in renal arterial and arteriolar endothelia, lymphatic vessel walls, peritubular capillaries, and glomerular compartments. In the interstitium, Cx43 was present as linear or punctate signals of the cell membrane of fibroblasts, in colocalization with pCx43. Cx43‐positive medullary fibroblasts co‐expressed COX‐2. No colocalization was detected between Cx43 and AQP1 or AQP2. Ultrastructural analysis showed Cx43 immunoreactivity at membrane contact zones between fibroblasts and in isolated cell processes of inner medullary interstitium. Cultured fibroblasts showed extensive, punctate Cx43 and pCx43 signals between adjacent cells. Signal was further distributed in non‐junctional areas of plasma membrane, suggesting Cx43 hemichannel functions. Lucifer yellow spread rapidly (10 min) between fibroblasts suggesting intercellular exchange via gap junctions, which was blocked by the gap junctional inhibitor, 18β‐glycyrrhetinic acid.ConclusionsCx43 in rat kidney shows prominent glomerular, vascular, and interstitial distribution, while epithelia, with exception of the podocytes, were negative. Focusing on interstitial fibroblasts, Cx43‐mediated information transfer was demonstrated. A constitutive nature of Cx43 phosphorylation was established in these cells. Our data suggest a cell‐specific heterogeneity of Cx43‐positive gap junctions mediating information exchange among renal cell types.

Gap Junctions Modulate The Effects Of Modafinil On Cocaine Self‐Administration Behavior In A Dopamine‐Independent Fashion In Rats

Modafinil and methylphenidate are among the few clinically available medications that block the dopamine (DA) transporter (DAT), a mechanism shared by abused psychostimulants like cocaine. Modafinil is FDA approved for the treatment of narcolepsy and other sleep disorders, while methylphenidate is approved as a medication for neurological disorders (e.g., attention deficit hyperactivity disorder). Both drugs have been reported for their non‐medical use as “smart drugs”, raising concerns regarding their potential for abuse by populations that may also be exposed to illicit substances. In this preclinical study, we compared the potential for abuse of modafinil with that of methylphenidate and their interactions with the reinforcing effects of cocaine in rats trained to self‐administer cocaine (0.03–1 mg/kg i.v., Fixed‐Ratio 5) with 5 doses assessed within each session. Since changes in DA levels in the nucleus accumbens shell (NAS) have been related to reinforcing effects of drugs, we monitored extracellular DA concentrations using microdialysis procedures in rats. Methylphenidate (0.03–1.0 mg/kg) maintained intravenous self‐administration behavior at comparable levels to active doses of cocaine (0.03–1.0 mg/kg). Modafinil (0.1–10 mg/kg) failed to maintain self‐administration behavior at any dose tested. However, pre‐session treatments (i.p.) with both modafinil (10–32 mg/kg) or methylphenidate (1.0–10 mg/kg) potentiated cocaine self‐administration behavior. Administration of cocaine, at self‐administered doses, produced dose‐related stimulation of NAS DA levels. Methylphenidate (1.0–10 mg/kg, i.p.), but not modafinil, (10–32 mg/kg, i.p.), enhanced cocaine‐induced stimulation of DA levels. These microdialysis results suggest that the effects of methylphenidate on cocaine actions are DA‐dependent, while those of modafinil are not. Modafinil is known to facilitate electrotonic coupling between cells by actions on gap junctions. Thus, we assessed the effects of carbenoxolone pretreatments, a gap junction inhibitor, on the potentiation of cocaine self‐administration produced by modafinil or methylphenidate. Carbenoxolone (1 mg/kg i.p.) attenuated modafinil, but not methylphenidate, potentiation of cocaine self‐administration behavior, suggesting that the facilitation of electrotonic coupling plays a role in modafinil’s potentiation of cocaine’s effects. In conclusion, modafinil shares with cocaine and methylphenidate important actions at the DAT, but the present data suggest a unique pharmacological stimulant profile, lacking abuse potential and facilitating electrotonic coupling. These results, together with clinical studies, further suggest a potential therapeutic use of modafinil in patients with cocaine use disorder.Support or Funding InformationThis research was supported by the Intramural Research Program of the National Institute on Drug Abuse, NIH, DHHS

Novel Contributions of Neutrophil‐derived Myeloperoxidase and Hypochlorous Acid to 20‐HETE Production that drives Post‐Ischemic Angiogenesis

Objective20‐hydroxy‐eicosatetraenoic acid (20‐HETE), an arachidonic acid metabolite produced by cytochrome P450 (CYP) 4A/F ω‐hydroxylases, was recently uncovered as a novel contributor to ischemia‐induced angiogenesis. The current study aims to determine whether neutrophil‐derived myeloperoxidase (MPO) and hypochlorous acid (HOCl) mediate post‐ischemic 20‐HETE increases that drive angiogenesis and its underlying mechanism.MethodsMice were subjected to a femoral artery ligation with the contralateral hindlimb acting as the non‐ischemic control. Hindlimb angiogenesis was assessed by laser doppler blood perfusion imaging (LDPI) as well as quantifying the microvascular density in the gracilis muscles where angiogenesis is taking place. Endothelial cells (EC) in culture were subjected to physiological concentrations of MPO and HOCl. Gracilis muscles and EC were analyzed for 20‐HETE production by LC/MS/MS. The mRNA expression of the 20‐HETE synthesizing enzyme, CYP4A11 was measured by real‐time PCR. Protein expression of CYP4A11 and HIF1a were analyzed using western blotting. The rate and mechanism of HOCl entry into EC was evaluated using live‐cell fluorescence microscopy with a HOCl‐specific indicator dye (FDOCl‐1) in the presence/absence of chloride channel blocker 9‐anthracenecarboxylic acid (9‐Ac) and/or gap junction inhibitor 18‐beta‐glycyrrhetinic acid (18β‐GA).ResultsIn animals that were selectively depleted of neutrophils, ischemia failed to induce 20‐HETE increases in the gracilis muscle compared to the controls (13 ± 1.5 vs 35 ± 5 pg/mg of protein). Additionally, MPO‐null mice failed to show increased post‐ischemic 20‐HETE (~2 ± .25 vs 35 ± 5 pg/mg of protein) as well. Both animals display a decreased post‐ischemic angiogenenic phenotype. In EC, the addition of MPO or HOCl markedly stimulated the expression of CYP4A11 and production of 20‐HETE (40 ± 12 vs 8 ± 5 pg/mg of protein). Upregulation of CYP4A11 transcript/protein (>2.5‐fold) and HIF1a protein expression (2‐fold) was observed as early as 15 minutes post‐HOCl exposure. The mechanism of HOCl entry was also exmained by FDOCl‐1 fluorescence quantification, which was markedly decreased in EC pre‐treated with 9‐Ac or 18β‐GA by approximately 40% and 60%, respectively.ConclusionOur study demonstrates for the first time the contribution of MPO and HOCl in promoting 20‐HETE production that drives post‐ischemic angiogenesis. Moreover, this study aims to further expand on the known mechanisms involved in regulating ischemia‐induced angiogenesis. These new findings have high clinical relevancy in studying vascular pathologies by identifing novel mechanisms that contribute to pathological angiogenesis.Support or Funding InformationThis study was supported by grants from AHA[11SDG6870004 (AMG) and 17GRNT33430003 (AMG)], the NIH [HL34300 (M.L.S.) and grant DK38226 (J.R.F.)]; & the R.A. Welch Foundation [GL625910 (J.R.F.)].

Connexin 43 Channels in Osteocytes Regulate Bone Responses to Mechanical Unloading.

Connexin (Cx) 43 forms gap junctions and hemichannels that mediate communication between osteocytes and adjacent cells or the extracellular environment in bone, respectively. To investigate the role of each channel type in response to mechanical unloading, two transgenic mouse models overexpressing dominant-negative Cx43 predominantly in osteocytes driven by a 10 kb dentin matrix protein 1 (Dmp1) promoter were generated. The R76W mutation resulted in gap junction inhibition and enhancement of hemichannels, whereas the Δ130–136 mutation inhibited both gap junctions and hemichannels. Both mutations led to cortical bone loss with increased endocortical osteoclast activity during unloading. Increased periosteal osteoclasts with decreased apoptotic osteocytes were observed only in R76W mice. These findings indicated that inhibiting osteocytic Cx43 channels promotes bone loss induced by unloading, mainly in the cortical area; moreover, hemichannels protect osteocytes against apoptosis and promote periosteal bone remodeling, whereas gap junctions modulate endocortical osteoclast activity in response to unloading.

Connexin 43: A novel ginsenoside Rg1-sensitive target in a rat model of depression

Our previous studies have shown that ginsenoside Rg1 (Rg1) exerts antidepressant-like effects in animal models of depression, accompanied by an improvement of astrocytic gap junction functions. However, whether connexin 43 (Cx43), the major connexin forming gap junctions between astrocytes, is the key regulator of Rg1-induced antidepressant-like effects is still unknown. In this study, we examine in vitro and in vivo the involvement of Cx43 in the antidepressant effects of Rg1. Corticosterone was used to establish an in vitro rat model of depression. Treatment with Rg1 1 h prior to corticosterone significantly improved the cell viability of astrocytes, which was significantly inhibited by carbenoxolone, a widely used gap junction inhibitor. Moreover, Rg1 treatment significantly ameliorated antidepressant-sensitive behaviours induced by infusion of carbenoxolone or Gap26, a selective inhibitor of Cx43, into the prefrontal cortex of the animals. Rg1 treatment increased the expression of Cx43 compared with Gap26 group. According to these results, the antidepressant-like effects of Rg1 were mainly mediated by Cx43-formed gap junctions.

Inhibition of Gap Junction–Mediated Intercellular Communication by Poly(I:C) in Cultured Human Corneal Fibroblasts

ABSTRACT Purpose/Aim Corneal stromal fibroblasts are connected to each other via gap junctions, which contribute to maintenance of corneal homeostasis. Viral infection of the corneal stroma can result in inflammation and scarring. The effects of polyinosinic-polycytidylic acid [poly(I:C)], an analog of viral double-stranded RNA, on gap junctional intercellular communication (GJIC) in cultured human corneal fibroblasts (HCFs) were examined. Materials and Methods Cultured HCFs were exposed to poly(I:C) in the absence or presence of inhibitors of mitogen-activated protein kinase (MAPK) signaling or the antioxidant N-acetyl-L-cysteine (NAC). Expression of the gap junction protein connexin 43 (Cx43) was examined by immunoblot and immunofluorescence analyses. The level of Cx43 mRNA or microRNA-21 or −130a was determined by quantitative reverse transcription–polymerase chain reaction analysis. GJIC was measured with a dye coupling assay. The amount of malondialdehyde and the activity of superoxide dismutase (SOD) were measured with assay kits. Results Exposure of HCFs to poly(I:C) resulted in down-regulation of Cx43 expression and GJIC activity as well as in up-regulation of microRNA-21 expression. Poly(I:C) increased the amount of malondialdehyde and reduced the activity of SOD in the cells, and these effects were prevented by NAC. The inhibitory effects of poly(I:C) on both Cx43 expression and GJIC activity were attenuated by NAC and by c-Jun NH2-terminal kinase (JNK) inhibitor II. Conclusions Poly(I:C) inhibited Cx43 expression and GJIC in cultured HCFs, possibly as a result of the associated up-regulation of microRNA-21. Poly(I:C) also increased oxidative stress in these cells, and such stress together with signaling by the MAPK JNK was implicated in the effects of poly(I:C) on Cx43 expression and GJIC activity. Down-regulation of GJIC activity among corneal fibroblasts by double-stranded RNA may thus contribute to the disruption of stromal homeostasis during viral infection of the cornea.

Gap junctions amplify TRPV4 activation-initiated cell injury via modification of intracellular Ca2+ and Ca2+-dependent regulation of TXNIP

ABSTRACT The elevated intracellular Ca2+ and oxidative stress are well-reported mechanisms behind renal tubular epithelial injury initiated by various insults. Given that TRPV4 and connexin43 (Cx43) channels are activated by a wide range of stimuli and regulate both intracellular Ca2+ and redox status, we speculated an involvement of these channels in renal tubular cell injury. Here, we tested this possibility and explored the potential underlying mechanisms. Our results demonstrated that exposure of renal tubular epithelial cells to aminoglycoside G418 led to cell death, which was attenuated by both TRPV4 and gap junction (Gj) inhibitor. Activation of TRPV4 caused cell damage, which was associated with an early increase in Cx43 expression and function. Inhibition of Cx43 with chemical inhibitor or siRNA largely prevented TRPV4 activation-induced cell damage. Further analysis revealed that TRPV4 agonists elicited a rise in intracellular Ca2+ and caused a Ca2+-dependent elevation in TXNIP (a negative regulator of the antioxidant thioredoxin). In the presence of Gj inhibitor, however, these effects of TRPV4 were largely prevented. The depletion of intracellular Ca2+ with Ca2+ chelator BAPTA-AM or downregulation of TXNIP with siRNA significantly alleviated TRPV4 activation-initiated cell injury. Collectively, our results point to a critical involvement of TRPV4/Cx43 channel interaction in renal tubular cell injury through mechanisms involving a synergetic induction of intracellular Ca2+ and oxidative stress. Channel interactions could be an important mechanism underlying cell injury. Targeting channels could have therapeutic potential for the treatment of acute tubular cell injury.

Precise control of ion channel and gap junction expression is required for patterning of the regenerating axolotl limb.

Axolotls and other salamanders have the capacity to regenerate lost tissue after an amputation or injury. Growth and morphogenesis are coordinated within cell groups in many contexts by the interplay of transcriptional networks and biophysical properties such as ion flows and voltage gradients. It is not, however, known whether regulators of a cell’s ionic state are involved in limb patterning at later stages of regeneration. Here we manipulated expression and activities of ion channels and gap junctions in vivo, in axolotl limb blastema cells. Limb amputations followed by retroviral infections were performed to drive expression of a human gap junction protein Connexin 26 (Cx26), potassium (Kir2.1-Y242F and Kv1.5) and sodium (NeoNav1.5) ion channel proteins along with EGFP control. Skeletal preparation revealed that overexpressing Cx26 caused syndactyly, while overexpression of ion channel proteins resulted in digit loss and structural abnormalities compared to EGFP expressing control limbs. Additionally, we showed that exposing limbs to the gap junction inhibitor lindane during the regeneration process caused digit loss. Our data reveal that manipulating native ion channel and gap junction function in blastema cells results in patterning defects involving the number and structure of the regenerated digits. Gap junctions and ion channels have been shown to mediate ion flows that control the endogenous voltage gradients which are tightly associated with the regulation of gene expression, cell cycle progression, migration, and other cellular behaviors. Therefore, we postulate that mis-expression of these channels may have disturbed this regulation causing uncoordinated cell behavior which results in morphological defects.

Pre-ovulatory intercellular regulation of miR-125a-3p within mouse ovarian follicles.

miR-125a-3p, a post-transcription regulator of Fyn kinase, is expressed in mouse pre-ovulatory follicles; its expression within the follicle decreases toward ovulation. Our aim was to follow the synthesis of miR-125a-3p and regulation of its expression in all follicular compartments, focusing on intercellular communication. Mural granulosa cells (GCs) or cumulus cells (CCs) were transfected with either scrambled-miR (negative control) or miR-125a-3p mimic. Freshly isolated GCs or CCs were incubated overnight in culture media conditioned by transfected cells. To examine a possible role of gap junctions in the regulation of miR-125a-3p, we incubated large antral follicles in the presence of carbenoxolone, a gap-junction inhibitor, and triggered them to mature with hGC. Levels of miR-125a family members in GCs, CCs, oocytes, and culture media were measured by qPCR. We showed that miR-125a-3p is synthesized by all follicular components, but is regulated within the follicle as a whole. It is secreted by mural-GCs and taken up by CCs, where it remains functional, and vice versa, mural-GCs can take up miR-125a-3p secreted by CCs. miR-125a-3p is transcribed and accumulated in oocytes throughout oogenesis. Transcriptionally quiescent GV oocytes utilize their accompanying follicular cells to monitor the level of miR-125a-3p within them, as indicated in an ex vivo follicle culture. Our study reveals that miR-125a-3p expression is modulated by a network of intercellular communications within pre-ovulatory follicles, thus enabling a coordinated decrease of miR-125a-3p toward ovulation.

Mind the gap (junction): cGMP induced by nitric oxide in cardiac myocytes originates from cardiac fibroblasts.

Background and PurposeThe intracellular signalling molecule cGMP, formed by NO‐sensitive GC (NO–GC), has an established function in the vascular system. Despite numerous reports about NO‐induced cGMP effects in the heart, the underlying cGMP signals are poorly characterized.Experimental ApproachTherefore, we analysed cGMP signals in cardiac myocytes and fibroblasts isolated from knock‐in mice expressing a FRET‐based cGMP indicator.Key ResultsWhereas in cardiac myocytes, none of the known NO–GC‐activating substances (NO, GC activators, and GC stimulators) increased cGMP even in the presence of PDE inhibitors, they induced substantial cGMP increases in cardiac fibroblasts. As cardiac myocytes and fibroblasts are electrically connected via gap junctions, we asked whether cGMP can take the same route. Indeed, in cardiomyocytes co‐cultured on cardiac fibroblasts, NO‐induced cGMP signals were detectable, and two groups of unrelated gap junction inhibitors abolished these signals.Conclusion and ImplicationWe conclude that NO‐induced cGMP formed in cardiac fibroblasts enters cardiac myocytes via gap junctions thereby turning cGMP into an intercellular signalling molecule. The findings shed new light on NO/cGMP signalling in the heart and will potentially broaden therapeutic opportunities for cardiac disease.