A 14‐3‐3 Mode‐1 Binding Motif Initiates Gap Junction Internalization During Acute Cardiac Ischemia

Altered phosphorylation and trafficking of connexin 43 (Cx43) during acute ischemia contributes to arrhythmogenic gap junction remodeling, yet the critical sequence and accessory proteins necessary for Cx43 internalization remain unresolved. 14‐3‐3 proteins can regulate protein trafficking, and a 14‐3‐3 mode‐1 binding motif is activated upon phosphorylation of Ser373 of the Cx43 C‐terminus. We hypothesized that Cx43^Ser373 phosphorylation is important to pathological gap junction remodeling. Immunofluorescence in human heart reveals the enrichment of 14‐3‐3 proteins at intercalated discs, suggesting interaction with gap junctions. Knockdown of 14‐3‐3τ in cell lines increases gap junction plaque size at cell–cell borders. Cx43^S373A mutation prevents Cx43/14‐3‐3 complexing and stabilizes Cx43 at the cell surface, indicating avoidance of degradation. Using Langendorff‐perfused mouse hearts, we detect phosphorylation of newly internalized Cx43 at Ser373 and Ser368 within 30 min of no‐flow ischemia. Phosphorylation of Cx43 at Ser368 by protein kinase C and Ser255 by mitogen‐activated protein kinase has previously been implicated in Cx43 internalization. The Cx43^S373A mutant is resistant to phosphorylation at both these residues and does not undergo ubiquitination, revealing Ser373 phosphorylation as an upstream gatekeeper of a posttranslational modification cascade necessary for Cx43 internalization. Cx43^Ser373 phosphorylation is a potent target for therapeutic interventions to preserve gap junction coupling in the stressed myocardium.      

β‐Adrenergic‐induced CD40 overexpression on gingival fibroblasts: Role of PGE2

CD40, a member of the tumour necrosis factor‐α receptor family, is constitutively expressed by cells of haematopoietic and non‐haematopoietic origin, including fibroblasts. Signalling through this receptor molecule regulates inflammatory mediator secretion by many cell types. The work has been performed in healthy subjects and the authors studied, by cellular culture, flow cytometric analysis and ELISA assay, the expression of CD40 and PGE₂ (prostaglandin E₂) generation on gingival fibroblasts stimulated by β‐AR (β‐adrenoceptor) agonists. Herein, the authors demonstrate that β‐AR subtype activation via their own specific agonists markedly increased CD40 expression on human gingival fibroblasts. This effect was prevented by β‐AR subtype‐specific antagonists. In addition, gingival fibroblast β‐AR stimulation resulted in an increase in PGE₂ generation. The inhibition of PLA₂ (phospholipase A₂) and COX‐1 (cyclo‐oxygenase‐1) but not COX‐2 impaired β‐AR increase of PGE₂, an effect that was restored by the addition of low concentrations of PGE₂, suggesting that PGE₂ generation is implicated in the mechanism underlying β‐AR‐agonist‐mediated CD40 overexpression. Our work has revealed an endogenous β‐AR mediator network involving gingival fibroblasts.     

Effects of the β‐agonist,isoprenaline, on the down‐regulation,functional responsiveness and trafficking of β2‐adrenergic receptors with N‐terminal polymorphisms

The β₂‐AR (β₂‐adrenergic receptor) is an important target for respiratory and CVD (cardiovascular disease) medications. Clinical studies suggest that N‐terminal polymorphisms of β₂‐AR may act as disease modifiers. We hypothesized that polymorphisms at amino acids 16 and 27 result in differential trafficking and down‐regulation of β₂‐AR variants following β‐agonist exposure. The functional consequences of the four possible combinations of these polymorphisms in the human β₂‐AR (designated β₂‐AR‐RE, β₂‐AR‐GE, β₂‐AR‐RQ and β₂‐AR‐GQ) were studied using site‐directed mutagenesis and recombinant expression in HEK‐293 cells (human embryonic kidney cells). Ligand‐binding assays demonstrated that after 24 h exposure to 1 μM isoprenaline, isoforms with Arg¹⁶ (β₂‐AR‐RE and β₂‐AR‐RQ) underwent increased down‐regulation compared with isoforms with Gly¹⁶ (β₂‐AR‐GE and β₂‐AR‐GQ). Consistent with these differences in down‐regulation between isoforms, prolonged isoprenaline treatment resulted in diminished cAMP response to subsequent isoprenaline challenge in β₂‐AR‐RE relative to β₂‐AR‐GE. Confocal microscopy revealed that the receptor isoforms had similar co‐localization with the early endosomal marker EEA1 following isoprenaline treatment, suggesting that they had similar patterns of internalization. None of the isoforms exhibited significant co‐localization with the recycling endosome marker Rab11 in response to isoprenaline treatment. Furthermore, we found that prolonged isoprenaline treatment led to a higher degree of co‐localization of β₂‐AR‐RE with the lysosomal marker LAMP1 (lysosome‐associated membrane protein 1) compared with that of β₂‐AR‐GE. Taken together, these results indicate that a mechanism responsible for differential responses of these receptor isoforms to the β‐agonist involves differences in the efficiency with which agonist‐activated receptors are trafficked to the lysosomes for degradation, or differences in degradation in the lysosomes.     

The role of β‐adrenergic receptors and p38MAPK signaling pathways in physiological processes of cardiosphere‐derived cells

The effects of β adrenergic receptors (β‐ARs) and p38 mitogen‐activated protein kinases (MAPK) pathways on cardiosphere‐derived cells (CDCs) are largely unknown. This study aimed to investigate the roles of β‐ARs and p38MAPK pathways on the proliferation, apoptosis, and differentiation capacity of CDCs. The CDCs were treated with β1‐AR blocker (Met group), β2‐AR antagonist (ICI group), and p38MAPK inhibitor (SB group), non‐selective β‐AR blocker (PRO group), and β‐AR agonist (ISO group). The viability, apoptotic rate and differentiation status of CDCs were determined by MST‐1 assay, flow cytometery, and Western blot, respectively. The CDCs viability significantly reduced in ICI group (all P < 0.05), and SB group had a significant high viability after 48 h treatment (P < 0.05). Compared with control group, all treated groups had a low apoptotic rate. After treatment for 72 h, ISO treatment elevated the expression of Nkx2.5, and could partially or fully attenuate the inhibitory effects of β‐AR antagonists and/or p38MAPK inhibitor. A similar overall trend of protein expression levels among all groups could be observed between protein pairs of cTnT and β1‐AR as well as c‐Kit and β2‐AR, respectively. These results suggested that β‐ARs and p38MAPK signaling pathways play crucial roles in the proliferation and differentiation of CDCs. Our findings should be helpful for better understanding the molecular mechanism underlying the physiological processes of CDCs.     

let‐7e replacement yields potent anti‐arrhythmic efficacy via targeting beta 1‐adrenergic receptor in rat heart

Beta‐adrenoceptor (β‐AR) exerts critical regulation of cardiac function. MicroRNAs (miRNAs) are potentially involved in a variety of biological and pathological processes. This study aimed to investigate the role of miRNA let‐7e in the up‐regulation of β₁‐AR and arrhythmogenesis in acute myocardial infarction (AMI) in rats. β₁‐AR expression was significantly up‐regulated and let‐7a, c, d, e and i were markedly down‐regulated in the infarcted heart after 6 and 24 hrs myocardial infarction. Forced expression of let‐7e suppressed β₁‐AR expression at the protein level, without affecting β₁‐AR mRNA level, in neonatal rat ventricular cells (NRVCs). Silencing of let‐7e by let‐7e antisense inhibitor (AMO‐let‐7e) enhanced β₁‐AR expression at the protein level in NRVCs. Administration of the lentivirus vector containing precursor let‐7e (len‐pre‐let‐7e) significantly inhibited β₁‐AR expression in rats, whereas len‐AMO‐let‐7e up‐regulated β₁‐AR relative to the baseline control level, presumably as a result of depression of tonic inhibition of β₁‐AR by endogenous let‐7e. Len‐negative control (len‐NC) did not produce significant influence on β₁‐AR expression. Len‐pre‐let‐7e also profoundly reduced the up‐regulation of β₁‐AR induced by AMI and this effect was abolished by len‐AMO‐let‐7e. Importantly, len‐pre‐let‐7e application significantly reduced arrhythmia incidence after AMI in rats and its anti‐arrhythmic effect was cancelled by len‐AMO‐let‐7e. Notably, anti‐arrhythmic efficacy of len‐pre‐let‐7e was similar to propranolol, a non‐selective β‐AR blocker and metoprolol, a selective β₁‐AR blocker. Down‐regulation of let‐7e contributes to the adverse increase in β₁‐AR expression in AMI and let‐7e supplement may be a new therapeutic approach for preventing adverse β₁‐AR up‐regulation and treating AMI‐induced arrhythmia.     

Gender differences in β‐adrenoceptor system in cardiac hypertrophy due to arteriovenous fistula

This study was undertaken to determine alterations in the β‐adrenoceptor (β‐AR) signaling system in male and female rats at 4 weeks after the induction of arteriovenous (AV) fistula or shunt. AV shunt produced a greater degree of cardiac hypertrophy and larger increase in cardiac output in male than in female animals. Increases in plasma levels of norepinephrine and epinephrine (EPI) due to AV shunt were also higher in male than females. While no difference in the β₁‐AR affinity was seen in males and females, AV shunt induced increase in β₁‐AR density in female rats was higher than that in males. Furthermore, no changes in basal adenylyl cyclase (AC) V/VI mRNA levels were seen; however, the increase in EPI‐stimulated AC activities was greater in AV shunt females than in males. AV shunt decreased myocardial β₁‐AR mRNA level in male rats and increased β₂‐AR mRNA level in female hearts; an increase in G_i‐protein mRNA was detected only in male hearts. Although GRK2 gene expression was increased in both sexes, an increase in GRK3 mRNA was seen only in AV shunt female rats. β‐arrestin1 mRNA was elevated in females whereas β‐arrestin 2 gene expression was increased in both male and female AV shunt rats. While these data demonstrate gender associated differences in various components of the β‐AR system in cardiac hypertrophy due to AV shunt, only higher levels of plasma catecholamines may account for the greater increase in cardiac output and higher degree of cardiac hypertrophy in males. J. Cell. Physiol. 226: 181–186, 2010. © 2010 Wiley‐Liss, Inc.     

β‐adrenergic blockade attenuates cardiac dysfunction and myofibrillar remodelling in congestive heart failure

Although β‐adrenoceptor (β‐AR) blockade is an important mode of therapy for congestive heart failure (CHF), subcellular mechanisms associated with its beneficial effects are not clear. Three weeks after inducing myocardial infarction (MI), rats were treated daily with or without 20 and 75 mg/kg atenolol, a selective β₁‐AR antagonist, or propranolol, a non‐selective β‐AR antagonist, for 5 weeks. Sham operated rats served as controls. All animals were assessed haemodynamically and echocardiographically and the left ventricle (LV) was processed for the determination of myofibrillar ATPase activity, α‐ and β‐myosin heavy chain (MHC) isoforms and gene expression as well as cardiac troponin I (cTnI) phosphorylation. Both atenolol and propranolol at 20 and 75 mg/kg doses attenuated cardiac hypertrophy and lung congestion in addition to increasing LV ejection fraction and LV systolic pressure as well as decreasing heart rate, LV end‐diastolic pressure and LV diameters in the infarcted animals. Treatment of infarcted animals with these agents also attenuated the MI‐induced depression in myofibrillar Ca²⁺‐stimulated ATPase activity and phosphorylated cTnI protein content. The MI‐induced decrease in α‐MHC and increase in β‐MHC protein content were attenuated by both atenolol and propranolol at low and high doses; however, only high dose of propranolol was effective in mitigating changes in the gene expression for α‐MHC and β‐MHC. Our results suggest that improvement of cardiac function by β‐AR blockade in CHF may be associated with attenuation of myofibrillar remodelling.     

Aliskiren‐attenuated myocardium apoptosis via regulation of autophagy and connexin‐43 in aged spontaneously hypertensive rats

There are controversies about the mechanism of myocardium apoptosis in hypertensive heart disease. The aim of this study was to investigate the relationship among autophagy, Cx43 and apoptosis in aged spontaneously hypertensive rats (SHRs) and establish whether Aliskiren is effective or not for the treatment of myocardium apoptosis. Twenty‐one SHRs aged 52 weeks were randomly divided into three groups, the first two receiving Aliskiren at a dose of 10 and 25 mg/kg/day respectively; the third, placebo for comparison with seven Wistar‐Kyoto (WKY) as controls. After a 2‐month treatment, systolic blood pressure (SBP), heart to bw ratios (HW/BW%) and angiotensin II (AngII) concentration were significantly enhanced in SHRs respectively. Apoptotic cardiomyocytes detected with TUNEL and immunofluorescent labelling for active caspase‐3 increased nearly fourfolds in SHRs, with a decline in the expression of survivin and AKT activation, and an increase in caspase‐3 activation and the ratio of Bax/Bcl‐2. Myocardium autophagy, detected with immunofluorescent labelling for LC3‐II, increased nearly threefolds in SHRs, with the up‐regulation of Atg5, Atg16L1, Beclin‐1 and LC3‐II. The expression of Cx43 plaque was found to be down‐regulated in SHRs. Aliskiren significantly reduced SBP, HW/BW%, AngII concentration and the expression of AT₁R. Thus, Aliskiren protects myocardium against apoptosis by decreasing autophagy, up‐regulating Cx43. These effects showed a dose‐dependent tendency, but no significance. In conclusion, the myocardium apoptosis developed during the hypertensive end‐stage of SHRs could be ameliorated by Aliskiren via the regulation of myocardium autophagy and maladaptive remodelling of Cx43.     

Enhanced connexin 43 expression delays intra‐mitoitc duration and cell cycle traverse independently of gap junction channel function

Connexins (Cxs) and gap junction (GJ)‐mediated communication have been linked with the regulation of cell cycle traverse. However, it is not clear whether Cx expression or GJ channel function are the key mediators in this process or at what stage this regulation may occur. We therefore tested the hypothesis that enhanced Cx expression could alter the rate of cell cycle traverse independently of GJ channel function. Sodium butyrate (NaBu) or anti‐arrhythmic peptide (AAP10) were used to enhance Cx expression in HeLa cells stably expressing Cx43 (HeLa‐43) and primary cultures of human fibroblasts (HFF) that predominantly express Cx43. To reduce GJ‐mediated communication, 18‐α‐glycyrrhetinic acid (GA) was used. In HeLa‐43 and HFF cells, NaBu and AAP10 enhanced Cx43 expression and increased channel function, while GA reduced GJ‐mediated communication but did not significantly alter Cx43 expression levels. Timelapse microscopy and flow cytometry of HeLa‐WT (wild‐type, Cx deficient) and HeLa‐43 cells dissected cell cycle traverse and enabled measurements of intra‐mitotic time and determined levels of G1 arrest. Enhanced Cx43 expression increased mitotic durations corresponding with a G1 delay in cell cycle, which was linked to an increase in expression of the cell cycle inhibitor p21^waf1/cip1 in both HeLa‐43 and HFF cells. Reductions in Cx43 channel function did not abrogate these responses, indicating that GJ channel function was not a critical factor in reducing cell proliferation in either cell type. We conclude that enhanced Cx43 expression and not GJ‐mediated communication, is involved in regulating cell cycle traverse. J. Cell. Biochem. 110: 772–782, 2010. © 2010 Wiley‐Liss, Inc.     

Phosphorylation of Cav1.2 on S1928 uncouples the L‐type Ca2+ channel from the β2 adrenergic receptor

Agonist‐triggered downregulation of β‐adrenergic receptors (ARs) constitutes vital negative feedback to prevent cellular overexcitation. Here, we report a novel downregulation of β₂AR signaling highly specific for Ca_v1.2. We find that β₂‐AR binding to Ca_v1.2 residues 1923–1942 is required for β‐adrenergic regulation of Ca_v1.2. Despite the prominence of PKA‐mediated phosphorylation of Ca_v1.2 S1928 within the newly identified β₂AR binding site, its physiological function has so far escaped identification. We show that phosphorylation of S1928 displaces the β₂AR from Ca_v1.2 upon β‐adrenergic stimulation rendering Ca_v1.2 refractory for several minutes from further β‐adrenergic stimulation. This effect is lost in S1928A knock‐in mice. Although AMPARs are clustered at postsynaptic sites like Ca_v1.2, β₂AR association with and regulation of AMPARs do not show such dissociation. Accordingly, displacement of the β₂AR from Ca_v1.2 is a uniquely specific desensitization mechanism of Ca_v1.2 regulation by highly localized β₂AR/cAMP/PKA/S1928 signaling. The physiological implications of this mechanism are underscored by our finding that LTP induced by prolonged theta tetanus (PTT‐LTP) depends on Ca_v1.2 and its regulation by channel‐associated β₂AR.     

Modulatory effects of connexin-43 expression on gap junction intercellular communications with mast cells and fibroblasts

The influence of mast cells upon aberrant wound repair and excessive fibrosis has supportive evidence, but the mechanism for these mast cell activities is unclear. It is proposed that heterocellular gap junction intercellular communication (GJIC) between fibroblasts and mast cells directs some fibroblast activities. An in vitro model was used employing a rodent derived peritoneal mast cell line (RMC-1) and human dermal derived fibroblasts. The influence of the expression of the gap junction channel structural protein, connexin 43 (Cx-43) on heterocellular GJIC, the expression of microtubule β-tubulin and microfilament α smooth muscle actin (SMA) were investigated. The knockdown of Cx-43 by siRNA in RMC-1 cells completely blocked GJIC between RMC-1 cells. SiRNA knockdown of Cx-43 within fibroblasts only dampened GJIC between fibroblasts. It appears Cx-43 is the only expressed connexin (Cx) in RMC-1 cells. Fibroblasts express other Cxs that participate in GJIC between fibroblasts in the absence of Cx-43 expression. Heterocellular GJIC between RMC-1 cells and fibroblasts transformed fibroblasts into myofibroblasts, expressing α SMA within cytoplasmic stress fibers. The knockdown of Cx-43 in RMC-1 cells increased β-tubulin expression, but its knockdown in fibroblasts reduced β-tubulin expression. Knocking down the expression of Cx-43 in fibroblasts limited αSMA expression. Cx-43 participation is critical for heterocellular GJIC between mast cells and fibroblasts, which may herald a novel direction for controlling fibrosis.     

Distinct roles for β‐arrestin2 and arrestin‐domain‐containing proteins in β2 adrenergic receptor trafficking

β‐arrestin 1 and 2 (also known as arrestin 2 and 3) are homologous adaptor proteins that regulate seven‐transmembrane receptor trafficking and signalling. Other proteins with predicted ‘arrestin‐like’ structural domains but lacking sequence homology have been indicated to function like β‐arrestin in receptor regulation. We demonstrate that β‐arrestin2 is the primary adaptor that rapidly binds agonist‐activated β₂ adrenergic receptors (β₂ARs) and promotes clathrin‐dependent internalization, E3 ligase Nedd4 recruitment and ubiquitin‐dependent lysosomal degradation of the receptor. The arrestin‐domain‐containing (ARRDC) proteins 2, 3 and 4 are secondary adaptors recruited to internalized β₂AR–Nedd4 complexes on endosomes and do not affect the adaptor roles of β‐arrestin2. Rather, the role of ARRDC proteins is to traffic Nedd4–β₂AR complexes to a subpopulation of early endosomes.     

Presence and Importance of Connexin43 During Myogenesis

We analyzed the expression of connexin(Cx)43 in proliferating and differentiating C₂C₁₂cells and in myoblasts obtained from newborn mice. Cx43 was present in both cell types and under both conditions. The functional role of gap junctional communication (GJC) during terminal differentiation was evaluated in C₂C₁₂myoblasts in the presence or absence of the gap junction blocker 18β-glycyrrhetinic acid (β-GA). Differentiation was temporally analyzed through myogenin expression, activity of creatine kinase (CK), and yield of multinucleated cells. In cells treated with β-GA, the CK activity and myotube formation were reversibly blocked. While in control cultures positive myogenin expression was seen in cell clusters, in β-GA treated cultures the myogenin immunoreactivity was detected in few, preferentially sparse cells. The role of Cx43 during terminal differentiation was evaluated in cultures of myoblasts obtained from Cx43^Cre-ER(T)/fltransgenic mice. Inducible deletion of Cx43 was obtained upon activation of Cre-ER(T) via 4-OH-tamoxifen applications. Cx43 deletion led to a drastic decrease in myogenin expression at 24 h of differentiation as compared to myoblasts from control mice. Our results indicate that Cx43-containing gap junctions are required for normal skeletal muscle terminal differentiation. These channels might provide a pathway for the intercellular transfer of signals involved in myogenesis.     

Syntheses and β‐adrenergic binding affinities of (R)‐ and (S)‐fluoronaphthyloxypropanolamines

The β‐adrenergic receptors mediate several physiological processes including heart rate (β₁), bronchodilation (β₂), and lipolysis (β₃). Therefore, selectivity is important for a possible therapeutic agent acting via these receptors. Aryloxypropanolamines are β‐receptor agonists or antagonists, depending on the aryl group and its substituents. We therefore hypothesized that fluorine substitution on the aromatic ring in this class could lead to significant biological effects because of the unique chemical characteristics of fluorine. Because the target compound has a chiral center, we set out to synthesize the two enantiomers so that effects of stereochemistry on biological activity could be evaluated. Syntheses of the enantiomers were performed starting with commercially available fluoronaphthalene and subsequent use of the chiral synthon (2R)‐ or (2S)‐glycidyl 3‐nitrobenzenesulfonate, depending on the desired enantiomer. High‐pressure liquid chromatography (HPLC) methods were used to characterize %ee. Each enantiomer was synthesized. They exhibited nanomolar binding activities on β‐adrenergic receptors. The (S)‐enantiomer was found to be up to 310 times more potent than the (R). It was also found to be about five‐fold more selective for β₂‐ than for β₁‐receptors. The current report demonstrates the importance of stereochemistry for the fluoroaromatic β‐receptor ligands. Chirality 11:144–148, 1999. © 1999 Wiley‐Liss, Inc.     

Functional formation of heterotypic gap junction channels by connexins-40 and -43

Connexin40 (Cx40) and connexin43 (Cx43) are co-expressed in the cardiovascular system, yet their ability to form functional heterotypic Cx43/Cx40 gap junctions remains controversial. We paired Cx43 or Cx40 stably-transfected N2a cells to examine the formation and biophysical properties of heterotypic Cx43/Cx40 gap junction channels. Dual whole cell patch clamp recordings demonstrated that Cx43 and Cx40 form functional heterotypic gap junctions with asymmetric transjunctional voltage (V_j) dependent gating properties. The heterotypic Cx43/Cx40 gap junctions exhibited less V_j gating when the Cx40 cell was positive and pronounced gating when negative. Endogenous N2a cell connexin expression levels were 1,000-fold lower than exogenously expressed Cx40 and Cx43 levels, measured by real-time PCR and Western blotting methods, suggestive of heterotypic gap junction formation by exogenous Cx40 and Cx43. Imposing a [KCl] gradient across the heterotypic gap junction modestly diminished the asymmetry of the macroscopic normalized junctional conductance – voltage (G_j-V_j) curve when [KCl] was reduced by 50% on the Cx43 side and greatly exacerbated the V_j gating asymmetries when lowered on the Cx40 side. Pairing wild-type (wt) Cx43 with the Cx40 E9,13K mutant protein produced a nearly symmetrical heterotypic G_j-V_j curve. These studies conclusively demonstrate the ability of Cx40 and Cx43 to form rectifying heterotypic gap junctions, owing primarily to alternate amino-terminal (NT) domain acidic and basic amino acid differences that may play a significant role in the physiology and/or pathology of the cardiovascular tissues including cardiac conduction properties and myoendothelial intercellular communication.     

Functional formation of heterotypic gap junction channels by connexins-40 and -43

Connexin40 (Cx40) and connexin43 (Cx43) are co-expressed in the cardiovascular system, yet their ability to form functional heterotypic Cx43/Cx40 gap junctions remains controversial. We paired Cx43 or Cx40 stably-transfected N2a cells to examine the formation and biophysical properties of heterotypic Cx43/Cx40 gap junction channels. Dual whole cell patch clamp recordings demonstrated that Cx43 and Cx40 form functional heterotypic gap junctions with asymmetric transjunctional voltage (V_j) dependent gating properties. The heterotypic Cx43/Cx40 gap junctions exhibited less V_j gating when the Cx40 cell was positive and pronounced gating when negative. Endogenous N2a cell connexin expression levels were 1,000-fold lower than exogenously expressed Cx40 and Cx43 levels, measured by real-time PCR and Western blotting methods, suggestive of heterotypic gap junction formation by exogenous Cx40 and Cx43. Imposing a [KCl] gradient across the heterotypic gap junction modestly diminished the asymmetry of the macroscopic normalized junctional conductance – voltage (G_j-V_j) curve when [KCl] was reduced by 50% on the Cx43 side and greatly exacerbated the V_j gating asymmetries when lowered on the Cx40 side. Pairing wild-type (wt) Cx43 with the Cx40 E9,13K mutant protein produced a nearly symmetrical heterotypic G_j-V_j curve. These studies conclusively demonstrate the ability of Cx40 and Cx43 to form rectifying heterotypic gap junctions, owing primarily to alternate amino-terminal (NT) domain acidic and basic amino acid differences that may play a significant role in the physiology and/or pathology of the cardiovascular tissues including cardiac conduction properties and myoendothelial intercellular communication.     

Desensitization of β‐adrenergic receptors in lung injury induced by 2‐chloroethyl ethyl sulfide,a mustard analog

2‐Choloroethyl Ethyl Sulfide (CEES) exposure causes inflammatory lung diseases, including acute respiratory distress syndrome (ARDS) and pulmonary fibrosis. This may be associated with oxidative stress, which has been implicated in the desensitization of beta‐adrenergic receptors (β‐ARs). The objective of this study was to investigate whether lung injury induced by intratracheal CEES exposure (2 mg/kg body weight) causes desensitization of β‐ARs. The animals were sacrificed after 7 days and lungs were removed. Lung injury was established by measuring the leakage of iodinated‐bovine serum albumin ([¹²⁵I]‐BSA) into lung tissue. Receptor‐binding characteristics were determined by measuring the binding of [³H] dihydroalprenolol ([³H] DHA) (0.5–24 nM) to membrane fraction in the presence and absence of DLDL ‐propranolol (10 μ M). Both high‐ and low‐affinity β‐ARs were identified in the lung. Binding capacity was significantly higher in low‐affinity site in both control and experimental groups. Although CEES exposure did not change K_D and B_max at the high‐affinity site, it significantly decreased both K_D and B_max at low affinity sites. A 20% decrease in β₂‐AR mRNA level and a 60% decrease in membrane protein levels were observed in the experimental group. Furthermore, there was significantly less stimulation of adenylate cyclase activity by both cholera toxin and isoproterenol in the experimental group in comparison to the control group. Treatment of lungs with 3‐isobutyl‐1‐methylxanthine (IBMX), an inhibitor of phosphodiesterase (PDE) could not abolish the difference between the control group and the experimental group on the stimulation of the adenylate cyclase activity. Thus, our study indicates that CEES‐induced lung injury is associated with desensitization of β₂‐AR. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:59–70, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20265     

Major involvement of connexin 43 in seminiferous epithelial junction dynamics and male fertility

In different epithelia, cell membranes contacting one another form intercellular junctional complexes including tight, adherens and gap junctions, which could mutually influence the expression of each other. We have here investigated the role of Cx43 in the control of adherens and tight junction proteins (N-cadherin, β-catenin, occludin and ZO-1) by using conditional Sertoli cell knockout Cx43 (SCCx43KO^−/−) transgenic mice and specific anti-Cx43 siRNA. Gap junction coupling and Cx43 levels were reduced in SCCx43KO^−/− as compared to Wild-type testes. Ultrastructural analysis revealed disappearance of gap junctions, the presence of tight and adherens junctions and persistent integrity of the blood-testis barrier in SCCx43KO^−/− testis. Occludin, N-cadherin and β-catenin levels were enhanced in SCCx43KO^−/− mice as compared to Wild-type animals whereas ZO-1 levels were reduced. Cx43 siRNA blocked gap junction functionality in Sertoli cells and altered tight and adherens protein levels. The Cx43 control of tight and adherens junctions appeared channel-dependent since gap junction blockers (glycyrrhetinic acid and oleamide) led to similar results. These data suggest that the control of spermatogenesis by Cx43 may be mediated through Sertoli cell Cx43 channels, which are required, not only in cell/cell communication between Sertoli and germ cells, but also in the regulation of other junctional proteins essential for the blood-testis barrier.     

HIF‐1α regulates EMT via the Snail and β‐catenin pathways in paraquat poisoning‐induced early pulmonary fibrosis

Paraquat (PQ) poisoning‐induced pulmonary fibrosis is one of the primary causes of death in patients with PQ poisoning. Hypoxia‐inducible factor‐1α (HIF‐1α) and epithelial‐mesenchymal transition (EMT) are involved in the progression of pulmonary fibrosis. Snail and β‐catenin are two other factors involved in promoting EMT. However, the relationship among HIF‐1α, Snail and β‐catenin in PQ poisoning‐induced pulmonary fibrosis is not clear. Our research aimed to determine whether the regulation of HIF‐1α in EMT occurs via the Snail and β‐catenin pathways in PQ poisoning‐induced pulmonary fibrosis. Sixty‐six Sprague–Dawley rats were randomly and evenly divided into a control group and a PQ group. The PQ group was treated with an intragastric infusion of a 20% PQ solution (50 mg/kg) for 2, 6, 12, 24, 48 and 72 hrs. A549 and RLE‐6TN cell lines were transfected with HIF‐1α siRNA for 48 hrs before being exposed to PQ. Western blotting, real‐time quantitative PCR, immunofluorescence, immunohistochemistry and other assays were used in our research. In vivo, the protein levels of HIF‐1α and α‐SMA were increased at 2 hrs and the level of ZO‐1 (Zonula Occluden‐1) was reduced at 12 hrs. In vitro, the transient transfection of HIF‐1α siRNA resulted in a decrease in the degree of EMT. The expression levels of Snail and β‐catenin were significantly reduced when HIF‐α was silenced. These data demonstrate that EMT may be involved in PQ poisoning‐induced pulmonary fibrosis and regulated by HIF‐1α via the Snail and β‐catenin pathways. Hypoxia‐inducible factor‐1α may be a therapeutic target for the treatment of PQ poisoning‐induced pulmonary fibrosis.