Cx43 mRNA Expression Research Articles

Role of gap junctions in the contractile response to agonists in the mesenteric artery of spontaneously hypertensive rats

To investigate the effects of hypertension on the changes in gap junctions between vascular smooth muscle cells (VSMCs) in the mesenteric artery (MA) of spontaneously hypertensive rats (SHRs). Whole-cell patch clamp, pressure myography, real-time quantitative reverse transcription PCR (qRT-PCR), western blot analysis and transmission electron microscopy were used to examine the differences in expression and function of the gap junction between MA VSMCs of SHR and control normotensive Wistar-Kyoto (WKY) rats. (1) Whole-cell patch clamp measurements showed that the membrane capacitance and conductance of in-situ MA VSMCs of SHR were significantly greater than those of WKY rats (P<0.05), suggesting enhanced gap junction coupling between MA VSMCs of SHR. (2) The administration of phenylephrine (PE) and KCl (an endothelium-independent vasoconstrictor) initiated more pronounced vasoconstriction in SHR versus WKY rats (P<0.05). Furthermore, 2-APB (a gap junction inhibitor) attenuated PE- and KCl-induced vasoconstriction, and the inhibitory effects of 2-APB were significantly greater in SHR (P<0.05). (3) The expression of connexin 45 (Cx45) mRNA and protein in the MA was greater in SHR versus WKY rats (P<0.05). The level of phosphorylated Cx43 was significantly higher in SHR versus WKY rats (P<0.05), although the expression of total Cx43 mRNA and protein in the MA was equivalent between SHR and WKY rats. Electron microscopy revealed that the gap junctions were significantly larger in SHR versus WKY rats. Increases in the expression of Cx45 and phosphorylation of Cx43 may contribute to the enhancement of communication across gap junctions between MA VSMCs of SHR, which may increase the contractile response to agonists.

Dietary omega-3 fatty acids attenuate myocardial arrhythmogenic factors and propensity of the heart to lethal arrhythmias in a rodent model of human essential hypertension

Hypertension-induced myocardial remodeling is known to be associated with increased risk for malignant arrhythmias and alterations in electrical coupling protein, connexin-43 (Cx43), may be involved. We investigated whether omega-3 fatty acids intake affects abnormalities of Cx43 as well as protein kinase C (PKC) signaling and myosin heavy chain (MyHC) profile at the early and late stage of hypertension in the context of the heart's susceptibility to ventricular fibrillation and ability to restore sinus rhythm. Untreated young and old male spontaneously hypertensive rats (SHRs) and age-matched normotensive rats were compared with animals supplemented by omega-3 (eicosapentaneoic acid + docosahexaneoic acid, 200 mg/kg body weight/day) for 2 months. Left ventricular tissues were taken for examination of subcellular integrity of gap junctions, Cx43 mRNA and protein expression, PKCε and PKCδ as well as MyHC determination. Electrically inducible ventricular fibrillation and sinus rhythm restoration (SRR) were examined on Langedorff-perfused heart preparation. Omega-3 intake significantly reduced cardiovascular risk factors, suppressed inducible ventricular fibrillation, and facilitated SRR in hypertensive rats. Supplementation attenuated lateralization and internalization of Cx43, suppressed elevated Cx43 mRNA, enhanced total Cx43 protein expression and/or expression of its functional phosphorylated forms as well as the expression of cardioprotective PKC-ε and suppressed pro-apoptotic PKC-δ isoform. Moreover, the omega-3 diet normalized MyHC profiles in SHR at early stage of disease and old nonhypertensive rats, but failed to do so in old SHR at late stage of disease. Findings suggest that amelioration of myocardial Cx43-related abnormalities, positive modulation of PKC pathways, and normalization of MyHC can significantly contribute to the antiarrhythmic effects of omega-3 in rat model mimicking human essential hypertension. Our results support the prophylactic use of omega-3 to minimize cardiovascular risk and sudden arrhythmic death.

Connexin-43 can delay early recurrence and metastasis in patients with hepatitis B-related hepatocellular carcinoma and low serum alpha-fetoprotein after radical hepatectomy

BackgroundWe studied the relationships among Cx43, CD105, and VEGF in specimens of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) with different serum AFP levels with respect to recurrence and metastasis.MethodsExpressions of Cx43, CD105, and VEGF in 234 HCC tissue specimens were examined using tissue microarray and immunohistochemistry. Cx43 mRNA expression was examined in 38 frozen HCC specimens using RT-PCR. Correlations between these expressions and tumor recurrence, metastasis, and prognosis were analyzed using Kaplan–Meier and Cox regression analyses.ResultsCx43 expression correlated with early tumor recurrence (P = 0.001), disease-free survival (P = 0.026), and overall survival (P = 0.000) in patients with serum AFP < 400 ng/ml, but not in those with serum AFP ≥ 400 μg/L. Cx43 expression is an independent predictor of later recurrence and longer overall survival and is inversely correlated with expression of CD105 and VEGF (P = 0.018 and 0.023, respectively), histological differentiation (P = 0.002), vessel tumor embolism (P = 0.029), and focal number (P = 0.017). Immunohistochemistry showed that Cx43 expression in patients with low AFP was lower in patients with distant metastases than in those with no metastasis or those with liver metastasis. Patients with early recurrence expressed less Cx43 mRNA than did those with no recurrence (χ2 = 9.827, P = 0.002).ConclusionsCx43 expression can delay early HCC recurrence, metastasis, and poor prognosis after radical hepatectomy in patients with HBV-related HCC and low AFP.

Roles of 3,4-methylenedioxymethamphetamine (MDMA)-induced alteration of connexin43 and intracellular Ca2+ oscillation in its cardiotoxicity

Although it is well known that 3,4-methylenedioxymethamphetamine (MDMA) can cause various cardiovascular abnormalities and even sudden death from cardiac arrhythmia, whether it has any effect on myocardial gap junctions, which might be one of the targets mediating MDMA-induced cardiotoxicity, remains unclear. ObjectiveTo test the hypothesis that MDMA may affect the myocardial gap junction protein connexin43 (Cx43) and induce cardiac dysrhythmia. Method(1) In vivo study: adult rats were treated with a single dose MDMA administration (20mg/kg, i.p.). Electrocardiogram detection and immunohistochemical analysis were performed to evaluate cardiac function and expression of Cx43, respectively; (2) in vitro study: cultured ventricular myocytes of neonatal rats were treated with MDMA (10, 100, 1000μmol/L) for 1h. Western blotting and real-time quantitative polymerase chain reaction (RT-qPCR) were performed to investigate the total Cx43 mRNA expression. Immunofluorescent analysis was used to evaluate the amount of junctional Cx43. The phosphorylation status of Cx43 at site Ser368 and intracellular Ca2+ oscillation were also studied. ResultsObvious changes in electrocardiographic patterns were found in rats following MDMA administration. They were characterized by prolonged QRS duration associated with increased amplitude of QRS complex. The heart rates in treated rats were significantly decreased compared to the rats in the control group. The immunohistochemical findings revealed a significant decrease in Cx43 expression. The in vitro study also showed a marked decline in total Cx43 protein associated with reduction of Cx43 mRNA, whereas the phosphorylated Cx43 at Ser368 was increased. Decrease of junctional Cx43 was found correlated with reduction in N-cadherin induced by high concentration of MDMA. Additionally, confocal microscopy findings revealed alteration of intracellular calcium oscillation patterns characterized by high frequency and increasing influx Ca2+. ConclusionsMDMA reduces expression of cardiac gap junction protein Cx43. The increase of phosphorylation status of Cx43 at Ser368 induced by MDMA is attributed, at least in part, to the Ca2+-dependent regulation of protein kinase C (PKC) activity. Our findings provide first evidence of MDMA-mediated changes in those cardiac gap junctions that may underlie MDMA-induced cardiac arrhythmia.

Cardiac fibroblasts inhibit β-adrenoceptor-dependent connexin43 expression in neonatal rat cardiomyocytes

Cardiac fibroblasts play an important role in adverse cardiac remodelling. As in many cardiac diseases connexin43 (Cx43) is altered, we wanted to elucidate whether fibroblasts may influence cardiac Cx43 expression. We used four different cell culture systems of neonatal rat cardiomyocytes (CM) and fibroblasts (FB): type 1, pure CM culture; type 2, co-culture of CM/FB; type 3, pure FB culture; type 4, Transwell® system: CM/FB co-cultured but separated by a microporous membrane. Stimulation of types 1-3 cell culture models with isoprenaline significantly enhanced Cx43-protein and Cx43-mRNA expression as well as phosphorylation of ERK and translocation of AP1 and CREB only in the CM cultures; whereas, the CM/FB co-cultures and the FB cultures did not respond to isoprenaline. Similarly, if CM and FB were separated by a microporous membrane (Transwell® system) the isoprenaline-induced increase in CM Cx43 was completely suppressed, suggesting the existence of a soluble factor responsible for the suppressant effect of FB. Angiotensin II determination in types 1 and 2 cell culture supernatants revealed that the CM/FB co-cultures exhibited a significant higher angiotensin II release than the CM cultures. Furthermore, we aimed to inhibit angiotensin II signal transduction pathway: blockade of AT1 receptors or PKC inhibition restored the responsiveness of CM/FB co-cultures to isoprenaline. Moreover, external addition of angiotensin II to CM cultures also resulted in suppression of isoprenaline-stimulated Cx43 expression in an AT1-receptor- and PKC-dependent manner. Thus, our study indicates that cardiac fibroblasts inhibit β-adrenoceptor-dependent Cx43 signalling in CM involving angiotensin II.

Expression of Connexin 43 and E-cadherin Protein and mRNA in Non-small Cell Lung Cancers in Chinese Patients

Connexin 43 (Cx43) and E-cadherin are important biomarkers related with cancer. Their expression at protein and mRNA levels was here investigated in 50 primary lung carcinoma tissues and 20 samples of adjacent normal tissue of Chinese patients with non-small cell lung cancer (NSCLC). Protein and mRNA expression were evaluated by ABC immunohistochemistry and RT-PCR. (1) The positive expression rates of Cx43 and E-cadherin protein were higher in the adjacent normal tissues than those in the primary lung carcinoma tissues; (2) the positive expression rates of Cx43 and E-cadherin protein decreased with NSCLC progression; (3) the expression of E-cadherin protein was not related with the pathological type of NSCLC; and (4) the relative quantity of the Cx43 or E-cadherin mRNA expression was correlated with the the histological type, clinical stage, cancer cell differentiation and the lymph node metastasis. The data suggested that the Cx43 and E-cadherin are reduced with NSCLC progression, and might be important biomarkers for judging the metastasis and prognosis.

17-β-Estradiol Counteracts the Effects of High Frequency Electromagnetic Fields on Trophoblastic Connexins and Integrins

We investigated the effect of high-frequency electromagnetic fields (HF-EMFs) and 17-β-estradiol on connexins (Cxs), integrins (Ints), and estrogen receptor (ER) expression, as well as on ultrastructure of trophoblast-derived HTR-8/SVneo cells. HF-EMF, 17-β-estradiol, and their combination induced an increase of Cx40 and Cx43 mRNA expression. HF-EMF decreased Int alpha1 and β1 mRNA levels but enhanced Int alpha5 mRNA expression. All the Ints mRNA expressions were increased by 17-β-estradiol and exposure to both stimuli. ER-β mRNA was reduced by HF-EMF but augmented by 17-β-estradiol alone or with HF-EMF. ER-β immunofluorescence showed a cytoplasmic localization in sham and HF-EMF exposed cells which became nuclear after treatment with hormone or both stimuli. Electron microscopy evidenced a loss of cellular contact in exposed cells which appeared counteracted by 17-β-estradiol. We demonstrate that 17-β-estradiol modulates Cxs and Ints as well as ER-β expression induced by HF-EMF, suggesting an influence of both stimuli on trophoblast differentiation and migration.

COMPARISON OF HUMAN AMNIOTIC FLUID-DERIVED AND UMBILICAL CORD WHARTON'S JELLY-DERIVED MESENCHYMAL STROMAL CELLS: CHARACTERISATION AND MYOCARDIAL DIFFERENTIATION CAPACITY

ObjectivesTo compare the characterisation and myocardial differentiation capacity of amniotic fluid-derived mesenchymal stromal cells (AF MSCs) and umbilical cord Wharton's Jelly-derived mesenchymal stromal cells (WJ MSCs).MethodsThe human AF MSCs were...

On the different roles of AT1 and AT2 receptors in stretch-induced changes of connexin43 expression and localisation

Cyclic mechanical stretch (CMS) and angiotensin II (ATII) play an important role in cardiac remodelling. Thus, we aimed to examine how ATII affects CMS-induced changes in localisation and expression of the gap junction protein connexin43 (Cx43). Neonatal rat cardiomyocytes cultured on gelatin-coated Flexcell cell culture plates were kept static or were exposed to CMS (110% of resting length, 1Hz) for 24h with or without additional ATII (0.1μmol/L). Moreover, inhibitors of ATII receptors (AT-R) were used (for AT(1)-R: losartan 0.1μmol/L, for AT(2)-R: PD123177 0.1μmol/L). Thereafter, the cardiomyocytes were investigated by immunohistology, PCR and Western blot. After 24h of CMS, cardiomyocytes were significantly elongated and orientated 75 ± 1.6° nearly perpendicular to the stretch axis. Furthermore, CMS significantly accentuated Cx43 at the cell poles (ratio Cx43 polar/lateral static: 2.32 ± 0.17; CMS: 10.08 ± 3.2). Additional ATII application significantly reduced Cx43 polarisation (ratio Cx43 polar/lateral ATII: 4.61 ± 0.42). The combined administration of ATII and losartan to CMS further reduced Cx43 polarisation to control levels, whilst the AT(2)-R blocker PD123177 restored polarisation. Moreover, CMS and ATII application resulted in a significant Cx43 protein and Cx43 mRNA up-regulation which could be blocked by losartan but not by PD123177. Thus, CMS results in a self-organisation of the cardiomyocytes leading to elongated cells orientated transversely towards the stretch axis with enhanced Cx43 expression and Cx43 accentuation at the cell poles. ATII enhances total Cx43 mRNA and protein expression probably via AT(1)-R (=inhibitory effect of losartan) and reduces Cx43 polarisation presumably via AT(2)-R, since PD123177 (but not losartan) inhibited the negative effects of ATII on polarisation.

Expression of connexin 32 and connexin 43 in acute myeloid leukemia and their roles in proliferation

Connexins (Cxs), a conserved family of trans-membrane proteins, function in the organization of cell-cell communicatin via gap junctions in multicellular organisms. However, the role of Cxs in acute myeloid leukemia (AML) is poorly understood. In this study, we investigated the relationship between cell proliferation and expression of connexin 43 (Cx43) and connexin 32 (Cx32) mRNA and proteins in acute myeloid leukemia in vitro. Proliferation was observed using a growth curve and the rate of proliferation was detected by MTT assay in the acute myeloid leukemia cell lines OCI-AML3 and OCIM2. Cell cycle and cell proliferation index were assessed by flow cytometry analysis. The mRNA expression of the gap junction genes Cx43 and Cx32 was detected by RT-PCR. The expression of Cx43 and Cx32 proteins in the cell lines was analyzed by western blot analysis and immunofluorescence. The doubling time of OCI-AML3 and OCIM2 was 48 h and 36 h, respectively. In OCIM2, the percentage of cells in the S phase fraction was 59.47±9.6%, and the proliferation rate was 78.12±8.9%; however, in OCI-AML3, the percentage of cells in the S phase was 24.95±5.8%, and the proliferation rate was 35.21±6.7%. At the mRNA level, both cell lines expressed Cx43 and Cx32, and there was no significant difference in the expression of Cx43 and Cx32 mRNA in the two cell lines. At the protein level, there was a significant difference in the expression of Cx43, but not of Cx32. The proliferation ability of OCIM2 was higher than OCI-AML3, and OCIM2 exhibited higher Cx43 western blot and fluorescence intensities compared with OCI-AML3. The results suggest that a higher expression of Cx43 in AML cells may play a significant role in the proliferation ability.

Contractile Protein and Extracellular Matrix Secretion of Cell Monolayer Sheets Following Cyclic Stretch

Cell therapy treatment of post-myocardial infarction patients aims to regenerate tissue to improve function and prevent scarring. Although scaffold-free cell sheets offer an attractive solution, the monolayers are limited currently in their integrity as the individual layers do not cyclically stretch and cannot match the mechanical force transduction of the myocardium. In this study, we have explored stretching HL-1 mouse cardiomyocytes and embryonic stem cells derived into cardiomyocytes cultured in monolayers, and on temperature-responsive substrates to increase contractile protein and extracellular matrix (ECM) secretion for the purpose of reinforcing the baseline proteins and ECM. Sheets of HL-1 cells were stretched at 3 conditions (5, 10, 19%) along with static, unstretched controls at 1 Hz for 24 h. Embryonic stem cells derived into cardiomyocytes were stretched at 0 and 10% at 1 Hz for 24 h. Contractile proteins α- and β-myosin heavy chain (αMHC and βMHC) and sarcomeric α-actinin exhibited a significant increase in protein expression at 10% elongation strain. Atrial natriuretic factor (ANF) and connexin43 (Cx43) mRNA expression increased between static and conditioned cells; however, mRNA and protein expression at 19% strain increased less than cyclically strained cells at 5% strain. ECM secretion was not found to increase with conditioning. Effective engineering of cell monolayers with improved mechanical integrity may require an optimal strain condition to achieve maximum expression.

Expression of Connexin 43 in the Porcine Foetal Gonads During Development

This study was designed to reveal connexin 43 (Cx43) mRNA and protein expression in porcine foetal gonads using RT-PCR, immunohistochemistry and Western blot analysis. Expression of Cx43 was investigated in porcine foetal ovaries and testes on days 50, 70 and 90 post coitum (p.c.). RT-PCR results indicated that Cx43 mRNA was expressed in both foetal ovaries and testes at all gestational ages examined. Cx43 protein was found in the foetal ovary but its distribution varied across ovarian compartments and changed during development. In foetal ovaries, Cx43 was localized between the interstitial cells surrounding egg nests on all investigated days of prenatal period. Moreover, Cx43 expression was observed between germ cells on day 50 p.c. as well as between pre-granulosa and granulosa cells of primordial and primary follicles on days 70 and 90 p.c. In the foetal testes, Cx43 protein was detected between neighbouring Leydig cells on all examined days of prenatal period and between adjacent Sertoli cells exclusively on day 90 p.c. The presence of Cx43 protein in all investigated foetal gonads was confirmed by Western blot analysis. Cx43 protein detection between pre-granulosa cells of primordial follicles suggests its role in regulation of the initial stages of follicle development. The Cx43 immunoexpression between neighbouring Leydig and between Sertoli cells indicates its involvement in controlling their functions. We propose that Cx43-mediated gap junctional communication is involved in the regulation of porcine foetal gonadal development.

In vitro-growth and Gene Expression of Porcine Preantral Follicles Retrieved by Different Protocols.

This study was conducted to determine how the isolation method of the porcine preantral follicles influenced the following follicular growth in vitro. Mechanical and enzymatical isolations were used for retrieving the follicles from prepubertal porcine ovaries, and in vitro-growth of the follicles and the expression of folliculogenesis-related genes were subsequently monitored. The enzymatic retrieval with collagenase treatment returned more follicles than the mechanical retrieval, while the percentage of morphologically normal follicles was higher with mechanical retrieval than with enzymatic retrieval. After 4 days of culture, mechanically retrieved, preantral follicles yielded more follicles with normal morphology than enzymatically retrieved follicles, which resulted in improved follicular growth. The mRNA expression of FSHR, LHR Cx43, DNMT1 and FGFR2 genes was significantly higher after culture of the follicles retrieved mechanically. These results suggest that mechanical isolation is a better method of isolating porcine preantral follicles that will develop into competent oocytes in in vitro culture.

Age‐related changes in gap junctional intercellular communication in osteoblastic cells

Aging demonstrates deleterious effects upon the skeleton which can predispose an individual to osteoporosis and related fractures. Despite the well-documented evidence that aging decreases bone formation, there remains little understanding whereby cellular aging alters skeletal homeostasis. We, and others, have previously demonstrated that gap junctions--membrane-spanning channels that allow direct cell-to-cell conductance of small signaling molecules--are critically involved in osteoblast differentiation and skeletal homeostasis. We examined whether the capacity of rat osteoblastic cells to form gap junctions and respond to known modulators of gap junction intercellular communication (GJIC) was dependent on the age of the animal from which they were isolated. We observed no effect of age upon osteoblastic Cx43 mRNA, protein or GJIC. We also examined age-related changes in PTH-stimulated GJIC. PTH demonstrated age-dependent effects upon GJIC: Osteoblastic cells from young rats increased GJIC in response to PTH, whereas there was no change in GJIC in response to PTH in osteoblastic cells from mature or old rats. PTH-stimulated GJIC occurred independently of changes in Cx43 mRNA or protein expression. Cholera toxin significantly increased GJIC in osteoblastic cells from young rats compared to those from mature and old rats. These data demonstrate an age-related impairment in the capacity of osteoblastic cells to generate functional gap junctions in response to PTH, and suggest that an age-related defect in G protein-coupled adenylate cyclase activity at least partially contributes to decreased PTH-stimulated GJIC.

Prostaglandin E2 maintains mouse ESC undifferentiated state through regulation of connexin31, connexin43 and connexin45 expression: Involvement of glycogen synthase kinase 3β/β‐catenin

Although previous reports have examined the function of prostaglandin E₂ (PGE₂) on gap junctions and undifferentiated stem cells, its effects on the reciprocal action of connexin (Cx) isoforms and undifferentiation in embryonic stem cells (ESCs) are unclear. Therefore, we investigated the role of PGE₂ on Cx isoforms and maintenance of mouse ESC undifferentiated state. We have analysed 10 Cx genes, but found nine of them. PGE₂ (50 μM) stimulated Cx31, Cx32, Cx40, Cx43 and Cx45 mRNA expression. Amongst them, PGE₂ maximally stimulated the Cx43 mRNA expression and gap junction inter-cellular coupling. Therefore, we investigated the effect of PGE₂ on Cx43 expression. PGE₂ activated cAMP/protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI3K)/Akt phosphorylation. In addition, treatments of adenylate cyclase activators increased Cx43 expression, but not PI3K/Akt phosphorylation. PGE₂ also inactivated GSK-3β and stimulated active-β-catenin. Furthermore, a ChiP assay demonstrated the association of β-catenin with the Cx26 (as control) and Cx43 promoter. Finally, down-regulation of PGE₂-induced Cx isoforms by AH 6809, Cx31-, Cx43-, Cx45 small interfering (si)RNA and 18α-glycyrrhetinic acid decreased levels of undifferentiated markers of ESCs, including Oct4, FoxD3, Sox2 and SSEA-1, but Nanog did not be down-regulated by Cx43 siRNA. PGE₂ stimulates Cx isoforms via GSK-3β/β-catenin via EP2-receptor-dependent cAMP/PKA and PI3K/Akt in mouse ESCs, thereby partially contributing to the maintenance of their undifferentiated state.

Effect of ramipril on the regulation of the expression of connexins 40 and 43 in a rabbit model of arterial balloon injury

Gap junctions (GJs) between the cells play a pivotal role in the transformation and proliferation processes of vascular smooth muscle cells (VSMCs). However, the expression of the component proteins of GJs, connexins40 and 43 (Cx40 and Cx43), are inconsistent in numerous cases. The aim of this study was to determine whether Cx40 and Cx43 play different roles in the renin-angiotensin system (RAS) involved in the remodeling of GJs in VSMCs under pathological conditions. A total of 28 male New Zealand white rabbits were divided medially into four groups: control, sham injury, injury and injury plus ramipril (0.5 mg/kg/day in the diet for two weeks). The animals were used to set up the rabbit model of arterial balloon injury. Transmission electron microscopy, western blotting, immunohistochemistry and reverse transcription‑polymerase chain reaction (RT-PCR) were performed on four samples of ballooned iliac arteries. Larger and more abundant GJs appeared in neointimal VSMCs and there were smaller and fewer GJs following ramipril treatment. mRNA and protein expression levels and level of immunostaining of Cx40 and Cx43 were consistently increased following injury. Although ramipril reduced the change in the levels of Cx43, no significant changes in Cx40 immunostaining, protein or mRNA levels were observed in the ramipril treatments. Ramipril may inhibit neointimal formation and downregulate the expression of Cx43 protein and mRNA, but the drug had no significant effect on the Cx40 protein and mRNA levels, suggesting that it was not Cx40 but Cx43 in GJs that contributes to the process of angiotensinII (Ang II)-converting enzyme inhibitors inhibiting the prolife-ration of VSMCs in balloon injury.

Involvement of connexin43 hemichannel in ATP release after -irradiation

Ionizing radiation induces biological effects not only in irradiated cells but also in non-irradiated cells, which is called the bystander effect. Recently, in vivo and in vitro experiments have suggested that both gap junction hemichannel connexin43 (Cx43) and extracellular adenosine triphosphate (ATP) released from cells play a role in the bystander effect. We have reported that γ-irradiation induces ATP release from B16 melanoma cells, which is dependent on the P2X7 receptor. However, the mechanism of ATP release caused by irradiation remains unclear. We here show the involvement of Cx43 in P2X7 receptor-dependent ATP release after 0.5 Gy γ-irradiation. Inhibitors of gap junction hemichannels and an inhibitory peptide for Cx43 (gap26), but not an inhibitory peptide for pannexin1 (Panx1), significantly blocked γ-irradiation-induced ATP release from B16 melanoma cells. We confirmed high expression of Cx43 mRNA in B16 melanoma cells. These results suggest involvement of Cx43 in radiation-induced ATP release. We found that after 0.5 Gy γ-irradiation tyrosine phosphorylation was significantly blocked by P2X7 receptor antagonist, but not gap26, suggesting that tyrosine phosphorylation is a downstream event from the P2X7 receptor. Since tyrosine kinase inhibitor significantly suppressed radiation-induced ATP release, tyrosine phosphorylation appears to play an important role in the Cx43-mediated ATP release downstream of the P2X7 receptor. In conclusion, the Cx43 hemichannel, which lies downstream of the P2X7 receptor, is involved in ATP release in response to radiation. Our results suggest a novel mechanism for radiation-induced biological effects mediated by both ATP and Cx43.

Effect of hypaconitine combined with liquiritin on the expression of calmodulin and connexin43 in rat cardiac muscle in vivo

To study the effects of hypaconitine used alone and combined with liquiritin on calmodulin (CaM) expression and connexin43 (Cx43) phosphorylation on serine368 (Ser368), as well as to investigate the intervention of liquiritin on these hypaconitine-induced effects. Adult Wistar rats were orally administered hypaconitine (0.23, 0.69, 2.07 mg/kg per day), liquiritin (20 mg/kg per day), or hypaconitine (2.07 mg/kg per day) plus liquiritin (20 mg/kg per day) for seven consecutive days. The mRNA expression levels of CaM and Cx43 in rat myocardial tissue were determined by real-time quantitative PCR. The protein contents of CaM and phosphorylated Cx43 (Ser368) were determined by Western blot. The results indicated that the mRNA and protein expression levels of CaM were significantly decreased by hypaconitine used alone and combined with liquiritin. Although CaM mRNA expression level was inhibited by liquiritin, its protein expression level was upregulated. Meanwhile, although no obvious effect on Cx43 mRNA expression was observed after the drug administration, the phosphorylation level of Cx43 (Ser368) was significantly inhibited. Furthermore, the coadministration of hypaconitine and liquiritin significantly reduced hypaconitine-induced inhibitory action on Cx43 (Ser368) phosphorylation. The study indicated that hypaconitine could inhibit CaM expression and Cx43 (Ser368) phosphorylation, and liquiritin could interfere with this kind of effect by synergistically inhibiting CaM expression and by antagonizing Cx43 (Ser368) dephosphorylation induced by hypaconitine.

Human amniotic fluid-derived c-kit+ and c-kit− stem cells: growth characteristics and some differentiation potential capacities comparison

Amniotic fluid (AF) contains heterogeneous and multipotential cell types. A pure mesenchymal stem cells group can be sorted from AF using flow cytometry. In order to evaluate a possible therapeutic application of these cells, the human AF-derived c-kit(+) stem cells (c-kit(+) AFS) were compared with the c-kit(-) (unselected) stem cells (c-kit(-) AFS). Our findings revealed that the optimal period to obtain c-kit(+) AFS cells was between 16 and 22weeks of gestation. Following cell sorting, c-kit(+) AFS cells shared similar morphological and proliferative characteristics as the c-kit(-) AFS cells. Both c-kit(+) and c-kit(-) AFS cells had the characteristics of mesenchymal stem cells through surface marker identification by flow cytometric and immunocytochemical analysis. Both c-kit(+) and c-kit(-) AFS cells could differentiate along adipogenic and osteogenic lineages. However, the myocardial differentiation capacity was enhanced in c-kit(+) AFS cells by detecting GATA-4, cTnT, α-actin, Cx43 mRNA and protein expression after myocardial induction; whereas induced c-kit(-) AFS cells were only detected with GATA-4 mRNA and protein expression. The c-kit(+) AFS cells could have potential clinical application for myogenesis in cardiac regenerative therapy.

Higher susceptibility of spontaneous and NNK-induced lung neoplasms in connexin 43 deficient CD1 × AJ F1 mice: Paradoxical expression of connexin 43 during lung carcinogenesis

Connexins (Cxs) are proteins that form the communicating gap junctions, and reportedly have a role in carcinogenesis. Here, we evaluated the importance of Connexin43 (Cx43) in spontaneous and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung carcinogenesis. Male wild-type (Cx43(+/+) ) and hemizygote (Cx43(+/-) ) CD1 × AJ F1 mice were injected with NNK or saline. After 60 weeks mice were euthanized; lung nodules were counted, measured, and fixed in formalin or snap frozen. Immunohistochemistry for Cx43 and Beta-catenin (β-catenin) was performed and Cx43 mRNA expression was evaluated by real-time PCR. Cx43 deletion significantly increased the incidence and number of spontaneous nodules in the CD1 × AJ F1 mice and the number of gross lesions and the aggressiveness of lesions in NNK-treated mice. Cx43 mRNA increased significantly and was correlated with the aggressiveness of tumors, although lesions from Cx43(+/-) mice expressed less Cx43 RNAm than their counterparts. Lung parenchyma presented a Cx43 immunostaining pattern with points or plaques between cells. In hyperplasias and adenomas, Cx43 was found in the membrane and in cytoplasm. Malignant lesions presented increased Cx43 in cytoplasm and a few membrane spots of immunostaining. β-catenin was weakly expressed in lung parenchyma. Though hyperplasias presented some cells with nuclear β-catenin, NNK-induced tumors contained a higher number of this staining pattern. Also, no difference in β-catenin occurred between both genotypes independently of the histological grade. In summary, our results indicate that Cx43 acts as a tumor suppressor gene in early lung tumorigenesis and loses this property in advanced carcinogenesis. Therefore, Cxs are better classified as conditional tumor suppressors.