To explore the role of connexin43 (Cx43) in gap junctional intercellular communication (GJIC) and propagated sensation along meridians, the expression of Cx43 in the rat epithelial cells and fibroblasts was studied both in vitro and in vivo. With the in vitro study, the rat epithelial cells and fibroblasts were cultured together, and the localization of Cx43 was detected by immunohistochemistry and indirect immunofluorescent cytochemistry and under confocal microscopy. And the expression of Cx43 on the surface of the cells was examined by flow cytometry. With the in vivo examination, 20 SD rats were randomized into control group (n = 10) and electrical acupuncture group (EA group, n = 10). EA ( 0.5-1. 5 V, 4-16 Hz , 30 min) was applied to "Zusanli" acupoint for 30 min at rat's hind paw, the localization of Cx43 was immunohistochemically detected. The immunohistochemical staining and indirect immunfluorescent cytochemistry showed that Cx43 was localized on the surface of the cells and in the cytoplasm. The relative expression level of Cx43 on the cellular membrane surfaces of the rat epithelial cells and fibroblasts, as determined by FACS, were 13.91% and 29.53% respectively. Our studied suggested that Cx43 might be involved in GJIC and propagated sensation along meridians.
Cells, Cultured ; Connexin 43/*biosynthesis ; Connexin 43/genetics ; Epithelial Cells/cytology ; Epithelial Cells/*metabolism ; Fibroblasts/cytology ; Fibroblasts/*metabolism ; Flow Cytometry ; Gap Junctions ; Meridians ; Microscopy, Confocal ; Random Allocation ; Rats, Sprague-Dawley

OBJECTIVETo explore the patterns of Cx43 and Pax3 protein expressions in the small intestinal muscular layers of human embryo during early development.

METHODSImmunohistochemistry with SABC method was employed to examine the expression of Cx43 and Pax3 proteins in the muscular layers of the small intestine in early human embryos in the second to fourth months of gestation.

RESULTSIn the second month of gestation, the muscle layer of the small intestine was negative for Cx43 and Pax3 protein expressions. In the third month, Cx43 and Pax3 expressions were negative in the inner circular muscle layer, but some positive cells were found in the longitudinal muscle layer and the myenteric plexus. In the fourth month, positive expression of Cx43 and Pax3 proteins were seen in the entire muscle layer.

CONCLUSIONCx43 and Pax3 proteins are closely related to the growth and development of the cells and tissues in the small intestinal muscle layer in human embryos.

Connexin 43 ; biosynthesis ; Embryo, Mammalian ; metabolism ; Humans ; Immunohistochemistry ; Intestine, Small ; embryology ; metabolism ; Muscle, Smooth ; embryology ; metabolism ; PAX3 Transcription Factor ; Paired Box Transcription Factors ; biosynthesis

OBJECTIVETo inhibit the expression of connexin43 (Cx43) in the human corpus cavernosum penis smooth muscle cells by small interfering RNA (siRNA) and detect the gap junction intercellular communication (GJIC), and to investigate the application of siRNA technology in the gap junction of corpus cavernosum penis smooth muscle cells and its role in the penile erection process.

METHODSWith the help of the software of Ambion Corporation, the specific recombinant plasmids with siRNA targeting human Cx43 gene were constructed. The recombinant plasmids having been stably transferred into human corpus cavernosum penis smooth muscle cells for 48 hours, semi-quantitive reverse transcription polymerase chain reaction (RT-PCR) and Western blotting techniques were used to examine the inhibitory effects of siRNA on the expressions of the Cx43 gene and protein, in comparison with the siRNA negative control and the blank control group, respectively. The GJIC was detected by scrape-loading and fluorescence dye transfer experiments through the fluorescence microscope.

RESULTSThe results of enzyme digestion analysis and DNA sequencing showed that the recombinant plasmid pSilencer 1.0-U6-siRNA-Cx43 was successfully constructed. The relative levels of Cx43 mRNA and protein expression in the smooth muscle cells were (0.45 +/- 0.08)% and (0.56 +/- 0.06)% after successful transfer of the recombinant plasmid. However, the expression levels of mRNA and protein were (0.72 +/- 0.04)% and (0.80 +/- 0.08)% in the negative siRNA transfer group, and (0.74 +/- 0.09)% and (0.77 +/- 0.11)% in the blank control, respectively, with a significant difference (P < 0.05). The GJIC also decreased significantly.

CONCLUSIONsiRNA can significantly inhibit the expression of Cx43 and block the GJIC in the human corpus cavernosum penis smooth muscle cells. siRNA technology plays an important role in penile erection and flaccidity.

Blotting, Northern ; Cells, Cultured ; Connexin 43 ; biosynthesis ; genetics ; Humans ; Intercellular Junctions ; Male ; Myocytes, Smooth Muscle ; physiology ; Penis ; cytology ; metabolism ; RNA, Small Interfering ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

OBJECTIVETo study the effects of extremely low frequency magnetic fields(ELF MF) on the amount and localization of connexin 43(Cx43) gap-junction protein in the Chinese hamster lung(CHL) cells, and to explore the mechanism of ELF MF suppression on gap-junctional intercellular communication(GJIC).

METHODSThe cells were irradiated for 24 h with 50 Hz sinusoidal magnetic field at 0.8 mT without or with 12-O-tetrade-canoylphorbol-3-acetate(TPA), 5 ng/ml for 1 h. The localization of Cx43 proteins were performed by indirect immunofluorescence histochemical analysis and detected by confocal microscopy. The second experiment was conducted to examine the quantity of Cx43 proteins level in nuclei or cytoplasm and detected by Western blotting analysis.

RESULTSThe cells exposed to TPA for 1 h displayed less bright labelled spots in the regions of intercellular junction than the normal cells. Most of Cx43 labelled spots occurred in the cytoplasm and aggregated near the nuclei. At the same time, the amount of Cx43 protein in cytoplasm were increased[(2.03 +/- 0.89) in ELF group, (2.43 +/- 0.82) in TPA group] as compared to normal control(1.04 +/- 0.17) (P < 0.01).

CONCLUSIONInhibition on GJIC function by ELF MF alone or combined with TPA may be related with the shift of Cx43 from the regions of intercellular junction to the cytoplasm.

Animals ; Cell Communication ; radiation effects ; Connexin 43 ; biosynthesis ; Cricetinae ; Cricetulus ; Cytoplasm ; metabolism ; Electromagnetic Fields ; adverse effects ; Gap Junctions ; radiation effects ; Lung ; metabolism ; radiation effects ; Tetradecanoylphorbol Acetate ; pharmacology

OBJECTIVE: To observe the change in expressions of connexin 32 and connexin 43 after the eradication of Helicobacter pylori (H.pylori) in patients with gastric precancerous lesion. METHODS: The expressions of connexin 32 and connexin 43 in gastric mucosa specimens biopsy under endoscopy were detected by immunohistochemistry. The expressions of connexin 32 and connexin 43 were detected before and after the eradication of H.pylori in 88 patients with gastric precancerous lesion, and in 33 patients with chronic superficial gastritis (CSG). RESULTS: The positive expression rates and the expressional intensity of connexin 32 and connexin 43 in patients with gastric precancerous lesions (51.1% and 54.5%) were lower than those in patients with CSG (100% and 93.9%, P < 0.05).In patients with gastric precancerous lesions,the positive expression rates and the expressional intensity of connexin 32 and connexin 43 in H.pylori positive group (41.4% and 44.8%) were lower than those in H.pylori negative group (70% and 73.3%, P < 0.05). In gastric precancerous lesions group, the positive expression rates of connexin 32 and connexin 43 in H.pylori positive group before the eradication therapy (41.4% and 44.8%, respectively) was lower than those after the eradication of H.pylori (97.9% and 91.7%, P < 0.05); in the eradication failure group, the positive expression rates of connexin 32 and connexin 43 were 40% and 50%. The eradication failure group before the treatment and after the treatment had no statistical significance(P > 0.05). CONCLUSION The expressions of connexin 32 and connexin 43 in patients with gastric precancerous lesions are low, and the eradication of H.pylori can upregulate their expressions.
Adult ; Aged ; Connexin 43 ; biosynthesis ; Connexins ; biosynthesis ; Female ; Gastritis ; metabolism ; microbiology ; Helicobacter Infections ; drug therapy ; metabolism ; Helicobacter pylori ; Humans ; Male ; Middle Aged ; Precancerous Conditions ; metabolism ; microbiology ; Stomach Neoplasms ; metabolism ; microbiology

OBJECTIVETo investigate the expressions of endothelial nitric oxide synthase (eNOS) and connexin 43 (Cx43) in the penile tissue of rats with diabetes mellitus induced erectile dysfunction (DMED) and their correlation with DMED.

METHODSSD rat models of DM were established by intraperitoneal injection of alloxan, and 8 weeks later, apomorphine was administered to induce ED in the DM models. The expressions of eNOS and Cx43 were measured by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively.

RESULTSAlloxan did not influence the expressions of eNOS mRNA and Cx43 mRNA in the penile tissue. Compared with the DM models, the expression of eNOS mRNA significantly decreased in the DMED group (0.155 +/- 0.157 vs 0.508 +/- 0.242, P < 0.01), while that of Cx43 mRNA markedly increased (0.993 +/- 0.157 vs 0.545 +/- 0.138, P < 0.01), with a negative correlation between the two expressions (r = -0.987). The same results were shown by immunohistochemistry in the penile smooth muscle cells of the DMED rats.

CONCLUSIONThe decrease of eNOS expression in the penile tissue might play a key role in the development of ED in diabetic patients, while the accompanying compensative elevation of the Cx43 level has yet to be further studied.

Animals ; Connexin 43 ; biosynthesis ; genetics ; Diabetes Mellitus, Experimental ; complications ; Erectile Dysfunction ; etiology ; physiopathology ; Immunohistochemistry ; Male ; Nitric Oxide Synthase Type III ; biosynthesis ; genetics ; Penis ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction

The relationship of connexin43 (Cx43) and bystander effect in ovarian tumor cells in herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy in vitro was explored and the effect of all-trans retinoic acid (RA) on the expression of Cx43 and bystander effect investigated. The Cx43 expression was detected by flowcytometry, Western blot, and immunofluorescence in two ovarian tumor cell lines OVCAR3, CaOV3 before and after RA treatment. Bystander effect was determined by the cells growth inhibitory rate with methyl thiazolyl tetrazolium. Following exposure to ganciclovir, there was much greater bystander killing in OVCAR3 than that in CaOV3 (P<0.05). The expression of Cx43 was detected in OVCAR3 by flowcytometry and Western blot, but it could not be detected in CaOV3. The expression of Cx43 in both cell lines could be induced by RA. Immunofluoresence staining showed that Cx43 protein of OVCAR3 was located on membrane surface, whereas CaOV3 in cytoplasm. RA could not change the location of Cx43 protein in both cell lines. There is relationship between Cx43 expression and HSV-TK/GCV bystander effect. HSV-TK/GCV bystander effect can be enhanced by RA in ovarian cancer.
Antiviral Agents ; pharmacology ; Bystander Effect ; Cell Line, Tumor ; Connexin 43 ; biosynthesis ; genetics ; Female ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; Genetic Therapy ; Humans ; Ovarian Neoplasms ; metabolism ; therapy ; Pregnancy ; Simplexvirus ; genetics ; Thymidine Kinase ; genetics ; Tretinoin ; pharmacology

OBJECTIVETo illuminate the regulating effect of all-trans retinoic acid (ATRA) on gap junctional intercellular communication (GJIC) and connexin 43 (Cx43) gene expression in glioma cells, which is tissue- and organ-specific.

METHODRat C6 glioma cells were exposed to ATRA at a concentration of 1, 10, 100 micromol/L and the GJIC function of the cells was examined with scrape-loading dye transfer assay 24 hours, 48 hours and 72 hours after ATRA treatment. The effect of ATRA on Cx43 gene expression was measured with semiquantitative reverse transcription polymerase chain reaction (RT-PCR) 24 hours after ATRA exposure.

RESULTSThe GJIC function of C6 glioma cells was significantly increased by ATRA at each concentration applied. The dye passed 4 to 5 rows of cells from the scraping edge in ATRA treated cells, but only 1 or 2 rows in the control. The augment effect was observed 24 hours after each concentration ATRA treatment, and lasted till 72 hours after treatment with 1 micromol/L and 10 micromol/L ATRA. Forty-eight hours after exposed to 100 micromol/L ATRA, the enhancement of GJIC was less obvious. There was no significant increase induced by ATRA on the transcription of Cx43 gene, as demonstrated by semiquantitative RT-PCR.

CONCLUSIONATRA turned out to be a potent enhancer on GJIC function in C6 glioma cells, andthe enhancement effect was most probable at post-transcriptional level.

Animals ; Antineoplastic Agents ; pharmacology ; Brain Neoplasms ; metabolism ; pathology ; Connexin 43 ; biosynthesis ; genetics ; Gap Junctions ; physiology ; Gene Expression ; Glioma ; metabolism ; pathology ; RNA, Messenger ; genetics ; Rats ; Tretinoin ; pharmacology ; Tumor Cells, Cultured

OBJECTIVE: To determine the effect of CagA(+) Helicobacter pylori(H.pylori)strain and anti-H.pylori drugs on the expression of connexin 43(Cx43) and cell proliferation of BGC-823 cells in vitro,and to investigate the relation between the changes of Cx43 expression, cell proliferation of BGC-823 cells and CagA(+)H.pylori. METHODS: BGC-823 cells were co-cultured with CagA(+) H.pylori strain(NCTC J99) or CagA(-) H.pylori strain(NCTC 12908)at bacteria/cells ratio of 20:1,100:1 and 500:1 for 24 hours and 48 hours respectively. anti-H.pylori drugs was given in the group co-cultured at bacteria/cells ratio of 100:1 after 16 hours. In the control group, BGC-823 cells were cultured for 24 hours and 48 hours respectively,but without H.pylori or antij H.pylori drugs. Immunocytochemical SABC method and the image analysis of the computer were applied to detect the changes of Cx43 expression in BGC-823 cells. The cell proliferation was examined by methyl tetrazolium (MTT) method. RESULTS: (1)The expression of Cx43 in the control group after cultivation for 48 hours was higher than that for 24 hours (P< 0.05). The expression of Cx43 in the groups co-cultured with CagA(+) H.pylori strain after cultivation for 48 hours was lower than that co-cultured for only 24 hours, and that of the groups co-cultured with CagA(+) H.pylori strain was lower than that of the control group for both 24 hours and 48 hours (P< 0.05). The expression of Cx43 in the groups at bacteria/cells ratio of 500:1 was lower than that at bacteria/cells ratio of 20:1 and 100:1 for both 24 and 48 hours (P< 0.05),and that at bacteria/cells ratio of 100:1 was lower than that at bacteria/cells ratio of 20:1 for 48 hours (P< 0.05).However, there was no significant difference in Cx43 expression between 24 and 48 hours in the groups co-cultured with CagA(-) H.pylori strain (P>0.05). Cx43 expression in the groups co-cultured with CagA(-) H.pylori strain at the ratio of 100:1 and 500:1 was lower than that in the control group, and Cx43 expression at the ratio of 500:1 was lower than that at the ratio of 20:1 for 24 hours and 48 hours. Cx43 expression increased after the intervention with anti-H.pylori drugs for 48 hours. (2) In the groups co-cultured with CagA(+)H.pylori strain, the optical density value of MTT indicated that the cell proliferation at the bacteria/cells ratio of 100:1 was higher than that in the control group, but no significant difference was found in other two groups co-cultured for 24 hours. After co-culturing for 48 hours, the cell proliferation at the bacteria/cells ratio of 20:1 and 100:1 was significantly accelerated, while the cell proliferation at 500:1 was inhibited. In the groups co-cultured with CagA(-) H.pylori strain,there was no change in the cell proliferation. Intervention with anti-H.pylori drugs could suppress the cell proliferation. CONCLUSION CagA(+) H.pylori can down-regulate the expression of Cx43 in BGC-823 cells,which is related to the reaction time and the density of H.pylori. Low density of CagA(+)H.pylori suspensions can accelerate the proliferation of BGC-823 cells, while high density can suppress the cell proliferation. The CagA(-) H.pylori has no effect on the cell proliferation. Intervention with anti-H.pylori drugs can up-regulate the expression of Cx43,and suppress the cell proliferation of BGC-823 cells.
Anti-Bacterial Agents ; pharmacology ; Antigens, Bacterial ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Connexin 43 ; biosynthesis ; Helicobacter pylori ; drug effects ; genetics ; metabolism ; Humans ; Immunohistochemistry ; Stomach Neoplasms ; metabolism ; microbiology ; pathology

OBJECTIVETo investigate the changes of expression of connexin-43 (Cx43) and the tight junction of microvessel endothelial cells (EC) to approach the effects in bleomycin (BLM) induced pulmonary fibrosis (PF).

METHODSForty healthy rats were equally and randomizedly divided into the control group and the experiment group. In both group, vWf in blood serum was measured with ELISA method on 3rd, 7th, 14th, 28th day after BLM treatment. Rats in each group were infused with lanthanum nitrate on 3rd, 7th, 14th, 28th day after BLM treatment. The lung samples were made and the tight junction and the distribution of the granules of lanthanum in the microvessel EC were observed with transmission electron microscopy in the control and experiment groups. The lung microvessel EC of the rats in each group were preserved by tissue culture methods at the same time, and the expression of Cx43 were observed by laser scanning confocal microscopy.

RESULTSThe serum vWf in the peripheral blood of the experiment group was significantly higher than that of the control group, and was the highest on 3rd day after BLM treatment (P < 0.01). The blood vessel EC of the control group were intact. The basement membrane was uninterrupted. Granules of lanthanum nitrate did not penetrate the tight junction of EC. The width of junction gap in the experimental group was increased and the lanthanum granules of high density were found deposited in the linear form in the gap junction. Low expression of Cx43 was observed in experiment group. The expression rate of Cx43 was 25%, 38%, 45% and 71% respectively on 3rd, 7th, 14th, 28th day, significantly less than those in the control group (P < 0.05).

CONCLUSIONIt may be the important pathological basis for the BLM induced abnormality of the interstitial tissues in the lung that the tight junction of EC is continuously in the open state, which causes the effusions of inflammatory cells and the corresponding cytokine secretion, and thus initiates the overproliferation of fibroblasts.

Animals ; Bleomycin ; pharmacology ; Connexin 43 ; biosynthesis ; Endothelial Cells ; drug effects ; metabolism ; Endothelium, Vascular ; cytology ; drug effects ; metabolism ; Female ; Lung ; blood supply ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Tight Junctions ; drug effects