The Effect Of Blockade Of Cx43 On The GJIC Between Cultured Unstable Detrusor Cells

Background and Objective: Intercellular communication is of important significance to multicellular organisms, which mediates the metabolic and electrical coupling and co-ordinates the behavior of cells within tissues and organs. A major pathway for intercellular communication is via gap junctional channels , which are plasma structures that form intercellular channels between adjacent cells . Through these channels , small molecules ( MW<1000Da), such as ions , second messengers , Ca2+, cAMP and cGMP can rapidly diffuse from cell to cell . Gap junction have been found in almost all mammalian tissues with the exception of circulating red blood cells and adult skeletal musle. Gap junctional channel is about 30? in diameter and consists of two hemi-channels ,each provided by one of the coupled cells. The hemichannel ,termed connexon , is composed of six specific protein subunits which belong to the gene family of connexins.. To date , over 20 connexins have been cloned and sequenced. All connexins share a common structural topology consisting of four transmembrane domains lingked by one cytoplasmic and two extracellular with cytoplasmic N-and C-termini. The intercellular communication mediated by gap junction called gap junctional intercellular communication(GJIC), which is involved in many biologic processes, such as the harmonious contraction of smooth muscle cells, exchange of nutrients and ions between connected cells, transfer of electrical signals, control of cell growth and pathways for signaling compounds. Among all the tissues, myocardium is the most completely one that the function of GJIC is explored. Gap junction channels mediate the rapid , orderly progagation of antion potentials that is required for synchronized myocardial contraction. As heart, bladder is also an organ that have spontaneous contractions, and the common feature of DI is the abnormal spontaneous contractions. So people speculate that , probably , there is some relationship between DI and the abnormal GJIC. Up to date, over ten connexin (Cx) proteins have been found in bladder detrusor tissue, and among which , Cx43 is the the widest distributed in the condition of DI, which is about 75 fold than that in normal bladder. But a precise physiologic role of Cx43 in bladder function is unknown because of a lack of specific functional inhibitors .To investigate the role of Cx43 in intercellular communication among unstable detrusor cells, we selectively modified the expression of Cx43 gene using antisense cDNA transisient transfections in culture and try to demonstrate the feasibility of resisting excitatory communication as the target of therapy for DI. Materials and methods: The mammalian cell expression vector PCI-neo was friendly given by Dr. PENG Yong . This plasmid contains a cytomegalovirus ( CMV ) promoter , multiple cloning sites , and a neomycin(neo) gene driven by SV40 in a PBS322 backbone. The pBluescript plasmid containing the entire coding sequence ( c DNA ) for rat Cx43 was a generous gift from Dr. E.C. Beyer(university of Chicago). The 1.1 kb Cx43 cDNA fragment was inserted downstream of a CMV promoter in reverse orientation , so that the antisense Cx43 cDNA expression was driven by the CMV promoter. This construct, named ASCx43 cDNA/pCI-neo , was confirmed by restriction digestion and sequence analysis of the joined parts. Female Wistar rats of 2～3 months , weighing from 170-200g were used. Proximal urethra were partial ligated to produce the rat bladder outlet obstruction (BOO) models. Intravesical pressure was measured 6 weeks later. Rats were divided into DI group and normal control group. Primary cell cultures were set up from fragments of rat bladder. The fresh bladder tissues were excised under sterile condition and rinsed with sterile saline. After removal of the serosa and mucosa, small tissue fragments were digested with collagenase I at 37 ℃for 2h. The cells were cultured in DMEM-F12 medium supplemented with 10% fetal bovine ( FBS ), 2mM L-glutamine, 100U/ml penicillin ,and 100 ug/ml streptomycin. Cells were maintained at 37 ℃with 5% CO2 in a humidified incubator and spit every 4 days at a 1:3 dilution . Cells between passages 2 and 3 were used in these experiments . The antisense Cx43 cDNA plasmid was transfected into cultured detrusor cells using Lipofectamine 2000 following the standard protocol of the manufacturer (GIBCO-BRL). Approximately 0.5×106 trypsinized detrusor cells were seeded on a 60-mm cell culture plate and incubated at 37 ℃with 5% CO2 in a humidified incubator for 24 hours until the cells were 90-95% confluent. The DNA-Lipofectamine 2000 mixture (8ug DNA and 20 ulLipofectamine 2000 diluted in 1 ml Opti-MEM) was prepared and incubated at room temperature . Replaced the complete medium with 4ml Opti-MEM and added the 1 ml mixture into the plate after 20 minutes . Incubated the cells at 37 ℃for 6 hours and then replaced the Opti-MEM with complete medium and still incubated at 37 ℃. Seventy-two hours later , cells were harvest for the following assay. To evaluate the expression change of connexin 43 mRNA in cultured detrusor cells , hemiquantitive RT-PCR technology was used . To detect the protein level change of connexin 43 , western blot technology was used . And to estimate the function of gap junction intercellular communication among cultured detrusor cells, scrap loading dye transfer(SLDT) analysis was used. In this research , cultured cells of passage two were loaded with a mixture of two fluorescent dyes: Lucifer yellow(LY),which passes through gap junctions, and rhodamine-dextran(RD) (MW 10000Da), which does not pass through because of its high molecule weight .This is a more accurate assay than the traditional studies using LY alone because it avoids false positives caused by cell division and cytoplasmic bridges that allow the passage of RD. Dye solution consisted of 0.05%LY and 0.75%RD in PBS were prepared before each experiment. Cells were cultured on 6-well plates at 0.5×106 cells per well. Cells subjected to scrape-loading when the cells were 90% confluent following a protocol adapted from Pepper,et al. Cells were washed three times with PBS , and 1.5ml dye solution was added to each well . Multiple thin scrapes across each well were made with a scalpel blue .Dye loading and diffusion were performed for 2 minutes in dark room and then quickly washed threee times with PBS, and followed by adding 4% paraformaldehyde in PBS, pH 7.4 , for 30 minutes to prevent further dye diffusion. Cells were again wshed three times with PBS before measurement of dye transfer. Fluorescence was visualized using a inverted optical fluorescent microscope. The average pixel intensity in the direction parallel to the scrape line as a function of diffusion distance was measured by the image analysis system of LSM510META laser confocal scanning microscopy. Results:1. By hemi-quantitive RT-PCR technology, the expression of Cx43 mRNA in DI group was much higher than that in normal group. 2. By western blot technology, the protein level of Cx43 in DI group was obviously higher that in normal group.3. SLDT analysis showed that the function of GJIC among cultured unstable cells was stronger than that among normal cells. 4. By hemi-quantitive RT-PCR technology, the expression of Cx43 mRNA in ASCx43/pCI-neo transfected group was obviously lower than that in pCI-neo transfected group. 5. By western blot technology, the protein level of Cx43 in ASCx43/pCI-neo transfected group was significantly lower than that in pCI-neo transfected group. 6. In SLDT tests, the function of GJIC among ASCx43/pCI-neo transfected cells was lower than that among pCI-neo transfected group. Conclusion: 1. The expression of Cx43 gene was much higher in DI group than that in normal group , whether it was at mRNA level or protein level. 2. The function of GJIC amng cultured unstable cells was much stronger than that among normal cells. 3. Cx43 and GJIC may have a close relationship with detrusor instablily. 4. The expression of Cx43 gene can be obviously blockaded by antisense Cx43 cDNA. 5. The function of GJIC between ASCx43/pCI-neo transfected cells was lower than that between pCI-neo transfected group. 6. Cx43 is probably one of the majorest connexins that make up the gap junction in unstable detrusor. 7. Cx43 gene may play an important role in the development of DI and may be a potential target for gene therapy of Detrusor Instability.