Effect Of Connexin 43 Phosphorylation In Detrusor Overactivity In Rat

Background and objective:Detrusor overactivity(DO) is a common clinical disease in which the bladder has a higher excitability than normal. The main symptoms of DO are urinary urgency and frequency, with or without urinary incontinence. The etiological factor and pathogenesis of DO are still unclear, and the treatment effect is not so satisfactory. Partial bladder outflow obstruction(PBOO) is the main reason for DO, its mechanism might involve the change in the detrusor innervation and myocyte excitability. Recently, changes in cell-cell communication were thought to be one of the important mechanism, and Connexin 43(CX43) play a key role in the development of DO. Compared to the control, the gap junction intercellular communication(GJIC) and the expression of CX43 were significant increased in DO group.CX43 is a phosphoprotein, the phosphorylation of CX43 can influence the GJIC, while whether the change of CX43 phosphorylation plays an important role on the pathogenesis of DO remains unknown.In our study, DO models were modeled to evaluate the effect of CX43 phosphorylation. In addition, the influence of PP1 and PP2 A on CX43 phosphorylation was preliminarily investigated.Methods:1. Establishing DO rat models: Sixty female rats(about 150g-180 g) were used in our study. Forty of them were anesthetized by intraperitoneal injecting phenobarbital(2%). The DO models were established by the operation of PBOO. Urodynamic evaluation was conducted to screen qualified model rats after 6 weeks. Sham-operated rats are regarded as control group.2. Western blotting: The proteins were extracted for western blotting as we reported previously. Proteins(50?g) were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membrane was blocked with 5% bovine serum albumin(BSA). The primary antibodies of CX43 polyclonal antibody, PP1, PP2 A, and β-actin were incubated at 4 ℃ over night. After incubated with secondary antibody conjugated to horseradish peroxidase, Immunolabeled proteins were detected by the ChemiDoc XRS+Image System. The relative expression levels were normalized with β-actin.3. Immunofluorescence staining: The bladders of six rats were sliced into 5 μm using freezing microtome. After attached on the glass slide, tissue sections were fixed in 4% paraformaldehyde(pH 7.4) for 30 min at 4°C and incubated in 1% BSA for 2 h at room temperature. Subsequently, the sections were incubated with CX43 monoclonal antibody, CX43 polyclonal antibody, PP1 antibody and PP2 antibody for 12 h at 4°C. The sections were washed with phosphate-buffered saline(PBS) for three times and incubated with the sencondary fluorescent antibodies conjugated with tetramethylrhodamine isothiocyanate(TRITC) and fluorescein isothiocyanate(FITC) for 1 h at room temperature. The sections were washed with PBS and incubated with the 4, 6-diamidino-2-phenylindole(DAPI) for 10 min at room temperature for nuclear staining. Finally, the sections were imaged using a laser confocal microscope.4. Isolation of bladder smooth muscle cells: The bladder was immersed into 5 ml digestion solution and cut into approximately 1-mm3 pieces. The digestion solution contains 2.0mg/mL type II collagenase, 2.0 mg/mL BSA, and 2.0 mg/mL trypsin inhibitor at 37°C. The tissue pieces were digested about 35 min and then 5 ml 10% BSA was added to terminate the digestion process. After centrifugation(1200 rpm/min for 5 min), the supernatant was discarded. Dulbecco’s modified Eagle’s media(DMEM) with 10% serum and 1% penicillin-streptomycin was added into tube to suspend the precipitate. The sample was dissociated about 300 times with the dropper. The cells were filtered away from the tissue pieces with a 200-mesh cell strainer(0.22μm) and seeded onto a glass coverslip at the bottom of a recording chamber. The cells were cultured in the incubator(37°C, 95% O2 and 5% CO2).5. Fluorescent bleaching recovery experiments(FRAP): The cells were washed 1 time with PBS, then 6-Carboxyfluorescein diacetate(6-CFDA, 10U/ml) was added into recording chamber. The cells were cultured in the incubator for 30 min and washed three times using PBS. Normal media was added into control group and DO group. Medium containing alkaline phosphatase(AP) was added into DO+AP group. Suitable cells were selected and bleached. Real-time images of fluorescence were collected for 5 min after bleaching.Results:1. DO model was successfully established. Total 23 rats were classified to the DO group.2. Compared with control group, the protein expressions of different phosphorylation status(P0, P1, P2) of CX43 are markedly increased(P<0.05), the PP2 A is reduced(P<0.05), the PP1 has no obvious change.3. The expression location of PP1, PP2 A is same with CX43.3. The GJIC of bladder smooth muscle is significantly enhanced in DO rats, while inhibited by AP.Conclusions:1. Increased CX43 protein phosphorylation level may play an important role in the progress of DO.2. Increased phosphorylation level of CX43 may be closely ralated to the decrease of PP2 A.