The Study On The Role Of Store Operated Calcium Channels In Contraction Of Bladder In Diabetic Rats

Objective: The aim of this study was to investigate the role of SOCCs in DCP with streptozocin?STZ?-induced diabetic rats.Specially,we established STZ induced DM rat model and study the effect of SOCCs on the contraction of DM bladder detrusor muscle strips in vitro,firstly.Then,we cultured the prmary detrusor cells and measured the changes of the concentration of intracellular Ca²⁺ under laser confocal microscopy?LCM?after activiation or inhibition the SOCCs.Lastly,we study the expression of STIM1?Orai1 m RNA and protein in DM rats at different stage of DCP.Methods: 1.In a serial experiment 70 of 6 to 8 week-old female S-D rats were matched by date of birth,and divided into 7 groups?n=10?.The normal control group?N group?was divided into 4 groups.The diabetes mellitus?DM group?group was divided into 3 groups.Diabetes was induced by a single intraperitoneal injection of 50 mg/kg streptozotocin dissolved in 0.1Mm citrate buffer solution,p H 4.5.The normal groups were treated with a similar volume of 0.1Mm citrate buffer solution only.DM groups:?1?the TM4 W group: the rats were sacrificed 4weeks after injection of STZ;?2?the TM8 W group: the rats were sacrificed 8 weeks after injection of STZ;?3?the TM12 W group: the rats were sacrificed 12 weeks after injection of STZ.N groups:?1?the N0 group: the rats were sacrificed 3 days after injection of citrate buffer;?2?the N1 group: the rats were sacrificed 4weeks after injection of citrate buffer;?3?the N2 group: the rats were sacrificed 8 weeks after injection of citrate buffer;?4?the rats were sacrificed 12 weeks after injection of citrate buffer.Three days after treatment,a blood sample was obtained by cutting the tail to estimate the random blood glucose level for three times.The bodyweight,blood glucose level and hematoxylin-eosin?H-E?staining were recorded.2.Bladder detrusor strips were prepared as 8*3*3mm from different groups and dipped in kreb's solution.The urothelium and submucosa layers were removed by using microinstruments under a microscope.SOCCs were activated by CPA andinhibited by SKF-96365 to research the effects of SOCCs on the contractive frequency and the amplitude of the strips.3.The enzyme digestion method was used to separate detrusor cells.Bladder tissue was digested with 4 mg/ml Collagen?and 4 mg/ml bovine serum albumin?BSA?,under 37?,5%CO2 condition for 40-60 min.Immunofluorescences was employing to detect the a-SMA special maker for identification bladder detrusor muscle cells?BDMCs?.4.BDMCs were pre-treated with Fluo-4AM after that the cells were cultured for 24 h.Using CPA or SKF-06365 to active or inhibit SOCCs to observe the changes of fluorescence intensity in different groups.The Ca²⁺ fluorescence intensity of DMC was detected in different groups.F1 was the measured value and F0 was the background value.F1/F0 was used to indicate the relative fluorescence intensity.5.RT-q PCR and Western Blot were used to detect the expressions of STIM1?Orai1 m RNA and protein in different groups.Results: 1.The blood glucose level of rats in DM groups is all higher than 16.7mmol/L.Contrast to the control group the bodyweight is significantly decreased?p<0.05?.Bladder dilatation and urine retention can be found.The blood glucose levels were significant higher in DM groups than N groups?p<0.05?.The H-E staining showed proliferation and hypertrophy of bladder smooth muscle cells in DM groups.The disordered fiber bundles and loose fibrous tissue were discovered as the diabetes progerssed.2.The changes of contractive frequency of the strips after CPA and SKF-96365 were used.The contractive frequency of strips in all experiment groups was increased after CPA was used.Compared to the control groups,the DM4 W and DM8 W was significant higher?p<0.05?.The contractive frequency of DM12 W was significant lower than DM4 W and DM8W?p<0.05?.There was no difference among the control groups.After inhibition SOCCs by SKF-96365,the contractive frequency of strips in all experiment groups was decreased.Contrast to the control groups,the DM4 W and DM8 W was still higher?p<0.05?.The contractive frequency of DM12 W was significant lower than DM4 W and DM8W?p<0.05?.There was no difference amongthe control groups.3.The changes of contractive amplitude of strips after CPA and SKF-96365 were used.The contractive amplitude of strips in all experiment groups was decreased with CPA and was rebounded to some extent with SKF-96365.But there were no significant differences among groups.4.We successfully obtained the primary detrusor muscle cells.The cells gradually grow to flake after 3 to 5 days.Be cultured for 7 days or so,the cells integration of 80% or more.The growth style of these cells was typical “peak-valley” manner.Immunofluorescences for ?-SMA were positive in these cells.5.The SOCCs was active with CPA.The fluorescence intensity in all experiment groups was increased.Compared to control groups,the DM4 W and DM8 W were significantly greater?p<0.05?.Among the three DM groups,DM4 W and DM8 W were higher than DM12W?p<0.05?.The similar results were obtained with SKF-96365.The fluorescence intensity in DM groups,DM4 W and DM8 W,were still higher than control groups.6.The expressions of Orai1 and STIM1 m RNA showed a similar trend at the three time points in SZT-induced diabetic rats,peaking at the 4th week and reaching its lowest level at the 12 th week.Compared with the control groups,the expressions of Orai1 m RNA were significantly higher in the DM4 W and DM8 W groups?p<0.05?,and the expressions of STIM1 m RNA were significantly higher in the DM4 W group?p<0.05?.Among DM groups,the expression of Orai1 m RNA in the 12 th week was significantly lower than the other two DM groups?p<0.05?.The expression of STIM1 m RNA in the 4th week was significantly higher than the other two DM groups?p<0.05?.The densitometric analysis of the bands for these proteins indicated that the expressions of Orai1 and STIM1 protein were similar with that of their m RNA levels in DM groups,which peaked in the 4th week and reached its lowest point in the 12 th week.Compared with control groups,the expressions of Orai1 and STIM1 protein were significantly higher in DM4 W and DM8 W groups?p<0.05?.Among DM groups,the expression of Orai1 protein at the 12 th week was significant lower than that at 4th and 8th weeks?p<0.05?.However,the expressions of STIM1 proteins were significantly different at all three time points?p<0.05?.Conclusions: 1.STZ-induced DM rat model was established successfully.SOCCs is involved in regulation of DM bladder detrusor strips contraction,and the contractive frequency of the strips in DCP was enhanced by SOCCs.2.Bladder detrusor muscle cells,for measurement the concentrate of intracellular Ca²⁺ under laser confocal microscopy?LCM?,were successfully cultured.SOCCs were involved in regulation of calcium concentration in bladder detrusor muscle cells of DM rats.The role of SOCCs in regulation of calcium concentration in bladder detrusor muscle cells in bladder were enhanced in DCP disease?4and 8weeks?.3.The expressions of STIM1 and Orai1 were enhanced at 4weeks in DCP.The expression of STIM1 m RNA and protein was changed in DM rats.