| Objective: To investigate gastric smooth muscle cell‘s large conductancecalcium-activated potassium channels (BKCa) of diabetic gastroparesis SDrats induced by STZ，and discuss the relevance between pathomechanismof diabetic gastroparesis and BKCa。Methods:1.Fifty healthy SD rats were randomly divided into two groups:control group (normal rats, n=20) and model group (diabetic rats, n=30).The rats of model group were induced by intraperitoneal injection of60mg／kg streptozotocin (STZ), the rats of control group received intraperitoneal injection of2ml/Kg buffer solution of citric acid. Rats were fedwith standard feedstuff regularly. The activity, appetite, the volume of urine and excrement, weight, concentration of blood glucose of every rat were observed and recorded.10weeks later, gastric emptying conduction velocity were detected. Rats were fasted but permitted to drink water freely for24hours before experiment, rats were injected into stomach with1mg/ml methylene blue solution0.4ml and killed30minutes later. The stomach residues were washed with4ml cold normal saline. The OD values of gastric residual pigment were detected with722spectrophotometer in640nm wavelength.2. Acute enzyme separation of SD rats gastric smooth muscle cells: Rats were fasted but permitted to drink water freely for24hours before being sacrificed experiment.single smooth muscle cell s of SD rats were enzymatically isolated in low calcium solution containing papain （2mg/mL）,DTT(2mg/mL),and bovine serum albumin(BSA,2mg/mL).Tissues were incubated at37℃in enzyme solution for15-40min and then suspended in low calcium solution..3. patch clamp single channel recording technique:The current was recorded on single myocyte in symmetric high potassium solution by patch clamp single channel recordingtechnique. Single channel current was amplified and filtered by patch clamp amplifier (CEZ-2200), then imported into the computer, pClamp6.0software was used to record the digital data and Clampfit10.1softwarewas used to analyze data.4.Observing the express of KCNMA and KCNMB1by using Immunohistochemistry: After fixing tissues by4%paraformaldehyde,cut tissues by Leica2245and check sections in microscope。detect KCNMA and KCNMB1According to the manual of antibody。Photograph the tissue section by （OLYMPUS1X71）and analyse graphic data by Image-Pro-Plus6.0。Results:1. The common situations of the rats in control group were obviously better than those in model group, typical symptom of diabetes mellitus appeared in model group rats, such as polyuria, polydipsia. The average weight of rats in model group is (190±23.71g) was significantly lower than that of rats in control group (428±25.05g g)，P<0.01. During this experiment, thirty diabetic rats and twenty control rats were chosen randomly to make experiment.. The average fasting concentration of blood glucose of rats in model group was （30.26±2.19mmol／L） was significantly higher than that of rats in control group (4.83±1.09mmol／L), P＜0.01. The rate of gastric residual pigmen of rats in model group （54.50%±3.92%） was significantly higher than that of rats in control group（40.69%±3.55%）,P＜0.01.2. Acute enzyme separation of SD rats gastric smooth muscle cells: The single gastric smooth muscle cell which was dissociated by papain can maintain its physiological functions, and itcan be used in patch clamp experiments3.(1) The properties of singlechannel currents of BKCachannels in SD rants smooth muscle cells were as follows:①S ingle channel currents ofBKCawere recorded in inside-out patch. The value of single channel conductance was220.10±10.90pS.②The channels had distinct voltage dependent and calcium dependentcharacteristics. When the [Ca2+]freeconcentration was0、10-7、5×10-7、10-6nM，the NPo of BKCa channel was0.018±0.007，0.0482±0.013,0.156±0.017,0.506±0.019。③In outside-out patch (Vm=+30mV),the activationof BKCachannel could be blocked by200nM IbTX completely.(2)In inside-out patch (Vm=+40mV)4. compered the single channel currents of control group SD rats BKCachannel with model group: in inside-outpatch (Vm=+40mV), the NPo of control group was0.018±0.006、Amp=7.9298±0.778pA、To=17.642±5.651ms、Tc=578.857±203.829ms，theNPo of model group was0.058±0.014、Amp=8.641±0.673、To=4.814±2.639、Tc=276.785±116.530. compered with control group,the NPo(t=9.294, P<0.01) and Amp(t=2.592,P=0.015) of model group significantly increased；the To（t=7.965,P<0.01） and Tc（t=7.965,P<0.01）significantly induced。5. Express of KCNMA and KCNMB1:the control group’s IOD of KCNMA was13558.2±2216.5，and KCNMB1was525875.5±65718.1；the model group’s IOD of KCNMA was29801.6±3123.1，KCNMB1was493504.6±27916.2。compared with control group，the KCNMB1’s IOD of model group significantly increased（t=9.484，P<0.01）. |
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