| Background Diabetes mellitus is an independent risk factor for coronary arterydisease, however, the underlying molecular mechanisms remain unclear. Largeconductance Ca2+-activated K+channel （BK channel） is an important ion channelmainly exclusively expressed in coronary vascular muscle cells, which is appreciatedin maintaining the membrane potential and contributing to the control of vasculartone by regulation of the excitation–contraction coupling process. Impaired BKchannel activities in smooth muscle cells may contribute to the development ofvascular dysfunction in diabetes mellitus, but the mechanisms underlying suchchanges have not been examined in detail.Objective Using patch clamp technique, western blot and fluorescent assaytechnique, we sought to investigate the effects of diabetes on BK channel andelucidate the molecular mechanisms of coronary dysfunction in diabetes.Methods （1） Streptozotocin-induced rat diabetic animal model was established byinjection intraperitoneally.（2） Coronary smooth muscle cells were isolated byenzyme digestion.（3） The BK currents in control and diabetic groups were recordedby patch clamp technique in single channel configuration.（4） BK channel proteinexpressions in normal and diabetic group were measured by Western blot.（5）Calcium concentrations in control group and diabetic group were measured byfluorescence assay.Results （1） Streptozotocin-induced rat diabetic animal model was establishedsuccessfully, blood glucose was markedly elevated（the diabetic group562.50±13.50mg/mL vs.121.80±3.17mg/dL in normal rats） and the successful rate was90%.（2）Isolated viable coronary smooth muscle cells were obtained, which remained thecontractile and electrophysiological properties. There were2030cells per field of vision in condition of magnification×100.（3） At1mol/L calcium concentration inexternal solution and test potentials at0mV,20mV,40mV,60mV,80mV,100mV,and120mV, the open probabilities （NP0） of BK channels in control group were0,0.0003±0.0001,0.0023±0.0007,0.0248±0.0043,0.0663±0.0369,0.3445±0.0445and1.2105±0.0481（n=5） respectively; and NP0were0,0.00002±0.00001,0.0003±0.0001,0.0026±0.0004,0.0257±0.0045,0.1361±0.0325and0.5217±0.1346（n=5） respectively in diabetic group. The NP0of BK channels in diabeticgroup were significantly decreased （P<0.05）.（4） Compared with control group, therewas no significant difference in-subunit protein expression in diabetic group（P>0.05）, however,1-subunit protein expression was remarkably reduced in diabeticgroup （P<0.05）.（5） Calcium concentrations in control group and diabetic group were（103±23） nmol/L （n=10） and19322nmol/L （n=6） respectively （P<0.05）.Conclusions （1） Intraperitoneal injection of streptozotocin can successfullyestablish diabetic rat animal model.（2） Coronary smooth muscle cells can be isolatedsuccessfully by enzyme digestion.（3） Downregulation of1-subunit and decrease ofBK current in smooth muscle cells is associated with increased vascular tone indiabetic coronary arteries.（4） Increase of cytosolic calcium concentration in smoothmuscle cells may be one of the important causes for coronary dysfunction in diabetes. |
