| Background: Diabetes mellitus(DM) is a well-known risk factor for coronary artery disease. Several guidelines on the treatment of cardiovascular risk factors base their recommendations on the assertion that diabetes mellitus is a coronary heart disease or cardiovascular disease risk equivalent. Large conductance Ca2+- activated K+ channel(BK channel) is an important ion channel mainly exclusively expressed in coronary vascular muscle cells, which is appreciated in maintaining the membrane potential and contributing to the control of vascular tone by regulation of the excitation–contraction coupling process.Impaired BK channel activities in smooth muscle cells may contribute to the development of vascular dysfunction in diabetes mellitus, but the mechanisms underlying such changes have not been examined in detail. This study will clarify relationship between diabetes mellitus and coronary heart disease based on the dysfunction of the BK channel.Objective: Using multi-channel myograph system, patch clamp technique and western blot technique, we sought to investigate the effects of diabetes on BK channels and elucidate the molecular mechanisms of coronary dysfunction in diabetes.Methods:(1) Streptozotocin-induced rat diabetic animal model was established by injection intraperitoneally. Coronary smooth muscle cells were isolated by enzyme digestion.(2) Potassium channels in normal coronary smooth muscle cells were identified by applications of different potassium blockers.BK currents were recorded by patch clamp in whole cell configuration. BK currents amplitude and conductance were calculated.(3)Regulation of vascular tension in normal and diabetic coronary arteries was evaluated by videomicroscopy system. Changes of vascular tension in normal and diabetic coronary arteries were assayed by multi-wire myograph system.(4) The BK currents in control and diabetic groups were recorded by patch clamp technique in single channel configuration and whole cell.(5) BK channel protein expressions in normal and diabetic group weremeasured by Western blot.Results:(1) Streptozotocin-induced rat diabetic animal model was established successfully, blood glucose was markedly elevated in the diabetic group(562.50 ±13.50mg/m L vs. 121.80 ±3.17 mg/d L in normal), and the successful rate was 90%. Isolated viable coronary smooth muscle cells were obtained, which remained the contractile and electrophysiological properties. There were 20~30 cells per field of vision in condition of magnification ×100.(2) BK currents in normal smooth muscle cells accounted for(65±4)% of total potassium currents.(3) More than 50%was contracted in inner diameters of coronary arteries when 100nmol/L IBTX, a specific BK channel blocker, was applied.When 100mmol/L KCl was washed out, vascular tensions of normal and diabetic coronary artery were(398±38)mg and(390±35)mg respectively(P>0.05); however, when100 nmol/L IBTX was added.the Vascular tensions of norlnal and diabetic coronary artery were(395±40)mg and(50±7)mg(P<0.05).(4) In comparison with normal group, the BK current densities in diabetic group significantly decreased by patch clamp technique in whole cell configuration when test potentials were more than 60 m V(P<0.05). The BK current densities at 150 m V in normal group and diabetic group were(275±40)p A/p F and(70±10)p A/p F respectively.(5) At 1μmol/L calcium concentration in external solution and test potentials at 0 m V, 20 m V, 40 m V, 60 m V, 80 m V, 100 m V, and 120 m V, the open probabilities(NP0) of BK channels in control group were 0, 0.0003 ± 0.0001, 0.0023 ±0.0007, 0.0248 ± 0.0043, 0.0663 ± 0.0369, 0.3445 ± 0.0445 and 1.2105 ± 0.0481(n=5) by patch clamp technique in single channel configuration respectively; and NP0 were 0,0.00002 ± 0.00001, 0.0003 ±0.0001, 0.0026 ±0.0004, 0.0257 ±0.0045, 0.1361 ±0.0325 and0.5217 ±0.1346(n=5) respectively in diabetic group. The NP0 of BK channels in diabetic group were significantly decreased(P<0.05).(6) Compared with control group, there was no significant difference in α-subunit protein expression in diabetic group(P>0.05),however, β1-subunit protein expression was remarkably reduced in diabetic group(P<0.05).Conclusion:(1) BK channels are widely distributed in normal coronary smoothmuscle cells and play an important role in regulation of coronary vascular tension.(2) BK channels in diabetic coronary artery are dysfunctional, BK currents decrease and vascular tensions increase.(3) Down-regulation of β1-subunit and decrease of BK current in smooth muscle cells is associated with increased vascular tone in diabetic coronary arteries, which may be one of the important causes for coronary dysfunction in diabetes. |
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