| Objective Docosahexaenic acid(DHA)is one of the major components of n-3 polyunsaturated fatty acids.It has been reported that it can reduce the incidence of cardiovascular diseases.Large conductance Ca2+-activated-K+ channel(BK channel)is one of the most important ion channels on coronary vascular smooth muscle cells and plays in a pivotal role in the regulation of vascular tension.However,the electrophysiological mechanisms of DHA on BK channels in coronary vascular smooth muscle cells are not fully understood.The purpose of this study was to investigate the electrophysiological effects of DHA on BK channels and intracellular calcium ion concentrations in rat coronary smooth muscle cells by patch clamp and fluorescent assay.In the meanwhile,we investigated the activation mechanisms of DHA with different concentrations on BK channels,and the results may provide theoretical evidences for prevention and treatment of cardiovascular diseases.Methods(1)Normal rat coronary smooth muscle cells were separated by"three-step" enzyme digestion method,i.e.,normal rat coronary arteries were separated first and then incubated in digestive enzyme ?(1 mg/ml bovine serum albumin),digestive enzymes ?(1 mg/ml bovine serum albumin,1.5 mg/ml papain and 1 mg/ml dithiothreitol)and digestive enzymes ?(1 mg/ml bovine serum albumin,0.25 mg/ml elastic protease and trypsin inhibitor 1 mg/ml)for about 3 minutes respectively.(2)Whole-cell K+ currents were recorded from freshly isolated rat coronary smooth muscle cells(SMCs)before and after DHA was applied,to investigate the activation effects of DHA on different K+channels,various K+ channel blockers were applied after baseline K+current recordings were obtained.(3)The effects of different concentrations of DHA on BK channel currents in normal rat coronary SMCs were recorded by whole-cell and single channel patch clamp technique,and the effects of different concentrations of DHA on BK channel currents in normal rat coronary SMCs before and after incubation of SKF525A were explored in the meanwhile.(4)The effects of different calcium concentrations on the opening probabilities of BK channels were measured by single channel patch clamp technique.Fura-2/AM was used to detect the effects of different concentrations of DHA on the changes of cytosolic calcium concentrations in normal rat coronary SMCs.Effects of DHA on the changes of intracellur calcium concentrations in normal rat coronary SMCs were observed with U73122 and 2-APB,an inhibitor of PLC and an IP3 receptor blocker.Results(1)Isolation of rat coronary SMCs:Large numbers of coronary SMCs were obtained by "three-step" enzyme digestion.The shapes of living coronary SMCs were usually long spindle,clear outline,smooth cell membrane,homogeneous cytoplasm,good refraction,and they adhere to the wall quickly.(2)The total K+current densities before and after 5?mol/L DHA applied were 69.8±6.9 pA/pF and 425.0±142.3 pA/pF with no inhibitors elicited from holding potential of-60 mV and test potential of 100 mV(n=5,P<0.05),13.9±2.7 pA/pF and 14.1±3.2 pA/pF(n=5,P>0.05)in the presence of TEA(10 mmol/L),25.1±5.6 pA/pF and 89.1±29.2 pA/pF(n=5,P<0.05)in the presence of IBTX(100 nmol/L),55.8±9.2 pA/pF and 380.6±125.5 pA/pF(n=5,P<0.05)in the presence of TRAM-34(200 nmol/L),56.5±8.7 pA/pF and 345.6±110.1 pA/pF(n=5,P<0.05)in the presence of 4AP(5 mmol/L),35.3±9.9 pA/pF and 326.6±97.6 pA/pF(n=5,P<0.05)in the presence of APA(1 mmol/L),68.6±6.8 pA/pF and 349.6±115.9 pA/pF(n=5,P<0.05)in the presence of GLY(10 mmol/L),23.0±7.2 pA/pF and 81.6±23.7 pA/pF(n=5,P<0.05)in the presence of IBTX(100 nmol/L)plus TRAM-34(200 nmol/L)and APA(1 mmol/L),and 20.9±3.8 pA/pF and 73.9±21.9 pA/pF(n=5,P<0.05)in the presence of IBTX(100 nmol/L)plus 4AP(5 mmol/L).(3)BK currents can be activated with the DHA at 0.1 mmol/L,0.3 mmol/L,and 1 mmol/L,furthermore,3 mmol/L DHA increased BK currents by 737±86%and 10 mmol/L DHA increased BK currents by 822±43%(n=6,P<0.05).Pre-treatment with SKF525A,baseline BK currents were the same as control group and DHA had no effects on BK currents at concentrations between 0.01 mmol/L and 1 mmol/L,but BK currents were increased by 327±81 and 627±32%at DHA concentrations of 3 mmol/L and 10 mmol/L,respectively(n=6,P<0.05).With 0 mmol/L,0.01 mmol/L,0.1 mmol/L,0.3 mmol/L and 1 mmol/L DHA in the bath solution and in the presence of 1 mmol/L free calcium,BK channel open probabilities at a membrane potential of+60 mV were 0.075±0.019,0.071±0.020,0.067×0.019,0.087±0.026,and 0.098±0.023,respectively(n=5,P>0.05 vs.no DHA).In the presence of 3 mmol/L,5 mmol/L,and 10 mmol/L DHA,BK channel open probabilities were 0.232±0.027,0.582±0.041,and 0.606±0.066,respectively(n=5,P<0.05 vs.no DHA).(4)Single BK channel currents were recorded from freshly isolated rat coronary SMCs in inside-out configuration.BK channel open probabilities with test potential+60 mV were 0.0006±0.0004,0.0010±0.0004,0.0013±0.0001,0.0100±0.0026,0.0510±0.0081,1.4563±0.0599,1.5935±0.0562 and 1.5444±0.1101 in the presence of 0.001 ?mol/L,0.01 ?mol/L,0.1 ?mol/L,0.5 ?mol/L,1?mol/L,5 ?mol/L,10 ?mol/L and 50?mol/L free calcium.DHA had no effects on cytosolic calcium concentrations at 0.001 mmol/L-0.01 mmol/L,but fluorescence intensity ratios were increased on exposure to DHA at higher concentrations with an 50%efficacy concentration of 0.037±0.01 mmol/L(n=5-8 cells for each concentration,P<0.05).Fluorescence intensity was significantly decreased after exposure to DHA in in rat coronary SMCs with U73122 and 2-APB,an inhibitor of PLC and an IP3 receptor blocker(n=5-8 cells,P<0.05 vs.control).Conclusion(1)Rat coronary SMCs can be successfully isolated by "three-step“enzyme digestion method.(2)DHA activates several K+channels in coronary SMCs,but predominantly BK channels.This finding indicates that the mechanism of vasodilatory effect by DHA.(3)Different concentrations of DHA can activate BK channels through different activation mechanisms.DHA-induced BK channel activation at low concentrations(<1 mmol/L)is mediated through CYP epoxygenase metabolites,while DHA at high concentrations(>1 mmol/L)directly activates BK channels.(4)DHA can activate BK channels through elevated cytosolic calcium via the PLC-IP3 pathway. |
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