Objective To explore the activity of Ca2 + -activated K+ ( KCa) inairwaysmoothmuscle cells( ASMCs) in a rat model of chronic obstructive pulmonary disease( COPD) , and to observe the effect of 11, 12-Epoxyeicosatrienoic acid( 11, 12-EETs) on the KCa channel of ASMCs. Methods Forty male Sprague-Dawley rats were randomly assigned to a COPD group and a normal control group. The rats in the COPD group were exposed to cigarette smoking in a relatively closed chamber to induce COPD. The ASMCs were isolated from small bronchi using an acute enzymatic digestion method. In the symmetrical high K+ solution,the KCa currents were separated with inside-out configuration using the patch clamp technique. The activity of KCa currents in ASMCs between the COPD group and the normal group were compared and the effect of 11, 12-EETs on KCa channel was recorded. The opening probability( Po) , opening time( To) and closing time ( Tc) of the KCa were measured. Results Compared with the normal group, Po of KCa in the COPD rats was much shorter ( 0. 084 ±0. 028 vs 0. 198 ±0. 029, P lt; 0. 01) , To was shorter [ ( 0. 732 ±0. 058) ms vs ( 1. 648 ±0. 152) ms, P lt; 0. 01] and Tc was longer[ ( 12. 259 ±2. 612) ms vs ( 6. 753 ±1. 237) ms, P lt;0. 01] . 11, 12-EETs can evoke the activity of KCa currents of ASMCs in the COPD rats while Po was increased( 0. 227 ±0. 059 vs 0. 084 ±0. 028, P lt; 0. 01) , To was much longer[ ( 2. 068 ±0. 064) ms vs ( 0. 732 ±0. 058) ms, P lt; 0. 01] , and Tc was shorter [ ( 4. 273 ±0. 978) ms vs ( 12. 259 ±2. 612) ms, P lt;0. 01] .Conclusions The results suggest that the decreasing of KCa activity plays an important role in the development of COPD. 11,12-EETs can directly evoke the activity of KCa channel in COPD rats, thus relax the airway smooth muscles.
Objective To investigate the role of Kv1. 5 in the pathogenesis of pulmonary hypertension simulated by hypobaria and hypoxia, and the effects of dichloroacetate ( DCA) on the Kv1. 5 expression in pulmonary arterial smooth muscle cells ( PASMCs ) and mean pulmonary arterial pressure ( mPAP) . Methods Twenty-four SD rats were randomly divided into a normal group ( N group) , a high altitude group ( HA group) , and a DCA treated group ( DCA group) . The N group were fed in normalconditions, the HA group and DCA group were fed in a hypobaria and hypoxia chamber simulated to an altitude of 5000 meters. In addition, the DCA group rats were gastric gavaged with DCA ( 70 mg · kg - 1 · d - 1 ) .Twenty-one days later, percentage of wall thickness ( WT% ) and percentage of wall area ( WA% ) of the pulmonary arteriole, mPAP, and the ratio of right ventricle / left ventricle and septum ( RV/ LV + S) were evaluated. Real-time PCR, immunohistochemistry and Western blot were carried out to detect the Kv1. 5 expression in PASMCs. Results In the HA group, WT% , and WA% of pulmonary arteriole, mPAP and RV/ ( LV + S) all increased significantly compared with the N group ( P lt;0. 01) . These changes in the DCA group were significantly lower than those in the HA group( P lt; 0. 01) . Furthermore, the protein and mRNA expression of Kv1. 5 in the PASMCs deceased significantly in the HA group compared with the N group( P lt;0. 01) , but recovered in the DCA group ( P lt;0. 01) . Conclusions The expression of Kv1. 5 in PASMCs is tremendously inhibited in rats fed in high altitude, which might be a important role of pulmonaryhypertension. DCA can inhibit the remodeling of pulmonary arterials probably by recovering Kv1. 5 expression.
Objective To investigate the role of mitochondrial adenosine triphosphatesensitive potassium channel(mitoKATP) in immature myocardial ischemic preconditioning, and to provide evidence for immature myocardial protection. Methods Langendorff isolated heart infused model was used in the experiment. Twentyfour rabbits (aged from 14 to 21 days) were randomly divided into 4 groups：ischemiareperfusion group（I/R group）, myocardial ischemic preconditioning group(E1 group), 5hydroxydecanoate(5-HD) group (E2 group) and Diazoxide (Diaz) group(E3 group). Hemodynamics recovery rate, myocardial water content(MWC), the leakage rates of serum creatine kinase and lactate dehydrogenase, adenosine triphosphate content, superoxide dismutase activity, malondialdehyde content, myocardial cell Ca2+ content and myocardial mitochondrial Ca2+ content, myocardial mitochondrial Ca2+-ATPase activity, the adenosine triphosphate(ATP) synthesizing ability of myocardial mitochondria were tested, and myocardial ultrastructure was observed via electron microscopy. Results The hemodynamics recovery rate, myocardial water content(P＜0.05), adenosine triphosphate content, superoxide dismutase activity, myocardial mitochondrial Ca2+-adenosine triphosphyatase(ATPase) activity and the ATP synthesizing ability of myocardial mitochondria of the rabbits in E1 and E3 group were significantly better than that in I/R group and E2 group(P＜0.05). Malondialdehyde content, the leakage rates of serum creatine kinase and lactate dehydrogenase, myocardial cell Ca2+ content and myocardial mitochondrial Ca2+ content of the rabbits in E1 group and E3 group were significantly lower than that in I/R group and E2 group (P＜0.05). The myocardial ultrastructure injury in E1 and E3 group were significantly reduced compared with that in I/R and E2 group. Conclusion Myocardial ischemic preconditioning has significant protective effects on immature myocardium. Its mechanism may be related to the activation of mitoKATP.
Objective To investigate the protective effects of ischemic postconditioning (IPo) on ischemiareperfusion (I/R) myocardium and the relationship with mitochondrial adenosine triphosphate (ATP) sensitive K+ channels (mitoKATP) and provide evidences to the development of druginduced postconditioning. Methods Langendorff models were established in 40 Wistar rats which were divided into 5 groups by random number table with 8 rats in each group. Normal control group(NC group): the rat hearts were continuously reperfused by KrebsHenseleit bicarbonate buffer (K-HB) for 100 min without any other treatment; I/R group: the rat hearts underwent a 40-min global ischemia followed by a 60-min reperfusion; IPo group: after a 40-min global ischemia, the process of 10-second reperfusion followed by a 10-second ischemia was repeated 6 times, then there was a continuous 58min reperfusion; 5-hydroxydecanoic acid(5-HD) group: after a 40min global ischemia, hearts with 5HD(100 μmol/L) K-HB were reperfused for 15min and then perfused without 5HD for 45min；IPo+5-HD group: after a 40-min global ischemia, the process that the isolated hearts with 5-HD(100 μmol/L) KHB were reperfused for 10second followed by a 10second ischemia was repeated 6 times, then the hearts with 5-HD(100 μmol/L) KHB were continuously [CM(159mm]perfused for 13-min followed by reperfusion without 5-HD(100 μmol/L) K-HB for 45-min. The cardiac function,coronary flow(CF), cardiac troponin I(cTnI) content in coronary effluent, the area of acute myocardial infarction (AMI) and myocardial ultrastructure were observed. Results Left ventricular developed pressure(74.3±3.3 mm Hg vs. 57.1±3.3 mm Hg,t=1300, P=0.000),+dp/dtmax(1 706.6±135.6 mm Hg/s vs. 1 313.3±96.2 mm Hg/s,t=6.28,P=0.000),－dp/dtmax(1 132.8±112.1 mm Hg/s vs. 575.7±67.7 mm Hg/s,t=13.48, P=0.000) and CF(6.49±0.30 ml/min vs. 3.70±0.24 ml/min,t=28.6,P=0.000) in IPo group were higher than those in I/R group. Left ventricular enddiastolic pressure(10.9±1.7mm Hg vs. 26.2±1.5 mm Hg,t=－19.21, P=0000)and cTnI content in coronary effluent (0.62±0.01 ng/ml vs. 0.71±0.01 ng/ml, t=-12.00,P=0.000) were lower than those in I/R group; the area of AMI decreased 20.8％ compared with that in I/R group (Plt;0.05). The myocardial protective effect in IPo+5HD group was similar with that in IPo group, but lower than that in IPo group. The electron microscope showed that IPo and IPo+5HD could reduce myocardial fiber damage and mitochondrial damage caused by I/R. Conclusion IPo can protect I/R myocardium, which is achieved mainly by activating mitoK-ATP channels.
Objective To investigate the effects of diazoxide （DIA）cardioplegic solution on the reduction of donor cardiomyocyte apoptosis, Methods In a Krebs-Henseleit （KH） solution perfused isolated rabbit heart Langendorff model, 32 rabbit hearts were divided into four groups with simple random sampling （8 rabbits in each group ）： DIA group （50μmol/L diazoxide mixed in KH solution）,STH group （ST, Thomas＇ solution）, 5-HD group （50μmol/L diazoxide and 100μmol/L 5-hydroxydecanoic acid mixed in KH solution）, KH group （KH solution）, The rabbit hearts of each group underwent 6 hours of hypothermic （4 C） storage in the corresponding cardioplegic solution. Left ventricular developed pressure （LVDP）, maximal values of positive rate of left ventricular pressure （＋dp/dtmax） were measured before and after storage, The post-storage values of LVDP and ＋dp/dtmax were expressed as the percentage of pre-storage control values. Apoptotic cardiomyocytes were detected by the TdT- mediated dUTP-biotin nick end labeling （TUNEL）. Malonaldehyde （MDA） contents and adenosine triphosphate （ATP） contents were also measured after storage. Results Recovery rates of LVDP, ＋dp/dtmax, and ATP contents in DIA group were higher than those of other 3 groups respectively（P〈0. 05）, Cardiomyocytes apoptosis percentage and MDA content were lower than other 3 groups respectively（P〈0. 05）, Conclusions Diazoxide cardioplegic solution can protect the isolated hearts and this may be relates to opening selective mitochondrial KATP channels. The selective mitochondrial KATP channel antagonist 5-hydroxydecanoic acid can block the cardioprotective effect of diazoxide.
Objective To observe the influences of depolarized arrest and hyperpolarized arrest on alternation of fluidity of myocardial cell membrane during cardiopulmonary bypass (CPB) and evaluate the protective effects on myocardium of hyperpolarized arrest. Methods Seventy-two felines were randomized into three groups, each group 24. Control group: 180 minutes of CPB was conducted without aortic and vena caval cross-clamping. Depolarized arrest group: hearts underwent 60 minutes of global ischemia after aortic cross-clamping (ACC) followed by 90 minutes of reperfusion. The cardioplegic solution consisted of St. Thomas solution (K+16mmol/L). Hyperpolarized arrest group: the protocol was the same as that in depolarized arrest group except that the cardioplegic solution consisted of St.Thomas solution with pinacidil (50 mmol/L,K+5mmol/L). Microviscosity, the reciprocal of fluidity of myocardial membrane was measured in all groups by using fluorescence polarization technique. (Results )Microvis cosity of myocardial cell in depolarized arrest group during ACC period was significantly higher than that before ACC and kept on rising during reperfusion period. Microviscosity of myocardial cell in hyperpolarized arrest group during ACC was trending up and reperfusion periods as well, but markedly lower compared to that in depolarized arrest group at corresponding time points(Plt;0.01). Conclusion Hyperpolarized arrest is more effective in protecting myocardial cells from ischemia-reperfusion injury than depolarized arrest during CPB by maintaining better fluidity of myocardial membrane.
Objective To investigate Kir4.1 expressions in Muuml;ller cells under high glucose conditions and treatment of pigment epitheliumderived factor (PEDF）. Methods Cultured rat Muuml;ller cells were divided into control group (5 mmol/L glucose), high glucose group (25 mmol/L glucose), PEDF treatment group (25 mmol/L glucose+100 ng/ml PEDF) and intervention control group（25 mmol/L glucose+phosphate buffer solution）. Kir4.1 expressions were measured by Western blot and real-time reverse transcription polymerase chain reaction (RT-PCR). Reactive oxygen species (ROS) productions were measured using 2prime;7prime;dichlorofluorescin diacetate and glutathione peroxidase （GPx）expressions were studied by real-time RT-PCR. Results By Western blot and real-time RT-PCR, it was found the expressions of Kir4.1 decreased obviously under high glucose conditions (real-time RT-PCR: t=4.12, P＜0.05; Western blot: t=3.53,P＜0.05); simultaneously, ROS generation was increased （t=3.76,P＜0.05）and GPx level was decreased （t=3.18,P＜0.05）. PEDF treatment inhibited the high glucose-induced Kir4.1 down regulation (real-time RT-PCR: t=3.66, P＜0.05; Western blot: t=6.43,P＜0.01) and decreased ROS generations （t=4.11,P＜0.05） and increased GPx levels (t=5.12,P＜0.01）. Conclusions The high glucose can supress Kir4.1 expressions in Muuml;ller cells by oxidative stress, and PEDF can ameliorate these effects.
Objective To observe the effects of high concentr at ion glucose on the calcium-activated potassium channel of rabbits′ retinal Müller cells. Methods The rabbits′retinal Müller cells were cultured in vitro under the condition of high concentration glucose, and identified by immunohistochemical staining and transmission electron microscopy. Patch-clamp technique was used to observe the changes of the calcium-activated potassium channel of retinal Müller cells caused by high concentration glucose at different time.Results High concentration glucose could inhibit the calcium-activated potassium channel of cultured retinal Müller cells in a time-dependent manner. Conclusion High concentration glucose may reduce the biological functions of Müller cells by inhibiting calcium-activated potassium channel. (Chin J Ocul Fundus Dis，2003，19：164-167)
ObjectiveTo observe the changes in open probability and protein expression of large conductance Ca2+-activated K+ (BK) channel in retinal vascular smooth muscle cells (RVSMCs) of diabetic rats. MethodsStreptozotocin (STZ)-induced rat diabetic animal model was established by STZ injection intraperitoneally.RVSMCs were isolated by enzyme digestion. The BK currents in control and diabetic groups were recorded by patch clamp technique in single channel configuration. BK channel protein expression in control and diabetic group were measured by Western blot. ResultsCompared with control group, the NP0 of BK channels in diabetic group were significantly increased (t=4.260, P < 0.05). Compared with control group, there was no significant difference inα-subunit protein expression in diabetic group in RVSMCs (t=10.126, P > 0.05); however, β1-subunit protein expression was remarkably increased in diabetic group (t=5.146, P < 0.05). ConclusionThe NP0 of BK channels andβ1-subunit protein expression are increased in RVSMCs of diabetic rats.