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Aim To investigate the effect of taurine-magnesium coordination compound (TMCC) on elec-trocardiogram of isolated guinea pig hearts, hoping to describe a primary research on its characteristic of anti-short QT syndrome. Methods The isolated guinea pig heart was retrograde perfused using Langendorff tech-nique. In order to determine the effects of TMCC on QT interval, transmural dispersion of repolarization, effective refractory period, instability of RR interval and instability of QT interval in the presence of potassi-um channel opener pinacidil, the electrocardiogram of isolated guinea pig hearts was recorded using Biopac physiological recorder. Results The shortened QT in-terval and the effective refractory period induced by pinacidil could be prolonged by TMCC; the increased transmural dispersion of repolarization induced by pinacidil could be decreased by TMCC; the increased instability of RR and QT interval induced by pinacidil could be decreased by TMCC. Conclusion TMCC has the effects of anti-SQT2 by prolonging the QT inter-val and the effective refractory period, reducing the transmural dispersion of repolarization and instability.
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Objective To evaluate the role of mitochondrial KATP (mito-KATP) channel in pinacidil postconditioning-induced reduction of myocardial ischemia-reperfusion (I/R) injury in rats.Methods SPF healthy male Sprague-Dawley rats,aged 16-20 weeks,weighing 250-300 g,were anesthetized with pentobarbital.Their hearts were excised and perfused in a Langendorff apparatus with K-H solution saturated with 95％ O2-5％ CO2 at 36.5-37.5 ℃.Thirty-two Langendorff-perfused hearts were divided into 4 groups (n =8 each) using a random number table:control group (group C),group I/R,pinacidil postconditioning group (group P) and 5-hydroxy decanoic acid plus pinacidil postconditioning group (group 5-HD+P).Myocardial ischemia was induced by interrupting perfusion for 40 min followed by 60 min reperfusion.Immediately after onset of reperfusion,hearts were perfused with K-H solution containing 50 μmol/L pinacidil for 2 min and then with K-H solution for 58 min in group P,hearts were perfused with K-H solution containing 100 μmol/L 5-HD for 5 min,with K-H solution containing 50 μmol/L pinacidil for 2 min and then with K-H solution for 53 min in group 5-HD+P.The heart rate (HR),left ventricular developed pressure (LVDP),left ventricular end-diastolic pressure (LVEDP) and the maximum rate of increase in left ventricular pressure (+dp/dtmax) were recorded at 20 min of equilibration (T1) and at the end of reperfusion (T2).Myocardial tissues were obtained at T2 for determination of myocardial infarct size and for examination of myocardial ultrastructure and Flameng scoring of the mitochondria was performed.Results Compared with group C,the HR,LVDP and +dp/dtmax were significantly decreased,and the LVEDP,myocardial infarct size and mitochondrial Flameng score were increased at T2 in group I/R (P＜0.05).Compared with group I/R,the HR,LVDP and +dp/dtmax were significantly increased and the LVEDP,myocardial infarct size and mitochondrial Flameng score were decreased at T2 (P＜0.05),and the pathological changes of myocardium were significantly attenuated in group P,and no significant change was found in the parameters mentioned above in group 5-HD+P (P＞0.05).Compared with group P,the HR,LVDP and + dp/dtmax were significantly decreased and the LVEDP,myocardial infarct size and mitochondrial Flameng score were increased at T2 (P＜O.05),and the pathological changes of myocardium were accentuated in group 5-HD+P.Conclusion The whole mechanism by which pinacidil postconditioning reduces myocardial I/R injury is related to promoting opening of mito-KATP channel in rats.
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Objective To observe the activation mechanism of Nrf2-ARE pathway in protective effect of ischemia and pinacidil postconditioning on isolated rat hearts.Methods The hearts of adult male Sprague Dawley rats were established ischemia-reperfusion injury model,and devided into six groups(n =8,each group),i.e.Normal group(Group N),ischemiareperfusion group (Group Con,I/R),ischemic postconditioning group (Group IPO),pinacidil postconditioning group (Group P50),N-(2-mercaptopropionyl)-glycine(MPG,2mmol/L) + IPO group(Group M + IPO),MPG + P50 group(Group M + P50).Rat hearts were perfused with Krebs-Henseleit(K-H) buffer for 20 minutes for equilibration.Subsequently,Group N was perfused with K-H buffer for 100 minutes after equilibration,Group Con was perfused with 4℃ ST.Thomas solution to stop the heart beating after equilibration,then the hearts were underwent 40 minutes global ischemia under 32℃,and followed by the K-H solution for 60 minutes.Group IPO after global ischemia period,the hearts were subjected to six 10-seconds cycles of ischemia/reperfusion at the beginning of reperfusion,then were reperfused for 58 minutes.Group P50 after global ischemia,rat hearts were perfused with K-H buffer containing pinacidil(50.μmol/L) for 2 minutes before reperfusion.Group M + IPO after global ischemia,the hearts were subjected to perfuse with K-H buffer containing MPG(2 mmol/L) for 3 minutes,and then underwent six 10-seconds cycles of ischemia/reperfusion before reperfusion.Group M + P50 after global ischemia,the hearts were perfused with K-H buffer containing MPG(2 mmol/L) for 3 minutes,and then subjected to perfuse with K-H buffer containing pinacidil(50 μmol/L) for 2 minutes before reperfusion.Cardiac function indexes(such as HR,LVDP,LVEDP,and the Max dp/dt) at the end point of equilibration and repeffusion were observed and recorded.The ultrastructure of myocardial tissue was observed by electron microscopy and the mitochondrial Flameng score was calculated.RT-PCR and western-blot were applied to detect the gene transcription and protein expression of HO-1,NQO1,SOD1,and Nrf2 in left ventricular myocardial tissue after reperfusion.Results The HR,LVDP and + dp/dtmax at the end of reperfusion:the cardiac function indexes are lower among each group compared with group N,group 1PO and group P50 are better than group Con (P ＜ 0.05).Compared with group IPO,there is no significant difference in group group P50,but group M + IPO is obviously decreased(P ＜ 0.05).Compared with group P50,group M + P50 index is decreased significantly(P ＜ 0.05).The LVEDP at the end of reperfusion is lower than that among each group as compared with group Con,which is significantly increased in group Con (P ＜ 0.05).Compared with group IPO,there is no significant difference in group P50,but group M + IPO is significantly increased(P ＜ 0.05).Compared with group P50,the group M + P50 is obviously decreased(P ＜ 0.05).The ultrastructure of myocardial tissue in group N is mostly normal,group Con presence serious damage.The ultrastructure damage of myocardial tissue is improved in group IPO and group P50 as compared with that in group Con,while group M + IPO is more serious than group IPO,group M + P50 is more serious group P50.The mitochondrial Flameng score is higher among each group as compared with group N (P ＜ 0.05),the score is lower in group IPO and group P50 as compared with group Con and corresponding nonblocking group (M + IPO,M + P50,P ＜0.05).The mRNA and the protein expressions of HO-1,NQO1,SOD1 and Nrf2 among each group are lower as compared with group N(P ＜0.05).Compared with those in group Con,the mRNA and the protein expressions in group IPO and group P50 are obviously increased(P ＜ 0.05),group IPO and group P50 are higher than those in group adding active oxygen scavenger(MPG) (P ＜ 0.05).Conclusion Ischemic postconditioning and pinacidil postconditioning have protective effect of myocardial tissue from ischemia reperfusion injury,while improve the cardiac function index.The cardiac protective effect of Ischemic and Pinacidil postconditioning methods may be involved the ROS in early reperfusion,which activate the Nrf2-ARE pathway,and up-regulate the expression downstream antioxidant protein and phase Ⅱ detoxifying enzyme,ultimately improve the cardiac function index during the reperfusion period.
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AIM:To investigate the protective effect of pinacidil postconditioning on rat myocardium suffering ischemia/reperfusion injury by mitochondrial proteomics .METHODS: Langendorff apparatus was used to establish the model of myocardial ischemia/reperfusion injury .Sprague-Dawley rats were randomly divided into 2 groups:pinacidil post-conditioning group (Pina group) and ischemia/reperfusion injury group (I/R group).After 20 min of perfusion with K-H solution, the perfusion was suspended for 40-min (global ischemia) follow by 60 min of reperfusion in I/R group.In Pina group at the end of 40 min global ischemia , the isolated hearts were perfused with K-H solution containing pinacidil ( 50μmol/L) for 2 min followed 58-min perfusion with regular K-H solution.Total proteins extracted from the mitochondria were applied to the two-dimensional gel electrophoresis (2-DE).The differentially expressed protein spots over 2 times were evaluated by a software .Then they were subjected to in-gel digestion , and analyzed by spectrometry .RESULTS:The expression levels of NDUFA10, NDUFS2 and NDUFV2 were elevated but those of IDHA and ECH 1 were decreased in Pina group compared with I/R group.Interestingly, 2 spots in the 2-DE map were identified as ATPase subunit δ.The ex-pression levels of one spot was elevated , while the other was decreased .CONCLUSION:Pinacidil postconditioning may decrease the degree of increased expression levels of NDUFA 10, NDUFS2 and NDUFV2, promote the expression of IDHA and ECH1, and induce the phosphorylation of ATPase subunit δ, which may be related to the protective mechanism of pinacidil postconditioning .
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Objective To evaluate the role of nuclear factor-E2 related factor 2 (Nrf2)-antioxidant response element (ARE) pathway in cardio-protection by ischemic or pinacidil postconditioning ( IP,PP) against ischemia-reperfusion (I/R) injury in isolated rat hearts.Methods Fifty-six male SD rats of both sexes weighing 200-250 g were anesthetized with intraperitoneal amobarbital sodium.The isolated rat hearts were perfused in a Langendorff apparatus with Krebs-Hensleit buffer (K-H).Fifty-six isolated rat hearts with I/R injury were randomly divided into 7 groups ( n =8 each):normal control group (group C) ; group I/R; group IP and group PP1-4 postconditioning with 4 different concentrations of pinacidil.After 20 min of equilibration,the perfusion was suspended for 40 min (global ischemia) followed by 60 min of reperfusion in group I/R.In group IP after 40 min of global ischemia,the isolated hearts underwent 6 cycles of 10 s reperfusion and 10 s ischemia followed by 58 min of reperfusion.In group PP1-4 at the end of 40 min of global ischemia,the isolated hearts were perfused with K-H containing pinacidil 5,10,30 and 50μmol/L for 5 min respectively followed by 55 min reperfusion with regular K-H.Left ventricular developed pressure (LVDP) and LVEDP were measured immediately before global ischemia and at the end of 60 min reperfusion.Myocardial specimens were obtained at the end of reperfusion for detection of Nrf2,quinopeoxidoreductase (NQO1),HO-1 and SOD1 mRNA (by RT-PCR) and protein (by Western blot) expression.Results I/R significantly up-regulated Nrf2,NQO1,HO-1 and SODI mRNA and protein expression,decreased LVDP and increased LVEDP in group I/R as compared with group C.IP and 30,50 μmol/L pinacidil postconditioning further significantly increased Nrf2,NQO1,HO-1 and SOD1 mRNA and protein expression and IP,5,10,30,50 μmol/L pinacidil postconditioning significantly increased LVDP and decreased LVEDP as compared with group I/R.Conclusion Ischemic or pinacidil postconditioning can attenuate I/R injury by activating Nrf2-ARE pathway in isolated rat hearts.
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ObjectiveTo investigate the effect ofpinacidil hyperpolarizaed arrest on p38 mitogen-activited protein kinase (p38MAPK) during myocardial ischemia-reperfusion (I/R) in isolated rat hearts.MethodsFortyeight male SD rats weighting 250-300 g were randomly divided into 6 groups( n =8 each): natural arrest group (group A) ; St.Thomas group (group B) ; pinacidil hyperpolarization arrest group (group C) ;5-hydroxydecanoate (5-HD) group (group D);HMR-1098 group(group E) and 5-HD + HMR-1098 group(group F).Langendorff reperfusion model was established and K-H solution was retrogradely perfused for 15 min.In group A the hearts were arrested naturally afar perfusion was stopped; in group B St.Thomas solution was perfused; in group C pinacidil hyperpolarization solution was perfused; in the other three groups,K-H solution was perfused to isolated rat hearts for 10 min followed by 5 min 5-HD (group D) or HMR-1098(group E) or 5-HD and HMR-1098(group F) perfusion,then hyperpolarization arrest solution was given in each group.Each hearts suffered 60 min ischemia after arrest followed by 30 min K-H solution reperfusion.Coronary flow(CF),HR,left ventricular developed pressure( LVDP),left ventricular systolic pressure(LVSP) and the maximum rate of pressure rise (dp/dtmax) were measured at the end of 15 min K-H solution perfusion and at 20 min of reperfusion.Myocardial phosphatic and nonphosphatic p38MAPK expression was determined by Western blot at 30 min of reperfusion.ResultsCompared with group C,CF,HR,LVDP,LVSP and dp/dtmax were significantly decreased at 20 min of reperfusion and phosphatic p38MAPK expression was down-regulated,non-phosphatic p38MAPK expression was up-regulated at 30 min of reperfusion in groups A,B,D,E and F (P ＜ 0.05).Compared with group E,CF,HR,LVDP,LVSP and dp/ dtmax were significantly decreased at 20 min of reperfusion and phosphatic p38MAPK expression was down-regulated,non-phosphatic p38MAPK expression was up-regulated at 30 min of reperfusion in groups D and F ( P ＜0.05).ConclusionHyperpolarized arrest induced by pinacidil can improve cardiac function following myocardial I/R injury by up-regulating phosphatic p38MAPK expression,down-regulating non-phosphatic p38 MAPK expression and mitochondrial potassium channel is more important than membranous one during the regulation of phosphatic p38MAPK expression.
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Objective To investigate the effect of heart preservation solution containing pinacidil on mitochondrial function in isolated rat hearts. Methods One hundred and twenty pathogen-free SD rats of both sexes weighing 250-350 g were used in this study. The animals were anesthetized with intraperitoneal pentobarbital 65 mg/kg. Their hearts were immediately removed and perfused in a Langendorff apparatus. Left ventricular enddiastolic pressure was measured from a fluid-filled latex balloon inserted in the left ventricle. The isolated hearts were randomized into 5 groups (n = 24 each):group Ⅰ was perfused with cardioplegic solution HTK; group Ⅱ with HTK containing pinacidil (a non-specific sarcKATP and mitoKATP channel opener) 0.5 mmol/L; group Ⅲ with HTK containing pinacidil 0.5 mmol/L + 5-HD (a selective mitoKATP channel blocker) 100 μmol/L; group Ⅳ with HTK containing pinacidil 0.5 mmol/L + HMR-1098 100 μmol/L (a selective sarcKATP channel blocker) and group Ⅴ with HTK containing pinacidil 0.5 mmol/L + 5-HD 100 μmol/L + HMR-1098 100μmol/L. The isolated hearts were perfused with simple HTK or HTK containing pinacidil or pinacidil + 5-HD and/or HMR 20 ml/kg at 10 ml/min and then removed from Langendorff apparatus and dipped into the same HTK solution for 8 h at 4 ℃followed by 60 min reperfusion. The respiratory function of mitochondria (respiratory control rate (RCR), the rate of oxygen consumption in state 3/state 4 and P/O) was measured at the end of equilibration (T1) after 8 hpreservation (T2) and at the end of 60 min reperfusion (T3). The CK-MB and LDH activities and cTnI expression in myocardium was detected at T1 and T3. The ultrastructure of myocardium was examined at T3. Results Perfusion suspension-reperfusion (PS/R) significantly decreased mitochondrial respiratory function (RCR, P/O and rate of O2 consumption in state 3) and increased myocardial cTnI concentration and CK-MB and LDH activities at T3 compared with baseline at T1 in group Ⅰ. Pinacidil significantly increased mitochondrial respiratory function (RCR, P/O and rate of O2 consumption in state 3) and decreased myocardial cTnI concentration and CK-MB and LDH activities in group Ⅱ as compared with group Ⅰ-indicative of protective effect of pinacidil on mitochondria against PS/R injury. The protective effect of pinacidil against PS/R injury was attenuated by 5-HD and/or HMR1098. The myocardial damage was slightest in group Ⅱ . Conclusion Both sarcolemmal and mitochondrial KATPchannel are involved in the protective effect of pinacidil against PS/R-induced myocardial damage during heart preservation.
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Objective To study the effects of Pinacidil on Ventricular Repolarization in LQT2 Rabbit Heart.Methods 30 New Zealand white rabbits weighted from 2.5~3.0 kg were randomly divided into 3 groups:group A(control),group B(pinacidil),group C(pinacidil + glibenclamide).Left ventricular endocardium and epicardium MAPs and volume-conducted ECGs in isolated Langendorff-perfused rabbit hearts were recorded simultaneously.To induce bradycardia,the AV nodes of rabbit hearts were damaged.EAD and TdP were induced by means of bradycardia in the presence of high concentration of d-sotalol(10-4M).Results In group B,pinacidil(5?mol/L)shortened the APD90 on endocardium from 445.48?54.31ms to 278.87?44.45ms after administration of pinacidil for 5 minutes(P0.05 vs.group A for all).Conclusion Pinacidil can decrease prolonged action potencial duration and TDR induced by bradycardia and high concentration of d-sotalol in rabbit hearts.Also,pinacidil could abolish the EAD and TdP related to delayed repolarization that may provide a novel and useful intervention in the clinical LQTS patients.
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Objectives: To examine effects of pinacidil on intracellular free calcium concentration of cardiomyocytes during hypoxia/reoxygenation. Methods:A cell culture model of neonatal rat cardiac myocytes was used. There were three groups, including control group, hypoxia/reoxygenation group and pinacidil group. Confocal microscope was used with Fluo 3/AM as calcium indicator to detect changes of intracellular free calcium concentration. Results:The intracellular fluoresence intensity of singular cardiomyocyte in hypoxia/reoxygenation group was significantly higher than that of the controls( P
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Objective To investigate the protective effect of pinacidil on the intestinal mucosa of scalded rats and the mechanism. Methods A total of 24 healthy SD rats were randomly divided into normal control group, burn group, and pinacidil treated group. Rats in burn group and pinacidil treated group were inflicted with 30% TBSA Ⅲ degree burn and resuscitated intraperitoneally with Ringer's solution immediately after burn. Pinacidil was injected intraperitoneally into rats in pinacidil treated group at the dose of 2 mg/kg. Mitochondrial respiratory function intestinal mucosa and levels of reactive oxygen species (ROS), maleic dialdehyde (MDA), and superoxide dismutase (SOD) in plasma were determined at 6 h after burn. Results Mitochondrial respiratory function control rate (RCR), ST3, and SOD levels in pinacidil treated group were evidently higher than those in the burn group. However, ST4, MDA, and ROS levels in pinacidil treated group were significantly lower than those in the burn group, but ST4 was not significantly different from that in the normal control. Conclusion Pinacidil can attenuate the damage of intestinal mucosa in scalded rats.
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AIM: To observe the effects of berberine (Ber) on left atria and trachea in guinea pigs. METHODS: The experiment was carried out with isolated left atria and trachea in guinea pigs, and the effects and interactions were compared between Ber and pinacidil (Pin). RESULTS: Ber concentration-dependent increased the force of contractile, while Pin decreased the force. Ber leaded a parallel rightward shift in accumulated response curve of depression contraction of left atria by Car, and it hardly changed the maximum response (E_(max)), while Pin leaded a parallel leftward shift in the same curve. In the combination of Ber and Pin, the dose-response curve hardly changed as the control one. In isolated guinea pig trachea, Ber caused a leftward shift in the dose-response curve of ACh, whereas Pin produced a rightward shift in the same curve without changing E_(max). When both Ber and Pin existed in the same container, there was no modification in the response to ACh. CONCLUSION: Ber shows the effect of blocking potassium channel.
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AIM: To study the effects of ischemia/reperfusion (I/R) on primary cultured sinoatrial node (SAN) cells and the influence of pinacidil (a K_(ATP) channel activator). METHODS: The SAN cells were isolated from newborn rats and purified. The 48 h cultured cells were cultivated in following mediums: simulated reperfusion solution as normal control, simulated ischemia/reperfusion solution (I/R), Pinacidil+I/R (P+I/R), 5-HD+P+I/R and 5-HD+I/R. Spontaneous action potentials were recorded by ruptured-patch whole-cell technique in current clamp ((I=0)) and the maximum diastolic potential (MDP), upstroke velocity (UV), action potential overshoot (APO), interbeat interval (IBI) and action potential durations at 50% repolarization (APD_(50)) were measured. RESULTS: Compared with control group, simulated ischemia/reperfusion shorten APD_(50), reduced UV, MDP and APO. Exposed to pinacidil, MDP of cells in I/R groups was hyperpolarized; IBI, UV and APO were increased; APD_(50) was shorten. 5-HD couldn't block the effects of pinacdil on APD_(50), IBI and MDP, but reversed its actions on increasing UV and APO. CONCLUSIONS: Pinacidil made changes of AP in I/R group by opening different K_(ATP) channels of SAN cells. The role of this changes on protection in SAN cells during ischemia/reperfusion requires further investigation. [
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A brief episode of ischemia and reperfusion termed 'ischemic preconditioning' has been established as rendering muscle tolerance to damage during a subsequent prolonged ischemia. The effects of ischemic preconditioning in the cardiac muscle are related to the stimulation of adenosine A1 receptor and the opening of KATP channel. The effect and mechanisms of ischemic preconditioning in the skeletal muscle are not known clearly. The superoxide radical injures the skeletal muscle during the ischemia and reperfusion. There are two types of SOD, which metabolizes the superoxide radicals to H2O2 and O2, in the cell. One of them is Cu, Zn-SOD in the cytoplasm and the other is Mn-SOD in the mitochondria. The activities of SOD are increased against the formation of superoxide radical during the reperfusion. The author performed the present study to investigate the effect and the mechanisms of ischemic preconditioning by measuring the expression of SOD mRNA on timely reperfused ischemic muscles. The healthy Sprague-Dawley rats weighing from 300 g to 350 g were used as experimental animals. Under pentobarbital (50 mg/kg) anesthesia, lower abdominal incision was done and left common iliac artery was occluded by vascular clamp for 2 hours. Rectus femoris muscles were obtained respectively at 3, 6, 12, 24 and 72 hours after reperfusion. The ischemic preconditioning group underwent three episodes of 5 minute occlusion and 5 minute reperfusion of common iliac artery followed by 2 hours of ischemia and timely reperfusion. Adenosine (50 microgram/kg) or pinacidil (1 mg/kg) was administered intravenously before ischemia. 8-cyclopentyl-1, 3-dipropylxanthine (15 mg/kg) or glibenclamide (0.5 mg/kg) was administered intravenously before ischemic preconditioning. Paraffin sections with 4 micrometer thickness in all groups were obtained. The expression of Cu, Zn- and Mn-SOD mRNA was observed by use of in situ hybridization. The results obtained were as follows. 1. The expression of SOD mRNA was seen only in small muscle fibers of the rectus femoris muscle of the rat. 2. Weak expressions of Cu, Zn- and Mn-SOD mRNA were observed in the normal control rat. 3. After 2 hours of ischemia, moderate expression of Cu, Zn-SOD mRNA was observed until 72 hours of reperfusion. Weak or moderate expression of Mn-SOD mRNA at 3 hours and 6 hours of reperfusion, weak or trace expression at 12 hours of reperfusion, moderate expression at 24 hours of reperfusion and weak or moderate expression at 72 hours of reperfusion were observed. 4. After ischemic preconditioning, moderate expressions of Cu, Zn-SOD mRNA were seen in the groups of 3, 6, 12 and 24 hours of reperfusion. Moderate expressions of Mn-SOD mRNA were seen in the group of 0, 3, 6 and 12 hours of reperfusion and strong expression was seen in the group of 24 hours of reperfusion after ischemic preconditioning. 5. After 2 hours of ischemia with ischemic preconditoining, moderate expressions of Cu, Zn-SOD mRNA were seen in the groups of 0, 3, 6, 12, 24 hours of reperfusion. Moderate expressions of Mn-SOD mRNA were observed in the groups of 0, 3, 6, and 12 hours of reperfusion and moderate or strong expression was seen in the group of 24 hours of reperfusion. 6. After 2 hours of ischemia with the pretreatment of adenosine, moderate expressions of Cu, Zn-SOD mRNA were seen in the group of 0, 3, 6, 12 and 24 hours of reperfusion. Moderate expression of Mn-SOD mRNA in the groups and 3 hours of reperfusion, strong expression in the group of 6 and 12 hours of reperfusion and moderate expression in the group of 24 hours of reperfusion were seen. 7. After 2 hours of ischemia with the pretreatment of pinacidil, moderate expressions of Cu, Zn-SOD mRNA were seen in the groups of 0, 3, 6 and 12 hours of reperfusion and those of Mn-SOD mRNA were seen in the groups of 3, 6, 12 and 24 hours of reperfusion. 8. After 2 hours of ischemia with ischemic preconditioning and the pretreatment of 8-cyclopentyl-1, 3- dipropylxanthine, moderate expression of Cu, Zn-SOD mRNA were observed in the groups of 0, 3, 6, and 12 hours of reperfusion and those of Mn-SOD were seen in the groups of 6, 12 and 72 hours of reperfusion. 9. After 2 hours of ischemia with ischemic preconditioning and the pretreatment of glibenclamide, moderate expressions of Cu, Zn- and Mn-SOD mRNA were seen in all groups of reperfusion. Consequently, these results suggest that the expression of Cu, Zn and Mn-SOD mRNA increases during 2 hours ischemia and reperfusion with or without ischemic preconditioning. The effects of ischemic preconditioning are closely related to the stimulation of adenosine A1 receptor and KATP channel.
Subject(s)
Animals , Rats , Adenosine , Anesthesia , Cytoplasm , Glyburide , Iliac Artery , In Situ Hybridization , Ischemia , Ischemic Preconditioning , Mitochondria , Muscle, Skeletal , Muscles , Myocardium , Paraffin , Pentobarbital , Pinacidil , Quadriceps Muscle , Rats, Sprague-Dawley , Receptor, Adenosine A1 , Reperfusion , RNA, Messenger , Superoxide Dismutase , SuperoxidesABSTRACT
The influences of specific protein phosphatase and protein kinase inhibitors on the ATP-sensitive K+ (KATP) channel-opening effect of pinacidil were investigated in single rat ventricular myocytes using patch clamp technique. In cell-attached patches, pinacidil (100 muM) induced the opening of the KATP channel, which was blocked by the pretreatment with H-7 (100 muM) whereas enhanced by the pretreatment with genistein (30 muM) or tyrphostin A23 (10 muM). In inside-out patches, pinacidil (10 muM) activated the KATP channels in the presence of ATP (0.3 mM) or AMP-PNP (0.3 mM) and in a partial rundown state. The effect of pinacidil (10 muM) was not affected by the pretreatment with protein tyrosine phosphatase 1B (PTP1B, 10 mug ml-1), but blocked by the pretreatment of protein phosphatase 2A (PP2A, 1 U ml-1). In addition, pinacidil (10 muM) could not induce the opening of the reactivated KATP channels in the presence of H-7 (100 muM) but enhanced it in the presence of ATP(1 mM) and genistein (30 muM). These results indicate that the KATP channel-opening effect of pinacidil is not mediated via phosphorylation of KATP channel protein or associated protein, although it still requires the phosphorylation of serine/threonine residues as a prerequisite condition.
Subject(s)
Animals , Rats , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine , Adenosine Triphosphate , Adenylyl Imidodiphosphate , Genistein , KATP Channels , Muscle Cells , Phosphorylation , Pinacidil , Protein Kinase Inhibitors , Protein Phosphatase 2 , Protein Tyrosine Phosphatase, Non-Receptor Type 1ABSTRACT
PURPOSE: Intracavernous injection of prostaglandin (PG) E1 or papaverine (PA) is widely used in the diagnosis and treatment of erectile dysfunction. Because these drugs are sometimes associated with insufficient erection in and side effects such as priapism, corporal fibrosis, and pain, there has been increasing interest in finding effective and safe alternatives. Recent studies demonstrated that pinacidil (PI) relaxes the smooth muscle. This study was performed to examine the efficacy of PI as an alternative or supplement to drugs such as PG, PA, or phentolamine (PT). MATERIALS AND METHODS: In 28 adult male cats, the maximal intracavernous pressure (ICPmax), time to ICPmax (T1/2) and duration of increased ICP (time) in response to intracavernous injection of PG, PA, or mixture of vasoactive drugs (PA + PT, PA + PG + PT) were compared with the responses to mixtures containing PI (PI + PA, PI + PA + PT,
Subject(s)
Adult , Animals , Cats , Humans , Male , Diagnosis , Erectile Dysfunction , Fibrosis , Muscle, Smooth , Papaverine , Penile Erection , Penis , Phentolamine , Pinacidil , Potassium Channels , PriapismABSTRACT
Objective To investigate the protective effects of ATP-sensitive potassium channel (K (ATP)) opener (Pinacidil) on cultured cortical neurons in newborn rats after anoxic/hypoglycemic damage. Methods Neurons were exposed to anoxic/hypoglycemic condition for 4 h,8 h,16 h, after being cultured for 10 days. All cultured neurons were divided into 4 groups: normal control group, model control group, Pinacidil (Pin) group and Pin plus Glibenclamide (Gli) group. The cell mortality, quantity of LDH and the percentage of neuron apoptosis in each group were determined. Results Pinacidil (10~(-6)mol/L ) reduced the percentage of cell death(4 h:21.42?3.68 vs 14.83?2.94; 8 h:36.58?6.14 vs 19.25?3.33; 16 h:49.17?8.3 vs 28.64?6.4, P
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The ischemia and reperfusion injury of skeletal muscle is caused by generation of reactive oxygen species. Recently, apoptosis has been associated with oxidative stress in a number of cell systems. The effects of ischemic preconditoining in cardiac muscle have been established as rendering muscle tolerance to ischemic reperfusion damage via opening of KAPT channel and activation of adenosine A1 receptor. The effects and mechanisms of ischemic preconditioning are not known clearly. The present study was performed to investigate the effect and the mechanisms of ischemic preconditioning by measuring the incidences of apoptosis on timely reperfused ischemic muscles. The healthy Sprague -Dawley rats weighing from 200 g to 250 g were used as experimental animals. Under pentobarbital (50 mg/kg) anesthesia, lower abdominal incision was done and left common iliac artery was ligated by using vascular clamp for 2 hours. Rectus femoris muscles were obtained at 0 hour, 1 hour, 2 hours, 6 hours, 12 hours and 24 hours of reperfusion. The group of ischemic preconditioning underwent three episodes of 5 minutes occlusion and 5 minutes reperfusion of common iliac artery followed by 2 hours of ischemia and timely reperfusion. Adenosine (50 microgram/kg) or pinacidil (1 mg/kg) were administered intravenously before ischemia and 2 hours of ischemia and timely reperfusion was done. 6 microM of paraffin sections were obtained. The incidencies of apoptosis were observed by use of in situ apoptosis detection kit. The results obtained were as follows. 1. The reactivities to apoptosis in the rectus femoris muscle increased after 2 hours of ischemia and timely reperfusion. 2. After 2 hours of ischemia and timely reperfusion with ischemic preconditioning and the treatment of pinacidil, the reactivities to apoptosis in all groups decreased markedly. 3. After 2 hours of ischemia and timely reperfusion with the treatment of adenosine, the reactivities to apoptosis in all groups were similar to those in the group of 2 hours of ischemia and reperfusion. Consequently, these results suggest that the reactivities to apoptosis decrease after 2 hours of ischemia and timely reperfusion with ischemic preconditioning. The effect of ischemic preconditioning is related to opening of KATP channel partly.
Subject(s)
Animals , Rats , Adenosine , Anesthesia , Apoptosis , Iliac Artery , Incidence , Ischemia , Ischemic Preconditioning , Muscle, Skeletal , Muscles , Myocardium , Oxidative Stress , Paraffin , Pentobarbital , Pinacidil , Quadriceps Muscle , Reactive Oxygen Species , Receptor, Adenosine A1 , Reperfusion Injury , ReperfusionABSTRACT
PURPOSE: Intracavernous injection of PGE1 or papaverine is widely used in the diagnosis and treatment of erectile dysfunction. However, these drugs have several side effects such as pain, priapism, and fibrotic lesions. In this study, we assessed the effects of pinacidil (a K+ -ATP - channel opener) as an alternative for inducing penile erection. METHODS: Using a feline model, the magnitude of penile erection caused by pinacicil was compared with that caused by other drugs, namely acetylcholine, PGE1 and L-arginine. The effects of K+ -channel blockers(4-aminopyridine, glibenclamide, and tetraethylammonium; TEA) and pinacidil on the induced erections were investigated. RESULTS: Intra-arterial injection of pinacicil increased the intracavernous pressure (ICP) in a dose-dependent fashion, and the increase in ICP induced by pinacicil plus acetylcholine, PGE1 or L-arginine was more pronounced than that induced by any of these drugs alone. Furthermore, pinacicil (10(-3)M/mL) effectively reversed the inhibitory effects of the K+-channel blockers on cavernous relaxation induced by acetylcholine, PGE1 or L-arginine (P<0.01). Notably, pinacidil induced cavernous relaxation even in cases refractory to a higher concentration <10(-1) M/mL) of erectics (n = 11; p<0.01). CONCLUSIONS: These results suggest that pinacidil is effective in relaxing feline erectile tissue in vivo, probably via increased K+ permeability and subsequent hyperpolarization. Further comparative studies with human erectile tissue and clinical testing are required to show whether K+-channel openers can be used in the diagnosis and treatment of erectile dysfunction.
Subject(s)
Humans , Male , Acetylcholine , Alprostadil , Arginine , Diagnosis , Erectile Dysfunction , Glyburide , Injections, Intra-Arterial , Papaverine , Penile Erection , Permeability , Pinacidil , Priapism , Relaxation , TetraethylammoniumABSTRACT
PURPOSE: Intracavernous injection of PGE1 or papaverine is widely used in the diagnosis and treatment of erectile dysfunction. However, these drugs have several side effects such as pain, priapism, and fibrotic lesions. In this study, we assessed the effects of pinacidil (a K+ -ATP - channel opener) as an alternative for inducing penile erection. METHODS: Using a feline model, the magnitude of penile erection caused by pinacicil was compared with that caused by other drugs, namely acetylcholine, PGE1 and L-arginine. The effects of K+ -channel blockers(4-aminopyridine, glibenclamide, and tetraethylammonium; TEA) and pinacidil on the induced erections were investigated. RESULTS: Intra-arterial injection of pinacicil increased the intracavernous pressure (ICP) in a dose-dependent fashion, and the increase in ICP induced by pinacicil plus acetylcholine, PGE1 or L-arginine was more pronounced than that induced by any of these drugs alone. Furthermore, pinacicil (10(-3)M/mL) effectively reversed the inhibitory effects of the K+-channel blockers on cavernous relaxation induced by acetylcholine, PGE1 or L-arginine (P<0.01). Notably, pinacidil induced cavernous relaxation even in cases refractory to a higher concentration <10(-1) M/mL) of erectics (n = 11; p<0.01). CONCLUSIONS: These results suggest that pinacidil is effective in relaxing feline erectile tissue in vivo, probably via increased K+ permeability and subsequent hyperpolarization. Further comparative studies with human erectile tissue and clinical testing are required to show whether K+-channel openers can be used in the diagnosis and treatment of erectile dysfunction.
Subject(s)
Humans , Male , Acetylcholine , Alprostadil , Arginine , Diagnosis , Erectile Dysfunction , Glyburide , Injections, Intra-Arterial , Papaverine , Penile Erection , Permeability , Pinacidil , Priapism , Relaxation , TetraethylammoniumABSTRACT
The effect of pinacidil, a K+ channel opener, on the contraction of rabbit carotid artery was investigated by using muscle contraction and Ca2= uptake experiments. Pinacidil reduced phenylephrine-induced contraction by dose dependent manner, which was antagonized by glibenclamide, a blocker of the ATP sensitive K+ channel. Phenylephrine-induced tonic contraction was more reduced by pinacidil than its phasic contraction. In the effect of pinacidil on the Ca2+ uptake of rabbit carotid artery, pinacidil decreased it at the resting state of tissue, dose-dependently. Phenylephrine-induced stimulation of Ca2+ uptake was also reduced by pinacidil. Pinacidil 10micrometer reduced high potassium-induced contraction, which was not reversed by glybenclamide 10micrometer. Threshold concentration of K+ increased by pinacidil pretreatment. Phorbol 12, 13-dibutyrate, an activator of protein kinase C, induced sustained contraction of rabbit carotid artery, which was reduced by pinacidil but not antagonized by glibenclamide. In Ca2+-free buffer, pinacidil also decreased phorbol 12, 13-dibutyrate-induced contraction. These results indicate that pinacidil reduces Ca2+ uptake of vascular smooth muscle by stimulation of K+ channel which could be antagonized by glibenclamide, and another mechanism of vasorelaxation which could not be antagonized by glibenclamide. It was indecated that pinacidil affects the contaction of smooth muscle by the inhibition of protein kinase C.