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组胺拮抗剂在小鼠 ATP-敏感性钾离子通道上的影响 朴伶华 1,姜圣男 3,柳贤德 2* (1. 海南医学院基础医学部,海口,海南 571199;2. 海南大学农学院,海口,海南 570228;3. 海南省 海口市人民医院,海口,海南 570109) 【摘要】目的:观察比较 3 种组胺拮抗剂对缺血性心肌细胞的 ATP-敏感性钾离子通道中的影响。方法: 利用急性酶解法分离小鼠心室肌细胞。结果:pyrilamine、chlorpheniramine 及 diphenhydramine 均可抑制 ATP-敏感性钾离子通道的活性,抑制程度为 pyrilamine > chlorpheniramine > diphenhydramine。组胺对 KATP 通道活性无影响。结论:第一代的组胺拮抗剂(pyrilamine 、 chlorpheniramine 及 diphenhydramine)对 KATP 通道活性有抑制作用,其抑制作用与膜上 H1 受体无关。 【关键词】 组胺拮抗剂;膜片钳;ATP-敏感性钾离子通道 【中图分类号】R332 【文献标识码】 【文章编号】YX2012231 Effect of Antihistaminic Agents on the ATP-sensitive Potassium Channel Activity in Isolated Mouse Ventricular Cardiomyocytes PIAO LINGHUA1, JIANG SHENGNAN 3, LIU XIANDE 2* (1Hainan medical university department of preclinical medicine, Haikou 571199; 2 Hainan university college of agriculture, Haikou, 570228; 3 Haikou people’s hospital department of nuclear medicine, Haikou, 570109) 【Abstract】 . Objective: The present study was undertaken to clarify the effect of the first generation histamine H1 receptor antagonists on the adenosine triphosphate-sensitive potassium (KATP) channel in the isolated mouse ventricular cardiomyocytes using excised inside-out and cell-attached patch clamp techniques. Methods: In the excised inside-out patch configuration, H1-antihistaminic agents (chlorpheniramine, pyrilamine and diphenhydramine), doses ranging from 1 to 100 μmol/L, inhibited KATP channel activity in a dose-dependent manner. The potency order reducing the channel activity was pyrilamine > diphenhydramine > chlorpheniramine. Results: All the antihistamines (100 μmol/L) also inhibited the pinacidil-induced KATP channel activity in the cell-attached patch configuration. The potency order of the 3 antihistamines inhibiting KATP channel activity is pyrilamine > chlorpheniramine > diphenhydramine in the 1 cell-attached configurations. Histamine did not affect the pinacidil-induced KATP channel activities by itself, in addition, did not influence the effects elicited by the 3 antihistamines on the pinacidil-induced KATP channel acitivities in the cell-attached patches. Conclusion: From these results, it concluded that the first generation histamine H1 receptor antagonists are involved in the regulation of the ATP-sensitive potassium channel activities in the mouse cardiac ventricular myocytes, and that the inhibitory actions of the antihistaminic agents on the channels are not dependent on the H1-receptors. 【Key words】antihistamine; patch clamp; ATP-sensitive potassium channel [作者简介] 朴伶华(1980−) ,女,研究方向:医学生理学。E-mail: [email protected]。 [通讯作者] 柳贤德(1979−) ,男,研究方向:动物医学。Email: [email protected]。 2 Introduction Numerous antihistaminic agents are used for the treatment of allergic diseases, but their use is sometimes restricted because of their adverse effects on the central nervous system, like somnolence and diminished alertness[1]. However, terfenadine, one of the most widely used antihistaminics, has been found that it exerts cardiovascular actions including life-threatening tachyarrythmias[2]. In addition, Jackman[3]reported that the prologation of QT interval precedes these arrhythmias, and therefore attention has been paid to the effects of the antihistaminic on the K+ currents that cause the action potential repolarization. In the cardiac muscles, adenosine triphosphate-sensitive K+ (KATP) channels are thought to play a role in the phenomenon of ischemic preconditioning in the heart, and the activation of these channels may improve recovery of regional contractile function of stunned myocardium by shortening action potential duration and attenuating membrane depolarization, thus decreasing contractility and preserving energy during ischemia. In addition, there are several types of K+ currents, which are involved in the action potential repolarization, such as the delayed rectifier IK1 and IKir. However, under pathological conditions of myocardial ischemia, a decrease in the intracellular adenosine triphosphate concentrations induces another K+ current IK,ATP within the plateau potential range of the ventricular action potentials[4,5]. Activation of this currents during acute ischemia shortens the action potential duration (APD) to protect the energy consumption and facilitates the ischemic preconditioning[6]. The K+ efflux due to these currents cause extracellular K+ accumulation[7]and may elicit arrythmia during cardiac ischemia. Despite the important roles of IK,ATP under pathological conditions, the effect of antihistaminics on the IK,ATP has not been clearly elucidated yet, and there also has been known that some antihistaminic agents are more likely to cause tachyarrhythmia in patients with underlying cardiac diseases, such as ischemic heart disease and congestive heart failure[2], indicating modulation of IK,ATP is involved in the cardiovascular side effects of the antihistaminics. Therefore, the aim of this study was to elucidate the relationships between the effects of antihistaminics and ATP-sensitive potassium channel activities in the enzymatically isolated mouse ventricular cardiac myocytes using inside-out and cell-attached patch clamp techniques. 1 Materials and Methods 1.1 Isolation of single mouse cardiac ventricular myocytes Single ventricular myocytes were isolated from adult mouse heart by the enzymatic dissociation procedure described previously[8]. In brief, the heart was cut from the ICR mouse which was kill by cervical disloation. And then after mounting the heart on a Langendorff apparatus, the organ was perfused for 5 min with a normally 1 mmol/L Ca2+-Tyrode's solution. The composition of Tyrode's solution was (in mmol/L) 137 NaCl, 5.4 KCl, 10 HEPES, 1 MgCl2, 0.33 NaH2PO4, 10 Glucose, 10 CaCl2 (pH 7.4 with NaOH). Ca2+-free Tyrode's solution was perfused for 10 min and Ca2+-free Tyrode's solution containing 1.5 mg/mL collagenase (Worthington Chemical Co.) was perfused for 25 min thereafter and then rinsed with a high K+, low Cl- kraftsbrühe (KB) solution contained (in 3 mmol/L) Taurine 20, L-Glutamic acid 70, KCl 25, KH2PO4 10, MgCl2 3, EGTA 0.5, HEPES 10, Glucose 10. The temperature of all the perfusion solutions was maintained at 37℃. The solution was bubbled with 100% O2. After the heart perfusion with the solution, the ventricular free wall was cut into small pieces, and isolated ventricular cells were stored in the KB solution then the isolated cells were observed under the microscope. 1.2 Electrophysiological recordings and solutions Single-channel recording was conducted in both the cell-attached and excised inside-out patch configurations[9]in the single mouse ventricular cardiac myocytes. Electrodes were fabricated from patch glass (Warner Instrument Co.) in two steps on an electrode puller (PP-83, Narishige Scientific Instruments). Pipette having a relatively high resistance (between 3 and 5 MΩ) were used to record as small number of channels in the patch as possible. The pipette was heat-polished immediately before use, and its shank was coated with Sylgard (Corning Co.). In this experiment, K-5 solution constituting (in mmol/L): KCl 118.5, MgCl2 2, KOH 21.5, EGTA 5, HEPES 10) was used for the bath and pipette recording solutions. S-(+)-Chlorpheniramine malate salt, dipheniramine hydrochloride, pyrilamine maleate salt, histamine, pinacidil, glibaenclamide and ATP were all obtained from Sigma Chemical Co. For stock solutions were initially dissolved in distilled water besides the pinacidil which was dissolved in DMSO. 1.3 Data acquisition and analysis In experiments, the currents data, obtained through a patch clamp amplifier (AxoPatch-1D, Axon Instruments), were filtered at 2 kHz, and stored on a video tape using a video data recorder. For the analysis of single channel activity, the data were transferred to a computer with pClamp (version 7.0, Axon Instruments) through an analog-to-digital converter interface (Digidata-1200, Axon Instruments). The open probability (Po) was calculated using the formula which was introduced by Spruce [10]: N Po=(Σtjj)/Td n j=1 where tj is the time spent at current levels corresponding to j=0, 1, 2... N channels in the open state. 4 Td is the duration of the recording and N is the number of channels active in the patch. Po is calculated for over 30-sec records. To measure the magnitude of the inhibition by the antihistaminic agents, the experimental results was fitted as follows, Relative activity (%) = (Po antihistamine / Po control) X 100 Relative activity 100% represents the channel activity for 30 secs of the control channel activity. 1.4 Quantification and Statistics Results are expressed as mean±S.E. of the N cells, The students t-test was used to determine the significance of differences between set of data. p<0.01 was considered statistically significant. 2 Results 2.1 Ascertain the ATP-sensitive K+ channel activity in the excised inside-out and cell-attached patches At -60 mV holding potential in excised inside-out patch configuration, channel activity appeared immediately after making excised inside-out patch, under the superfusion of ATP-free bath solution and 1 mmol/L ATP inhibited the channel activity almost completely. Channel activities resumed when the ATP was wash out. Glibenclamide (50 μmol/L), a KATP channel blocker, inhibited the channel activities again (Fig. 1). 图 1. ATP(1 mM)及格列本脲(20 μM)在膜内向外方式中对小鼠心室肌细胞 KATP 通道的 影响 Figure 1. A trace showing the typical ATP-sensitive K+ channel activities in the excised inside-out configuration at -60mV holding potential (HP) in isolated mouse ventricular myocytes. Channel activity appeared immediately after making excised inside-out patch and ATP (1 mM) inhibited the channel activity almost comletely. Channel activity revived when ATP was washed out, and Glibenclamide (20 μM) inhibited the channel activity again. At -60 mV holding potential in the cell-attached patch configuration, scanty channel activities 5 were developed in cell-attached patch configuration under the superfusion of ATP-free bath solution. Pinacidil (200 μmol/L), a K+ channel opener, induced a plenty of channel activities and the pinacidil-induced channel activities were reduced by glibenclamide (50 μmol/L) (Fig. 2). 图 2.格列本脲(50 μM)在细胞吸附法中对小鼠心室肌细胞 KATP 通道的影响 Figure 2. A trace showing the typical KATP channel activities in the cell-attached patch at -60mV holding potential (HP) in the isolated mouse ventricular myocytes. Pinacidil (200 μM) induced the channel activity and glibenclamide (50 μM) inhibited the channel activity. From the results of these experiments, it was ascertained that the channel activities in the present study are ATP-sensitive K+ channel activities in the excised inside-out and cell-attached patches respectively. 2.2 Effect of antihistamines on the KATP channel activity in the excised inside-out patches The antihistaminic agents, used in the present study, generally inhibited KATP channel activity in the excised inside-out patches under the superfusion of ATP-free solution. The relative activities of the channel by 1, 10 and 100 μmol/L chlorpheniramine were 48.3±7.91, 16.5±2.13, 3.3±1.80%, respectively (Fig. 3). 图 3. chlorpheniramine 在膜内向外方式中对小鼠心室肌细胞 KATP 通道的影响 6 Figure 3. Effect of chlorpheniramine (CPM) on the KATP channel activity in the excised inside-out patch configuration at -60 mV holding potential (HP). A: CPM attenuated KATP channel activities in a dose-dependent manner. B: Histogram shows its inhibition at different concentration. Relative activity 100% represents the channel activity for 30 secs of the control channel activity. (* p<0.01 significant difference from the control.) Diphenhydramine (DPH) also attenuated the KATP channel activity in a dose-dependent manner in the excised inside-out patch. Relative activities by 1, 10 and 100 ?mol/L DPH were 42.3±6.99, 22.8±4.60 and 5.2±1.29%, respectively (Fig. 4). 图 4. diphenhydramine 在膜内向外方式中对小鼠心室肌细胞 KATP 通道的影响 Figure 4. Effect of diphenhydramine (DPH) on the KATP channel activities in the excised inside-out patch configuration at -60 mV holding potential (HP). A: DPH attenuated KATP channel activity in a dose-dependent manner. B: Histogram shows its inhibition at different concentration. Relative activity 100% represents the channel activity for 30 secs of the control channel activity. (* p<0.01 significant difference from control). An another histamine antagonist pyrilamine (PYL) inhibited KATP channel activity in the excised inside-out patch at -60 mV holding potential, similar fashion to those of the CPM and DPH, a relative activities by 1, 10 and 100 ?mol/L were 35.7±11.05, 10.9±3.25 and 1.0±0.29% respectively (Fig. 5). The potency rank order of the 3 antihistamines inhibiting KATP channel activity in the excised inside-out patch was PYL > DPH > CPM (Fig. 6). 7 图 5. pyrilamine 在膜内向外方式中对小鼠心室肌细胞 KATP 通道的影响 Figure 5. Effect of pyrilamine (PYL) on the KATP channel activities in the excised inside-out patch configuration at -60 mV holding potential (HP). A: PYL attenuated KATP channel activity in a dose-dependent manner. B: Histogram shows its inhibition at different concentration. Relative activity 100% represents the channel activity for 30 secs of the control channel activity. (* p<0.01 significant difference from control). 图 6. 比较 3 种抗组胺药物对 KATP 通道的影响程度 Figure 6. Dose-response curves of antihistamines for the inhibition of KATP channel activities in the excised inside-out patch configuration at -60 mV of holding potential. The potency order of antihistamines (10 and 100 μM) was PYL>CPM>DPH but was PYL > DPH > CPM at 1 μM in the mouse cardiac ventricular myocytes. 8 2.3 Effect of Antihistamines on the KATP channel activity in the cell-attached patches In the cell-attached patch configuration, KATP channel activities were not spontaneously developed at -60 mV holding potential. The pinacidil artificially opens the channel by altering its sensitivity to ATP, and therefore pinacidil (200 μmol/L) was used for activition of the KATP channel. All the 3 antihistamines (CPM, DPH and PYL; 100 μmol/L) inhibited the pinacidil-induced KATP channel activity in the cell-attached patches, and the rank order of potency was PYL > CPM > DPH, as seen in the relative channel activities 3.9±2.27, 10.8±9.09 and 29.0±8.44% respectively (Fig. 7 and 8). 图 7. 3 种抗组胺药物(chlorpheniramine,diphenhydramine 及 pyrilamine)在细胞吸附法中对 小鼠心室肌细胞 KATP 通道的影响 Figure 7. Effects of antihistamines on the KATP channel activity in the cell-attached patch at -60 mV holding potential in the mouse cardiac ventricular myocytes. Chlorpheniramine (CPM), diphenhydramine (DPH) and pyrilamine (PYL) inhibited pinacidil-induced KATP channel activities. 9 图 8. 3 种抗组胺药物在细胞吸附试验中对 KATP 通道影响程度比较,p<0.01 Figure 8. Effects of antihistamine (100 μM) on the pinacidil-induced KATP channel activity in the cell-attached configuration at -60 mV holding potential. Diphenhydramine (DPH), chlorphenhydramine (CPM) and pyrilamine (PYL) inhibited the pinacidil-induced channel activities. Relative activity 100% represents the channel activity for 30 secs of the control channel activity. (* p<0.01 significant difference from control.) 2.4 Influence of histamine on the antihistamins' inhibitory effects of pinacidil-induced channel activity in the cell-attached patches It was examined whether the antihistamine's inhibitory effects of KATP channel activity was through the histamine receptors or not. Firstly, the histamine was added to the bath solution which contains pinacidil. Histamine itself did not affect the pinacidil-induced channel activities (Fig. 9), and the antihistamines (CMP, DPH and PYL; 100 μmol/L), added to the bath solution, still inhibited the pinacidil-induced channel activities even in the presence of histmaine (Fig. 10). Relative activity by the 3 antihistamines (CMP, DPH and PYL; 100 μmol/L) under both histamine and pinacidil superfusion was 13.3±11.73, 7.6±2.49 and 3.0±1.79%, respectively (Fig. 11). 10 图 9. 组胺在细胞吸附法中对小鼠心室肌细胞 KATP 通道的影响 Figure 9. Effect of histamine (100 μM) on the pinacidil-induced KATP channel activity in the cell-attached configuration at -60 mV holding potential in isolated mouse ventricular myocytes. Histamine have no effect on the pinacidil-induced channel activity. 图 10. 组胺和 3 种抗组胺药物在细胞吸附法对心室肌细胞 KATP 通道影响关系 Figure 10. Influence of histamine on the antihistamins' inhibitiory effect of the pinacidil-induced channel activity at -60 mV holding potential in the cell-attached patch configuration in isolated mouse ventricular myocytes. Histamine did not affect the antihistamins' inhibitiory effect on the pinacidil-induced channel activities. 11 图 11. 组胺和 3 种抗组胺药物在细胞吸附法对心室肌细胞 KATP 通道影响关系比较,p<0.01 Figure 11. Effects of histamine on the pinacidil-induced channel activity in the cell-attached patch configuration. Histamine (100 μM) has no effect on the channel activity inhibitory effect of the antihistamins. Relative activity 100% represents the channel activity for 30 secs of the control channel activity. (* p<0.01 significant difference from control.) 3 Discussion The present study demonstrates that the first generation antihistaminics (chlorpheniramine, diphenhydramine and pyrilamine) inhibited the ATP-sensitive potassium (KATP) channel activities in the mouse ventricular myocytes, indicating antihistaminic agents are involved in the ischemia pathophysiology. This is the first result to show that antihistaminics could inhibit the KATP channel activities at both inside and outside of the cells. During the ischemia and cardiac failure, the activation of the KATP channels by deletion of ATP would shorten the action potential duration (APD). Simultaneously, the hyperpolarization of membrane might reduce Ca2+ influx into the metabolically the disturbed cell, and limit further myocardial damages. These actions would contribute to the prevention of calcium overload related arrhythmias[11]. Antihistaminics could inhibit KATP channel activities, and therefore inhibited the K+ efflux, making the plateau phase prolonged and increase of the Ca2+ influx. This would be harmful effect for the ischemic myocardium, due to the positive inotropic and chronotropic action of antihistaiminics. In addition to the result of present study, other recent evidence suggests that mitochondrial KATP channels mediate cardioprotection in ischemic preconditioning[12,13]. In the present study, it was found that the rank order of potency inhibiting KATP channel 12 activities by the 3 antihistaminics was PYL > DPH > CPM in the case of excised inside-out patch configuration, and was PYL > CPM > DPH in the case of cell-attached patch configration respectively. The reason why the potency order of the 3 antihistaminics is not just same in the two patch configurations could not be understood only with this study, but it indicates that the subcellular action mechanisms inhibiting KATP channel activity in the two configurations are not the same. As seen in the present study, antihistamines inhibited the KATP channel activities in general. However, it is clear whether this antihistaminics' effect on the KATP channel regulation is from the action through the histamine receptor or not. Not only histamine did not affect the KATP channel itself but also did not affect the antihistaminics' KATP channel inhibitory effects, indicating no connection with the H1-receptors. It could be supposed that the antihistaminics' KATP channel inhibition is not from the action through the histamine receptor, but rather, from the action directly to the channel moiety. It has been reported that terfenadine, a histamine antagonist, blocks pancreatic β-cell KATP channels via binding to the cytoplasmic side of the pore-forming subunit[14]. In this experiment, the antihistaminics inhibited the KATP channel in the excised inside-out and cell-attached patches in the isolated mouse ventricular myocytes. Though it is not defined the effect of the IK,ATP activation on the arrhythmias either early or delayed afterdepolarization[15,16], the prolongation of action potential due to the IK,ATP blockage in the ischemic zone would attenuate the dispersion of the refactory period. It has been also reported that blockade of IK,ATP contributed to the antihistaminic actions of antiarrhythmic agents[17,18]. Although IK,ATP play a key role in the ischemic preconditioning[6,19], some patient had porarrhythmic events and sudden death were developed in some patients, with some of antihistaminic agents, inhibiting IK,ATP[20,21]. In summary, some first generation antihistmainic agents are involved in the regulation of ATP-sensitive potassium channel activity in the myocardium, and the inhibitory effects of the agents on the KATP channel are not from the actions through the classical H1-histamine receptors, would be harmful to ischemic myocardium. It was examined the effects of first generation H1 histamine receptor antagonists (chlorpheniramine, pyrilamine and diphenhydramine) on the ATP-sensitive potassium (KATP) channel activity in the isolated mouse ventricular cardiac myocytes. 13 Single cardiac myocytes were isolated from the ventricle of adult mouse with enzymatic dissociation, and the excised insede-out and cell-attached patch clamp techniques were used to measure the ATP-sensitve K+ channel activities. In the excised inside-out patch configuration, H1-antihistaminic agents (chlorpheniramine, pyrilamine and diphenhydramine), doses ranging from 1 to 100 μmol/L, inhibited KATP channel activity respectively in a dose-dependent manner and the potency order of these is pyrilamine > diphenhydramine > chlorpheniramine. All the antihistamines (100 μmol/L) also inhibited the pinacidil-induced KATP channel activity in the cell-attached patch configuration. The potency order of the 3 antihistamines inhibiting KATP channel activity is pyrilamine > chlorpheniramine > diphenhydramine in the cell-attached patches. Histamine did not affect the pinacidil-induced KATP channel activities by itself, in addition, histamine did not affect the effects by the 3 antihistamines on the pinacidil-induced KATP channel acitivities in the cell-attached patches. 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