Selective modulation of the ATP-sensitive K+ channel by nicorandil in guinea-pig cardiac cell membrane

Summary Effects of a vasodilator, nicorandil (2-nicotinamidoethyl nitrate) on four kinds for cardiac K⁺ channels were investigated in guinea pig ventricular and atrial cells using inside-out patch recording combined with oilgate concentration jump method.Nicorandil of 300 mol/l failed to affect the inward-rectifier K⁺ channel and the Na⁺-activated K⁺ channel. The open probability of the muscarinic K⁺ channel, when activated by the application of GTP, was not changed by the drug. Nicorandil selectively increased the open probability of the ATP-sensitive K⁺ channel that was partly suppressed by intracellular ATP. The median effective concentration (EC50) of nicorandil was 74 mol/l and Hill coefficient was 1.32 in the concentration-open probability relationship. The closing rate of the K⁺ channel by ATP was markedly delayed by the drug, whereas the open rate on removal of ATP was scarcely affected. Nicorandil had only little effect on this channel after run-down. It was concluded that nicorandil selectively activates the ATP-sensitive K⁺ channel mainly by modulating the ATP-dependent gate.Send offprint requests to M. Takano at the above address     

碘化N-正丁基氟哌啶醇对豚鼠心房肌细胞乙酰胆碱敏感性钾通道的影响

目的研究碘化N正丁基氟哌啶醇(F2)对豚鼠心房肌细胞乙酰胆碱敏感性钾通道(KACh)的影响,探讨其对KACh的作用机制。方法采用膜片钳全细胞记录方法,测定F2对原代培养的豚鼠心房肌细胞乙酰胆碱敏感性钾电流IK(ACh)的影响。结果细胞外给予F2对豚鼠心房肌细胞IK(ACh)呈可逆性、浓度依赖性的阻断作用。细胞内添入抗水解的GTP类似物GTPγS后,结果同前。细胞内给予50μmol·L-1F2对IK(ACh)无作用。结论F2是豚鼠心房肌细胞KACh的一种快速通道阻断剂,发挥作用部位在细胞膜外侧,作用位点在钾通道本身,与乙酰胆碱受体无关。     

ATP-sensitive K+ channels in the kidney

ATP-sensitive K⁺ channels (K_ATP channels) form a link between the metabolic state of the cell and the permeability of the cell membrane for K⁺ which, in turn, is a major determinant of cell membrane potential. K_ATP channels are found in many different cell types. Their regulation by ATP and other nucleotides and their modulation by other cellular factors such as pH and kinase activity varies widely and is fine-tuned for the function that these channels have to fulfill. In most excitable tissues they are closed and open when cell metabolism is impaired; thereby the cell is clamped in the resting state which saves ATP and helps to preserve the structural integrity of the cell. There are, however, notable exceptions from this rule; in pancreatic -cells, certain neurons and some vascular beds, these channels are open during the normal functioning of the cell.In the renal tubular system, K_ATP channels are found in the proximal tubule, the thick ascending limb of Henle's loop and the cortical collecting duct. Under physiological conditions, these channels have a high open probability and play an important role in the reabsorption of electrolytes and solutes as well as in K⁺ homeostasis. The physiological role of their nucleotide sensitivity is not entirely clear; one consequence is the coupling of channel activity to the activity of the Na-K-ATPase (pump-leak coupling), resulting in coordinated vectorial transport. In ischemia, however, the reduced ATP/ADP ratio would increase the open probability of the K_ATP channels independently from pump activity; this is particularly dangerous in the proximal tubule, where 60 to 70% of the glomerular ultrafiltrate is reabsorbed.The pharmacology of K_ATP channels is well developed including the sulphonylureas as standard blockers and the structurally heterogeneous family of channel openers. Blockers and openers, exemplified by glibenclamide and levcromakalim, show a wide spectrum of affinities towards the different types of K_ATP channels. Recent cloning efforts have solved the mystery about the structure of the channel: the K_ATP channels in the pancreatic -cell and in the principal cell of the renal cortical collecting duct are heteromultimers, composed of an inwardly rectifying K⁺ channel and sulphonylurea binding subunit(s) with unknown stoichiometry. The proteins making up the K_ATP channel in these two cell types are different (though homologous), explaining the physiological and pharmacological differences between these channel subtypes.     

ATP敏感性钾通道的阻断剂与开放剂研究进展

ATP敏感性钾通道是高血压、心绞痛、糖尿病及缺血性脑损伤等多种疾病的治疗靶点之一。通过对调节KATP通道药物的选择性、可逆性、作用位点以及相互调节的研究，可以对一些临床用药进行重新评估，并指导开发高选择性的KATP通道阻断剂和开放剂。     

丹参素对大鼠心室肌动作电位、L-型钙电流和ATP敏感性钾电流的作用

目的研究丹参素对单个大鼠心室肌细胞动作电位、L-型钙电流和ATP敏感性钾电流（IKAVP）的影响,探讨丹参素在离子通道水平的药理机制。方法胶原酶急性酶解法分离单个大鼠心室肌细胞,采用全细胞膜片钳的方法,记录丹参素对动作电位、L-型钙电流和IKATP的影响。结果丹参素可影响心室肌细胞动作电位时程（APD）,并能使APD25、APD50和APD90显著缩短;丹参素能够抑制三．型钙电流;丹参素可使IKA卯外向电流增大,此效应呈浓度依赖性。结论丹参素的心肌保护作用机制与抑制L-型钙电流和部分激活IKA口外向电流有关。     

Preparation and Physicochemical Characterization of Aqueous Dispersions of Coenzyme Q10 Nanoparticles

The present study describes a novel pharmaceutical formulation of coenzyme Q₁₀, viz. submicron-sized dispersions of the substance prepared by emulsification of molten coenzyme Q₁₀ in an aqueous phase. Photon correlation spectroscopy reveals mean diameters of 60 to 300 nm depending on process parameters. Coenzyme Q₁₀ nanoparticles remain stable on storage for more than 30 months. Lipophilic drugs can be incorporated into the nanoparticles demonstrating their potential use as a drug carrier system. Transmission electron micrographs of freeze-fractured replica show spherical particles with an amorphous core. Cryo-electron microscopy reveals the coexistence of small unilamellar vesicles in phospholipid stabilized dispersions. Thermoanalysis and X-ray studies indicate that the dispersed and emulsified coenzyme Q₁₀ does not recrystallize even at 4°C over 30 months. These agree with ¹H NMR data which demonstrate that coenzyme Q₁₀ molecules have a high mobility when formulated as nanoparticles and that colloidally dispersed coenzyme Q₁₀ remains in the state of a supercooled melt. Despite the high melting point of the bulk material, coenzyme Q₁₀ dispersions represent no suspensions but O/W emulsions according to the IUPAC definition (1).     

Modulation of the delayed K+ current by histamine in guinea pig ventricular myocytes

Summary The effects of histamine on delayed K⁺ current (I_K) were investigated in patch-clamped single guinea pig ventricular myocytes. Histamine increased I_K with a maximal fractional response of 2.7 and a k_d of 9.4 × 10^–7 mol/l. At a concentration of 10^–8 mol/l, histamine did not increase I_K significantly, but increased I_Ca by 52% ± 12%. The voltage-dependence of I_K activation, the reversal potential and the time course of the I_K tail decay were not changed by histamine. Under pretreatment with 10^–4 mol/l of ranitidine, neither histamine (10^–6 mol/l) nor 2-pyridylethylamine (10^–4 mol/l) caused any sizable increase in I_K. When the cell was pretreated with a saturating dose of isoproterenol (10^–6 mol/l), histamine did not additively enhance I_K. The I_K enhancement by 3 × 10^–7 mol/l histamine was partially antagonized by concurrent exposure to 5 × 10^–6 mol/l carbachol. Whereas, use of a higher concentration of histamine (10^–6 mol/l) obscured the inhibitory effect of carbachol. It is concluded that histaminergic action of I_K is attributed exclusively to H₂ receptor-mediated reactions involving G_s protein and adenylate cyclase. Send offprint requests to Y. Habuchi at the above address     

ATP敏感性钾通道在乳化异氟醚心肌保护效应中的作用

目的探讨ATP敏感性钾通道(KATP)是否参与乳化异氟醚(EI)对心肌细胞的保护作用。方法原代培养乳鼠离体心肌细胞,建立缺氧/复氧(H/R)损伤模型,随机分为6组,正常组(N组)、缺氧/复氧组(H/R组)、H/R+脂肪乳组(F组)、H/R+10mmol.L-1格列本脲组(G组)、H/R+1.68mmol.L-1EI组(EI组)和H/R+1.68mmol.L-1EI+10mmol.L-1格列本脲组(EIG组),n=6。各组取细胞培养上清液测定LDH活力与cTnI含量,心肌细胞匀浆后测定SOD活力与MDA含量;用倒置显微镜观察心肌细胞生长特性及形态的变化。结果与N组比较,各组的LDH、MDA、cTnI均升高(P<0.05),SOD下降(P<0.05)。与H/R组比较,F组的SOD下降(P<0.05),LDH、MDA、cTnI的差异均无统计学意义(P>0.05),G组的SOD、LDH、MDA、cTnI的差异均无统计学意义;EI组和EIG组的LDH、MDA、cTnI均降低(P<0.05),SOD升高(P<0.05)。与F组比较,EI组和EIG组的LDH、MDA、cTnI均下降(P<0.05),SOD升高(P<0.05)。与EI组比较,EIG组的LDH活力、MDA和cT-nI含量均升高,SOD活力降低(P<0.05)。结论乳化异氟醚对原代培养心肌细胞缺氧/复氧损伤的保护作用可能与其激活ATP敏感性钾通道有关。     

Tolbutamide- and diazoxide-sensitive K+ channel in neurons of substantia nigra pars reticulata

Summary Single-channel K⁺ currents were recorded in cell-attached patches from slices of rat substantia nigra. On the somata of neurons in the caudal half of the substantia nigra pars reticulata a K⁺ selective channel with a unitary conductance of 71 pS (154 mmol/l K⁺ in pipette filling solution) was identified. The channel was activated both by application of diazoxide (300 mol/l) and by energy-depleting conditions (200 mol/l cyanide) and was reversibly blocked by tolbutamide (0.1–1 mmol/l). It is concluded that neurons in the substantia nigra pars reticulata of the rat contain a typical ATP-sensitive K⁺ channel the activity of which can be modulated by diazoxide and sulfonylureas.Correspondence to: C. Schwanstecher at the above address     

Action of nicorandil on ATP-sensitive K+ channel in guinea-pig ventricular myocytes.

1. Patch-clamp techniques were used to study the effects of nicorandil (2-nicotinamiodethyl nitrate) on the adenosine 5'-triphosphate (ATP)-sensitive K+ channel current (IK.ATP) in guinea-pig ventricular myocytes. 2. Nicorandil activated the time-independent outward current. This effect was dependent on intracellular ATP concentration ([ATP]i) showing a larger effect at 2 mM than at 10 mM [ATP]i. The nicorandil-induced outward current was inhibited by application of 0.3 microM glibenclamide. 3. In the inside-out patch configuration, 0.3-1.0 mM nicorandil increased the open-stage probability of IK.ATP without a change in its conductance value (about 90pS). This effect was inhibited by glibenclamide. Analysis of the open and closed time distributions showed that nicorandil had no effect on open and closed distributions shorter than 5 ms. On the other hand, nicorandil increased the life time of bursts and decreased the interburst intervals. 4 The inward rectifier K+ channel current was not influenced by internal application of nicorandil. 5 Therefore, we conclude that IK.ATP is the only K+ current activated by nicorandil, and the main effect of nicorandil is on the kinetics of the IK.ATP bursting behaviour. These actions are similar to that of pinacidil on this preparation.     

Actions of ZD0947, a novel ATP-sensitive K+ channel opener, on membrane currents in human detrusor myocytes

BACKGROUND AND PURPOSE: ATP-sensitive K+ channels (K(ATP) channels) play important roles in regulating the resting membrane potential of detrusor smooth muscle. Actions of ZD0947, a novel KATP channel opener, on both carbachol (CCh)-induced detrusor contractions and membrane currents in human urinary bladder myocytes were investigated. EXPERIMENTAL APPROACH: Tension measurements and patch-clamp techniques were utilized to study the effects of ZD0947 in segments of human urinary bladder. Immunohistochemistry was also performed to detect the expression of the sulphonylurea receptor 1 (SUR1) and the SUR2B antigens in human detrusor muscle. KEY RESULTS: ZD0947 (> or = 0.1 microM) caused a concentration-dependent relaxation of the CCh-induced contraction of human detrusor, which was reversed by glibenclamide. The rank order of the potency to relax the CCh-induced contraction was pinacidil > ZD0947 > diazoxide. In conventional whole-cell configuration, ZD0947 (> or = 1 microM) caused a concentration-dependent inward K+ current which was suppressed by glibenclamide at -60 mV. When 1 mM ATP was included in the pipette solution, application of pinacidil or ZD0947 caused no inward K+ current at -60 mV. Gliclazide (< or =1 microM), a selective SUR1 blocker, inhibited the ZD0947-induced currents (Ki = 4.0 microM) and the diazoxide-induced currents (high-affinity site, Ki1 = 42.4 nM; low-affinity site, Ki2 = 84.5 microM) at -60 mV. Immunohistochemical studies indicated the presence of SUR1 and SUR2B proteins, which are constituents of KATP channels, in the bundles of human detrusor smooth muscle. CONCLUSIONS AND IMPLICATIONS: These results suggest that ZD0947 caused a glibenclamide-sensitive detrusor relaxation through activation of glibenclamide-sensitive KATP channels in human urinary bladder.     

Effects of isoflurane and ketamine on ATP-sensitive K channels in rat substantia nigra

Whole cell recordings were made using midbrain slices to examine the effects of two different anaesthetics on ATP-sensitive K (K_ATP) channels in principle neurons of rat substantia nigra pars compacta. When neurons were dialyzed with an ATP-free pipette solution during perfusion with a glucose-free external solution, a hyperpolarization and an outward current developed slowly in a tolbutamide-inhibitable manner. The volatile anaesthetic 3% isoflurane slightly depolarised the neurons in the presence of ATP in the pipette solution and glucose in the external solution, but it did not affect the hyperpolarization or outward current in response to omission of ATP and glucose. Ketamine, an intravenous anaesthetic, did not change the membrane potential when ATP and glucose were included; however, it reversibly inhibited the hyperpolarization and outward current induced by intracellular ATP depletion in a dose-dependent manner. These effects of ketamine were not mimicked by AP-5, an NMDA receptor antagonist, or indatraline, an inhibitor of catecholamine uptake. These findings suggest that these anaesthetics have no stimulatory action on K_ATP channels in these neurons when intracellular ATP is preserved and that ketamine but not isoflurane inhibits K_ATP channels when the channels were activated by low intracellular ATP.     

辅酶Q10注射液正交函数——分光光度测定法

本文报告了用正交函数法直接测定辅酶Q₁₀注射液含量的方法。以无水乙醇为溶剂,选择波长范围为268～292nm,间隔为12nm,用3点求二次正交多项式的P₂值,可消除吐温-80的干扰。辅酶Q₁₀回收率为98.60±0.66%。本法操作简单,准确度和精密度均较满意。     

人血浆中辅酶Q10的HPLC测定法及其动态研究

目的：建立人血浆中辅酶Q₁₀的高效液相色谱检测法,以测定人体内辅酶Q₁₀的经时变化过程。方法：血浆经无水乙醇沉淀蛋白后,以正己烷提取,进行高效液相色谱法检测。色谱柱为Spherisorb C₁₈ 10 μm 25 cm×4.6 mm ID,流动相为无水乙醇—水—冰醋酸(98∶2∶0.7),检测波长为275 nm,内标为辅酶Q₉。结果：在0.2～4.0 μg.ml^-1浓度范围内峰面积比与浓度呈良好的线性关系,γ=0.9998,方法重现性好,提取回收率大于90%;以本法测定了8名男性健康受试者服用辅酶Q₁₀制剂前后血浆中辅酶Q₁₀的浓度经时变化过程。结论：用本法测定人血浆中辅酶Q₁₀浓度结果满意;人血浆中内源性辅酶Q₁₀浓度为(763.3±86.3) ng.ml^-1,且受饮食及运动量的影响。     

Application of derivative spectrophotometry for determination of coenzyme Q10 in pharmaceuticals and plasma

The use of derivative spectrophotometry is proposed in this work for determination of coenzyme Q₁₀ in formulations and in human plasma. The spectrophotometric procedure is simpler and less expensive than chromatographic techniques commonly used for the analysis of coenzyme. The active compound can be determined in the range 0.25–10 ppm for standard solutions and pharmaceuticals and 0.05–1.5 ppm in plasma. The proposed method was applied for coenzyme determination in real samples. The results agree well with declared value and with these obtained by HPLC.     

Analysis of CoQ10 in rat serum by ultra-performance liquid chromatography mass spectrometry after oral administration

A UPLC-MS method for determining Coenzyme Q(10) (CoQ(10)) levels in rat serum was developed. CoQ(10) was quantitatively extracted into 2-propanol using a fast extraction procedure. The separation of CoQ(10) was performed on a Waters Acquity UPLCtrade mark BEH C(18) column (1.7 microm, 1.0 mm x 50 mm) with the mobile phase containing acetonitrile, 2-propanol, and formic acid (90:10:0.1) over 5 min. The sensitivity of this method allows for the quantitation of 50 ng/mL CoQ(10) in serum (S/N=10). The linearity of this method was found to be from 50 to 20,000 ng/mL. The precision was less than 10% (intra- and inter-day), and the average extraction recovery was between 90 and 105%. This procedure provides a precise, sensitive and direct assay method for the determination of CoQ(10) in rat serum after oral administration. This method could be applied to further pharmacokinetic studies of CoQ(10).     

Role of coenzyme Q(10) as an antioxidant and bioenergizer in periodontal diseases

Periodontal disease is an inflammatory disease process resulting from the interaction of a bacterial attack and host inflammatory response. Arrays of molecules are considered to mediate the inflammatory response at one time or another, among these are free radicals and reactive oxygen species (ROS). Periodontal pathogens can induce ROS overproduction and thus may cause collagen and periodontal cell breakdown. When ROS are scavenged by antioxidants, there can be a reduction of collagen degradation. Ubiquinol (reduced form coenzyme Q(10)) serves as an endogenous antioxidant which increases the concentration of CoQ(10) in the diseased gingiva and effectively suppresses advanced periodontal inflammation.     

辅酶Q10静脉注射乳的制备及其体外释放研究

目的制备辅酶Q10静脉注射乳,并研究其体外释放行为。方法对处方组成进行响应面优化,确定最佳处方。用激光粒度仪测定乳剂粒径、Zeta电位,透射电镜观察乳滴形态,初步考察乳剂稳定性及体外释放行为。结果油水比与磷脂用量对其稳定性系数Ke的影响较大,采用最佳处方制备的乳剂平均粒径为172±19.2 nm,Zeta电位为-33.1±2.9 m V。体外药物的释放试验中,反透析法比透析法稍快。结论所制备的辅酶Q10静脉注射乳质量可控,稳定性良好,具一定缓释作用。     

Effects of fluoroquinolones on insulin secretion and beta-cell ATP-sensitive K+ channels

Although fluoroquinolones are used widely in the treatment of various infectious diseases, some of the drugs are known to cause hypoglycemia as a side-effect. We have investigated the effects of three fluoroquinolone derivatives, levofloxacin, gatifloxacin, and temafloxacin, on insulin secretion and pancreatic beta-cell ATP-sensitive K(+) channel (K(ATP) channel) activity. While levofloxacin had only a small effect on insulin secretion and K(ATP) currents, gatifloxacin and temafloxacin stimulated insulin secretion and inhibited K(ATP) channel currents in a dose-dependent manner. We also determined the site of action of gatifloxacin and temafloxacin on the K(ATP) channel. In a reconstituted system, gatifloxacin and temafloxacin inhibited Kir6.2 Delta C26 channels, which function in the absence of the SUR subunit, indicating direct action of the drugs on the Kir6.2 subunits. These results suggest that stimulation of insulin secretion by inhibition of pancreatic beta-cell K(ATP) channels underlies the hypoglycemia caused by certain fluoroquinolones.