ATP-sensitive potassium channels are altered in ventricular myocytes from diabetic rats

The affinities of Factor XIII (FXIII), Factor XIIIa (FXIIla), and cellular transglutaminase (Tg) for fibrinogen (Fgn), fibrin (Fbn), and fibronectin (Fn) were compared using a solid-phase binding assay. Initial rates of binding were as follows: FXIII bound Fbn 3-fold more than Fgn. FXIII did not bind Fn till 20 min. Increasing the ligands concentrations and binding time, resulted in weak binding of FXIII to Fn. FXIIla bound Fbn 2-fold more than Fgn and 28-fold more than Fn. Tg bound Fn 130-fold more than either Fgn or Fbn. At equilibrium, the extent of binding was determined to be as follows: FXIII bound Fbn 3–15-fold more than Fgn and 8-fold more than Fn. FXIIIa bound Fgn and Fbn equally and 12–25-fold more than Fn. FXIIla bound Fgn or Fbn 2-fold and 25-fold greater than FXIII-Fbn and FXIII-Fgn interactions, respectively. Tg bound about equally to Fgn and Fbn and 10–20-fold less than Fn. The K _d s for FXIIla binding to Fn, Fgn, and Fbn were 100, 23, and 19 nM, respectively. The K _d for Tg binding to Fn was 6.5 nM. The binding hierarchies are: [Tg-Fn]>[FXIIIa-Fgn]=[FXIIIa-Fbn]>[FXIII-Fbn]>[FXIIIFgn]=[FXIIIa-Fn]>[Tg-Fbn]=[Tg-Fgn]>[FXIII-Fn]. Such hierarchies could regulate the cross-linkings by FXIIIa and Tg during hemostasis, wound healing, and cell adhesion.Abbreviations Tg cellular transglutaminase - FXIII coagulation factor XIII - FXIIla factor XIIIa (thrombin-activated FXIII) - Fgn human plasma fibrinogen - Fn human plasma fibronectin - Fbn human plasma fibrin (thrombin-cleaved fibrinogen) - ECM extracellular matrix     

血小板活化因子对豚鼠心室肌细胞动作电位和钾通道的影响

应用全细胞膜片钳技术研究了血小板活化因子(platelet activatingfactor,PAF)对豚鼠心室肌细胞动作电位和钾电流的影响.结果发现,当电极内液ATP浓度为5 mmol/L(模拟正常条件)时,1 μmol/L PAF使APD90由对照的225.8±23.3 ms延长至352.8±29.8ms(n=5,P＜0.05);使IK尾电流在指令电压 30 mV由对照的173.5±16.7 pA降至152.1±11.5 pA(P＜0.05,n=4);使Ikl在指令电压为-120 mV时由对照组的-6.1±1.3 nA降至-5.6±1.1 nA(P＜0.05,n=5);但PAF在生理膜电位范围(-90mV～ 20mV)对IK1没有影响.当电极内液ATP浓度为0mmol/L时,IK·ATP开放(模拟缺血条件),1 μmol/LPAF却显著缩短APD90,由对照的153±24.6 ms缩短至88.2±19.4 ms(n=5,P＜0.01).而用1 μmol/L格列本脲(IK·ATP的特异阻断剂)预处理后,恢复了PAF可显著延长动作电位时程的作用.结果提示,PAF可能扩大缺血心肌和正常心肌细胞动作电位时程的不均一性,是缺血/再灌注性心律失常发生的重要原因.     

A role for ATP-sensitive potassium channels in male sexual behavior

ATP-sensitive potassium (K⁺_ATP) channels regulate cell excitability and are expressed in steroid-responsive brain regions involved in sexual behavior, such as the preoptic area (POA) and medial basal hypothalamus (MBH). We hypothesized that K⁺_ATP channels serve as a mechanism by which testosterone can control the electrical activity of neurons and consequently elicit male sexual responsiveness. RT-PCR analysis indicated that castration induces, while testosterone inhibits, mRNA expression of the K⁺_ATP channel subunit Kir6.2 in both the POA and MBH of adult male rats. Intracerebral infusion of the pharmacological K⁺_ATP channel inhibitor tolbutamide increased the proportion of long-term castrates displaying sexual behavior and restored mount frequency, intromission frequency, and copulatory efficacy to values observed in testes-intact animals. Infusions of tolbutamide, but not vehicle, also decreased latencies to mount and intromit in castrated males. Unilateral tolbutamide infusion directly into the POA significantly reduced mount latency of castrates; however, it did not affect other copulatory measures, suggesting that blockade of K⁺_ATP channels in additional brain regions may be necessary to recover the full range of sexual behavior. These data indicate that blockade of K⁺_ATP channels is sufficient to elicit the male sexual response in the absence of testosterone. Our observations are consistent with the hypothesis that testosterone modulates male sexual behavior by regulating K⁺_ATP channels in the brain. Decreased channel expression or channel blockade may increase the excitability of androgen-target neurons, rendering them more sensitive to the hormonal, chemical, and somatosensory inputs they receive, and potentially increase secretion of neurotransmitters that facilitate sexual behavior.     

核苷酸对ATP-敏感性钾通道开放剂吡那地尔舒血管效应的调节作用

目的: 在正常Wistar大鼠离体主动脉上,研究核苷酸类物质ATP,ADP,UDP,GTP及其代谢产物腺苷(Ade)对ATP敏感性钾通道(ATP-sensitive potassium channel,KATP)开放剂吡那地尔(pinacidil,Pin)舒血管效应的影响,以评价其对动脉平滑肌KATP功能的调节作用.方法: 大鼠离体主动脉去内皮血管环以核苷酸或核苷酸与格列本脲(Gli)孵育10 min后,再以KCl 20 mmol*L-1诱发收缩,观察Pin的血管舒张效应的变化.结果: 离体去内皮大鼠主动脉标本经ATP,ADP,UDP和GTP 4种核苷酸和Ade在100 μmol*L-1浓度下预孵育后,Pin舒血管作用发生变化:①ATP可减弱Pin对KATP的开放作用,但增强Gli对KATP的阻断作用.②ADP既减弱Pin对KATP的开放作用,又减弱Gli对KATP的阻断作用.③Ade对KATP功能的调节与ADP相似,既减弱Pin对KATP的开放作用,又减弱Gli对KATP的阻断作用.④UDP 100 μmol*L-1可增强Pin对KATP的开放作用,但减弱Gli对KATP的阻断作用.⑤GTP可增强Pin激活KATP开放的作用,但对Gli阻断KATP开放的作用无影响.结论: 不同的核苷酸和腺苷均可调节血管平滑肌KATP的功能,但其调节作用的药理学特征并不相同.     

组胺拮抗剂对小鼠ATP-敏感性钾离子通道的影响

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。结果组胺拮抗剂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。     

新型钾通道开放剂对心血管ATP-敏感性钾通道基因表达的调节作用

目的：研究脂肪胺类的新型钾通道开放剂（KCO）埃他卡林（Ipt）和氰胍类的KCO吡那地尔（Pin）对大鼠心血管ATP-敏感性钾通道（KATP）的亚基SUR1、SUR2、Kir6．1和Kir6．2等在mRNA水平的调节作用。方法：SD大鼠给药1周后处死并取组织,提取总RNA,利用反转录-聚合酶链式反应（RT-PCR）研究以上基因在mRNA水平的改变。结果：与正常对照相比,心脏组织中,Ipt和Pin对KATP的4个亚基在mRNA水平均无显著影响;主动脉平滑肌上,Ipt对4个亚基的mRNA表达无显著影响,但Pin可显著上调SUR2的mRNA表达;尾动脉平滑肌上,Ipt对Kit6．1／Kit6．2、Pin对SUR2／Kir6．1均有显著下调的作用。结论：心肌、大动脉平滑肌和小动脉平滑肌KATP基因表达的调控不同,Ipt选择性调节小动脉平滑肌Kit6．1／Kit6．2;Ipt对心血管KATP基因表达的调节作用不同于Pin。     

Triphenylphosphonium salts of 1,2,4-benzothiadiazine 1,1-dioxides related to diazoxide targeting mitochondrial ATP-sensitive potassium channels

The present work aims at identifying new ion channel modulators able to target mitochondrial ATP-sensitive potassium channels (mitoK_ATP channels). An innovative approach should consist in fixing a cationic and hydrophobic triphenylphosphonium fragment on the structure of known K_ATP channel openers. Such phosphonium salts are expected to cross the biological membranes and to accumulate into mitochondria.Previous works revealed that the presence of an (R)-1-hydroxy-2-propylamino chain at the 3-position of 4H-1,2,4-benzothiadiazine 1,1-dioxides K_ATP channel openers increased, in most cases, the selectivity towards the pancreatic-type (SUR1/Kir6.2) K_ATP channel. In order to target cardiac mitoK_ATP channels, we decided to introduce a triphenylphosphonium group through an ester link on the SUR1-selective (R)-7-chloro-3-(1-hydroxy-2-propyl)amino-4H-1,2,4-benzothiadiazine 1,1-dioxide. The new compounds were found to preserve an inhibitory activity on insulin secretion (SUR1-type K_ATP channel openers) while no clear demonstration of an impact on mitochondria from cardiomyocytes (measurement of oxygen consumption, respiratory parameters and ATP production on H9C2 cells) was observed. However, the most active (inhibition of insulin release) compound 17 was found to penetrate the cardiac cells and to reach mitochondria.     

ATP-sensitive potassium channels and vascular function

Fluorescence-based approaches provide powerful techniques to directly report structural dynamics underlying gating processes in Shaker K_V channels. Here, following on from work carried out in Shaker channels, we have used voltage clamp fluorimetry for the first time to study voltage sensor motions in mammalian K_V1.5 channels, by attaching TMRM fluorescent probes to substituted cysteine residues in the S3-S4 linker of K_V1.5 (A397C). Compared with the Shaker channel, there are significant differences in the fluorescence signals that occur on activation of the channel. In addition to a well-understood fluorescence quenching signal associated with S4 movement, we have recorded a unique partial recovery of fluorescence after the quenching that is attributable to gating events at the outer pore mouth,¹ that is not seen in Shaker despite significant homology between it and Kv1.5 channels in the S5-P loop-S6 region. Extracellular potassium is known to modulate C-type inactivation in Shaker and K_V channels at sites in the outer pore mouth, and so here we have measured the concentration-dependence of potassium effects on the fluorescence recovery signals from A397C. Elevation of extracellular K⁺ inhibits the rapid fluorescence recovery, with complete abolition at 99 mM K⁺, and an IC₅₀ of 29 mM K⁺_o. These experiments suggest that the rapid fluorescence recovery reflects early gating movements associated with inactivation, modulated by extracellular K⁺, and further support the idea that outer pore motions occur rapidly after K_V1.5 channel opening and can be observed by fluorophores attached to the S3-S4 linker.     

Properties of inwardly rectifying K+ channels in ventricular myocytes

Summary The voltage- and time-dependent properties of whole-cell, multi-channel (outside-out), and single channel inwardly-rectifying K⁺ currents were studied using adult and neonatal rat, and embryonic chick ventricular myocytes. Inward rectification of the current-voltage relationship was found in the whole-cell and single channel measurements. The steady-state single channel probability of opening decreased with hyperpolarization from E_K, as did the mean open time, thereby explaining the time-dependent inactivation of the macroscopic current. Myocytes dialysed with a Mg⁺⁺-free K⁺ solution (to remove the property of inward rectification) displayed a quasi-linear current-voltage relationship. The outward K⁺ currents flowing through the modified inward rectifier channels were able to be blocked by the local anesthetic and anti-arrhythmic agent, lidocaine.     

Stretch activated ion channels in ventricular myocytes

Patch-clamp recordings from ventricular myocytes of neonatal rats identified ionic channels that open in response to membrane stretch caused by negative pressures (1 to 6 cm Hg) in the electrode. The stretch response, consisting of markedly increased channel opening frequency, was maintained, with some variability, during long (>40 seconds) stretch applications. The channels have a conductance averaging 120 pS in isotonic KCl, have a mean reversal potential 31 mV depolarized from resting membrane potential, and do not require external Ca⁺⁺ for activation. The channels appear to be relatively non-selective for cations. Since they are gated by physiological levels of tension, stretch-activated channels may represent, a cellular control system wherein beat-to-beat tension and/or osmotic balance modulate a portion of membrane conductance.Abbreviations SACs stretch-activated channels - HEPES 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid     

西洛他唑对人心房肌细胞瞬间外向钾电流的影响

目的:观察西洛他唑对人心房肌细胞瞬间外向钾电流(Ito1)的影响,探讨该药抗心律失常作用的机制.方法:二步酶解法分离人单个右心房肌细胞,应用全细胞膜片钳技术记录人心房肌细胞Ito1.结果:在保持电位-50 mV和去极化脉冲为 50 mV条件下,30 μmol/L西洛他唑显著降低Ito1,使Ito1幅值由加药前(8.16±0.70)pA/pF降至(4.84±0.60)pA/pF(P<0.01).西洛他唑在1～50 μmol/L范围内呈浓度依赖性的抑制Ito1,1 μmol/L时即产生作用,50 μmol/L时达最大效应(降低51.09%±3.00%),IC50为(13.18±2.60)μmol/L.此外,该药对Ito1的电压依赖性激活和失活曲线以及恢复曲线均无显著影响.结论:本实验结果表明西洛他唑浓度依赖性地阻滞人心房肌细胞的Ito1.     

Cytoplasmic acidosis induces multiple conductance states in ATP-sensitive potassium channels of cardiac myocytes

We studied the effect of cytoplasmic acidosis on the ionic conducting states of ATP-sensitive potassium channels in heart ventricular cells of guinea pigs and rabbits by using a patch-clamp technique with inside-out patch configuration. Under normal conditions (pH 7.4), the channel alternated between a closed state and a main open state in the absence of nucleotides on the cytoplasmic side. As internal pH was reduced below 6.5, the single channel current manifested distinct subconductance levels. The probability of the appearance of these subconductance levels was pH dependent with a greater probability of subconductance states at lower pH. A variance-mean amplitude analysis technique revealed two subconductance levels approximately equally spaced between the main open level and the closed level (63 and 33%). A current-voltage plot of the two subconductance levels and the main level showed that they had similar reversal potentials and rectification properties. An intrinsic flickering gating property characteristic of these ATP-sensitive channels was found unchanged in the 63% subconductance state, suggesting that this subconductance state and the main conductance state share similar ion pore properties (including ion selection and block) and similar gating mechanisms. The appearance of the subconductance states decreased as ionic strength was increased, and the subconductance states were also slightly voltage dependent, suggesting an electrostatic interaction between the protons and the negative surface charge in the vicinity of the binding sites, which may be close to the inner entrance of the ion pore. Proteolytic modification of the channel on the cytoplasmic side with trypsin did not abolish the subconductance levels. External acidosis did not induce subconductance levels. These results suggest that protons bound to the negatively charged group at the inner entrance of the channel ion pore may induce conformational changes, leading to partially reduced conductance states.     

Minoxidil prevents 3,4-methylenedioxymethamphetamine-induced serotonin depletions: role of mitochondrial ATP-sensitive potassium channels, Akt and ERK

Preconditioning has emerged as a valid strategy against different neurotoxic insults. Although the mechanisms underlying preconditioning are not fully understood, the activation of ATP-sensitive potassium (KATP) channels has been proposed to play a pivotal role in neuronal preconditioning. In the present work we examine whether minoxidil a KATP channel activator protects against the long-term toxicity caused by the amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA) in rats. Our data show that intrastriatal administration of minoxidil prevents MDMA-induced long-term indole depletions in the rat striatum. This effect was not related to an effect on core temperature, as pre-treatment with minoxidil did not significantly alter MDMA-induced hyperthermia. Taking into account that minoxidil opens both sarcolemmal and mitochondrial KATP channels, we examined the role of each type of channels in the protective effects of minoxidil using specific inhibitors. The administration of HMR-1098, a blocker of the sarcolemmal KATP channels, along with minoxidil did not affect the protection afforded by the latter. On the contrary the selective mitochondrial KATP channel blocker 5-hydroxydecanoic acid completely reversed the protection afforded by minoxidil, thereby implicating the involvement of mitochondrial (but not sarcolemmal) KATP channels. Furthermore our data show the participation of Akt and extracellular signal-regulated kinases in minoxidil-afforded protection. Intrastriatal administration of wortmannin or PD98059 (inhibitors of phosphatidylinositol-3-kinase and mitogen-activated protein kinase/extracellular regulated protein kinase, respectively), along with minoxidil abolished the protective effect of minoxidil against the serotonergic toxicity caused by MDMA. These results demonstrate that minoxidil by opening mitochondrial KATP channels completely prevents MDMA toxicity and that Akt and MAP kinases are involved in minoxidil-afforded neuroprotection.     

Identification of two types of ATP-sensitive K+ channels in rat ventricular myocytes

The ATP-sensitive K(+) (K(ATP)) channels are known to provide a functional linkage between the electrical activity of the cell membrane and metabolism. Two types of inwardly rectifying K(+) channel subunits (i.e., Kir6.1 and Kir6.2) with which sulfonylurea receptors are associated were reported to constitute the K(ATP) channels. In this study, we provide evidence to show two types of K(ATP) channels with different biophysical properties functionally expressed in isolated rat ventricular myocytes. Using patch-clamp technique, we found that single-channel conductance for the different two types of K(ATP) channels in these cells was 57 and 21 pS. The kinetic properties, including mean open time and bursting kinetics, did not differ between these two types of K(ATP) channels. Diazoxide only activated the small-conductance K(ATP) channel, while pinacidil and dinitrophenol stimulated both channels. Both of these K(ATP) channels were sensitive to block by glibenclamide. Additionally, western blotting, immunochemistry, and RT-PCR revealed two types of Kir6.X channels, i.e., Kir6.1 and Kir6.2, in rat ventricular myocytes. Single-cell Ca(2+) imaging also revealed that similar to dinitrophenol, diazoxide reduced the concentration of intracellular Ca(2+). The present results suggest that these two types of K(ATP) channels may functionally be related to the activity of heart cells.     

Stilbene disulfonates block ATP-sensitive K+ channels in guinea pig ventricular myocytes

Effects of stilbene disulfonates on single K_ATP channel currents were investigated in inside-out and outside-out membrane patches from guinea pig ventricular myocytes. All drugs tested, 4,4′-diisothiocyanatostilbene, 2,2′-disulfonic acid (DIDS), 4-acetamido0-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS), 4,4′-dinitrostilbene-2,2′-disulfonic acid (DNDS), and 4,4′-diaminostilbene-2,2′-disulfonic acid (DADS), inhibited the K_ATP channel when they were applied to the intracellular, but not extracellular side of the membrane patch. Inhibitory actions of DIDS and SITS were irreversible, whereas those induced by DNDS and DADS were reversible. K_ATP channel inhibition was concentration dependent with an order of potency of DIDS>SITS ≈ DNDS > DADS; the Hill coefficient was close to unity for each drug. No change in channel conductance was observed during exposure to DIDS or DNDS; however, channel kinetics was altered. Distribution of the open time within bursts and that between bursts could be described by a single exponential relation in the absence and presence of DIDS or DNDS. The time constant of the open time within bursts was not altered, but that between bursts was decreased by DIDS (from 40.0±8.1 to 29.8±6.7 msec, P< 0.05) and by DNDS (from 43.1±9.3 to 31.9±7.1 msec, P<0.05). Distributions of closed time within bursts were also fitted to a single exponential function both in the absence and presence of drugs, while those of the closed time between bursts were fitted to a single exponential function in the absence of drugs, but a double exponential function was required in the presence of drugs. The rates of onset and development of channel inhibition by DIDS and DNDS appeared to be concentration dependent; a longer time was required to reach a new steady-state of channel activity as drug concentration was decreased. Inhibition by DIDS or DNDS was regulated by intracellular pH; inhibition was greater during acidic conditions. For DIDS (0.1 mm), the open probability (P _o) expressed as a fraction of the value before drug application was 42.9±8.3% at pH 7.4 and 8.2±6.6% at pH 6.5 (P<0.01); corresponding values for DNDS (1 mm) were 39.6±17.6 and 8.9 ±5.8%, respectively (P<0.01). From these data, we conclude that stilbene disulfonates block the K_ATP channel by binding to their target site with one-to-one stoichiometry. Similar to glibenclamide, the binding of stilbene disulfonates may reflect interpolation in an “intermediate lipid compartment” between the cytosolic drug and the site of drug action.     

Ion channels are linked to differentiation in keratinocytes

Summary In vivo and in vitro, keratinocyte differentiation is linked with increased extracellular Ca²⁺. In order to correlate ion channels with cell differentiation and investigate keratinocyte membrane responses to Ca²⁺, keratinocyte single channel currents were studied using the patch-clamp technique. The most frequently observed channel was a 14 pS nonspecific cation channel. This channel was permeable to Ca²⁺ and activated by physiological concentrations of Ca²⁺. We also found a 35 pS Cl^– channel whose open probability increased with depolarization. Finally, a 70 pS K⁺ channel was seen only in cell-attached or nystatin-permeabilized patches. We correlated channel types with staining for involucrin, an early marker of keratinocyte differentiation. While the nonspecific cation channel and Cl^– channel were seen in both involucrin positive and involucrin negative cells, all channels in which the K⁺ channel activity was present were involucrin positive. Membrane currents through these channels may be one pathway by which signals for keratinocyte proliferation or differentiation are sent.This work was supported in part by a National Institutes of Health grant K08 AR01853-03 and a National Science Foundation grant DCB-9009915 (to T.M.M.); National Institutes of Health Research Career Development Award K04 ARO 1803 and AR 39031 (to R.R.I.) and a National Institutes of Health grant GM-44840 (to P.A.P.).     

Time-dependent alteration in cromakalim-induced relaxation of corpus cavernosum from streptozocin-induced diabetic rats

The purpose of the present study was to investigate the relaxant responses to the ATP-sensitive potassium (K(ATP)) channel opener cromakalim in corpus cavernosum strips from 1-, 2-, 4-, 6-, and 8-week streptozocin-induced diabetic rats. Cromakalim (1 nM-0.1 mM) produced concentration-dependent relaxation in phenylephrine (7.5 microM)-precontracted isolated rat corporal strips. Compared with age-matched control animals, a significant enhancement in cromakalim-induced relaxation of corpus cavernosum was observed in 2-week diabetic animals, whereas the relaxant responses to cromakalim were decreased in 6-and 8-week diabetic animals. However, the cromakalim-induced relaxation was not altered in either 1-week or 4-week rat corporal strips in comparison with corresponding age-matched non-diabetic groups. Preincubation with the K(ATP) channel blocker glibenclamide (10 microM) significantly inhibited the cromakalim-induced relaxation in both non-diabetic and diabetic rat corpus cavernosum, but neither the voltage-dependent K(+) channel (K(V)) antagonist 4-aminopyridine (1 mM) nor the calcium-activated K(+) channel (K(Ca)) antagonist charybdotoxin (0.1 microM) had significant effect on cromakalim-induced relaxation in both control and diabetic rat corporal strips. Relaxation responses to the nitric oxide donor sodium nitroprusside (1 nM-0.1 mM) in diabetic rat corpus cavernosum were similar to that of age-matched controls. These data demonstrated that the relaxant responses to cromakalim were altered in diabetic cavernosal strips in a time dependent manner, suggesting that the period of diabetes mellitus may play a key role in the K(ATP) channels function in rat corpus cavernosum.