Specific antagonism by glibenclamide of negative inotropic effects of potassium channel openers in canine atrial muscle

The mode of antagonism by glibenclamide, a potassium channel blocker, of the negative inotropic effects of potassium channel openers, cromakalim, pinacidil and nicorandil, was investigated in canine atrial muscle. Glibenclamide shifted the concentration-negative inotropic effect curves for cromakalim, pinacidil and nicorandil to the right without affecting the basal force of contraction. Schild analysis yielded uniform pA2 values of 6.06-6.35 for glibenclamide against the three potassium channel openers. The force of contraction of atrial muscles previously reduced by cromakalim was also antagonized by increasing concentrations of glibenclamide. Glibenclamide affected neither the concentration-negative inotropic effect curves for carbachol, an opener of the muscarinic receptor-coupled potassium channel, nor those for nifedipine, a calcium channel blocker. From these results, it became evident that glibenclamide behaved as a pharmacological antagonist of cromakalim, pinacidil and nicorandil in cardiac inotropy. The antagonism seems to involve competition of glibenclamide and these potassium channel openers, presumably at the ATP-sensitive channel in canine right atrial muscles.     

Effects of potassium channel openers on single potassium channels in mouse skeletal muscle

Summary The patch-clamp technique was used to study the effects of the potassium channel openers cromakalim, pinacidil, RP 49356 and diazoxide on single potassium channels in mouse skeletal muscle.In excised patches in the inside-out configuration, one type of potassium channel, the ATP-sensitive potassium channel, could be activated by internally applied RP 49356 even in the absence of internal ATP. At a concentration of 0.4 and 0.8 mmol/l, RP 49356 increased the open-probability of the channels by a factor of 2.7 and 17.4 respectively. The stimulating effect of cromakalim (0.2–0.8 mmol/l) and pinacidil (0.4 mmol/l) depended on the presence of ATP (0.1 mmol/l) at the cytoplasmic side of the patch membrane. The two drugs were able to restore the open-probability of the channels blocked by internal ATP (0.1 mmol/l) to 50–90% of its value in ATP-free solution. No channel reactivation could be observed at a higher ATP concentration (1 mmol/l). Diazoxide (0.4 mmol/l) had almost no effect. None of these channel openers could stimulate the other prominent type of potassium channel in skeletal muscle, the large-conductance Ca²⁺-activated potassium channel.The results show that cromakalim, pinacidil and RP 49356 are specific openers of ATP-sensitive potassium channels in skeletal muscle. It is suggested that the drugs displace the channel blocker ATP and that RP 49356 in addition recruits inactive channels. Send offprint requests to B. Neumcke at the above address     

Clinical pharmacology of potassium channel openers.

Opening of K+ channels in cell membranes with resulting increase in K+ conductance, shifts the membrane potential in a hyperpolarizing direction towards the K+ equilibrium potential. Hyperpolarization reduces the opening probability of ion channels involved in membrane depolarization and excitation is reduced. K+ channel openers are believed to hyperpolarize smooth muscle cells by a direct action on the cell membrane. The best known members of the group are cromakalim, nicorandil and pinacidil, but several new compounds are being evaluated. In addition, it has recently been shown that also clinically well-known drugs like, e.g. diazoxide and minoxidil exhibit K+ channel opening properties. Nicorandil and new compounds containing nitro groups have a dual mechanism of action, also activating guanylate cyclase, an effect that contributes to their cardiovascular effect profile. K+ channel openers have a wide range of effects. Some of their properties and actions are summarized, and their present applications and/or potential for future application, in e.g. hypertension, angina pectoris, asthma, bladder instability, and several other disorders are discussed. It is concluded that K+ channel openning represents an interesting pharmacological principle with many potential clinical applications. However, most available drugs do not seem to have a sufficient tissue selectivity to be useful therapeutic alternatives. Before the potential of the new members of the group on clinical trials can be properly evaluated, clinical experiences are needed.     

Modulation of intracellular calcium by potassium channel openers in vascular muscle

Summary We investigated two putative K⁺ channel openers, pinacidil and BRL34915 (cromakalim), and demonstrated their vasorelaxant effectiveness on rat artery contractions induced by K⁺, tetraethylammonium (TEA), or norepinephrine. The K⁺ channel opener-induced decrease in tension was rapid, even when tension was stimulated by 100 mmol/l K⁺. Measurements of intracellular free Ca⁺⁺ (activity) by ultra-high sensitivity digital imaging microscopy was carried out by briefly loaded fura2 (fluorescence ratio) quantitation in isolated, contracting cells of rat azygos vein. Submicron resolution was achieved by measuring cytoplasmic Ca⁺⁺-sensitive fluorescence at each pixel, and size and intensity of areas with high Ca⁺⁺ concentrations, called hot spots, were determined by a computer-generated, 3 algorithm. Hot spots, which most likely represent the sites of Ca⁺⁺ release and re-uptake by Ca⁺⁺-regulatory organelles, increased in size and intensity upon addition of K⁺ or norepinephrine, reaching an early peak prior to the whole cell average peak in cytoplasmic Ca⁺⁺ activity. Both norepinephrine and K⁺-induced stimulation resulted in Ca⁺⁺ activity increases that were primarily due to Ca⁺⁺ release from storage sites. Reduction of free Ca⁺⁺ activity to resting or lower levels occurred upon addition of pinacidil or cromakalim. Intracellular Ca⁺⁺ decreases due to K⁺ channel openers appeared abruptly beginning at the central portions of the cells, resulting in a pronounced early drop in central Ca⁺⁺ activity while elevated Ca⁺⁺ levels persisted at the periphery. While this late stage residual of peripheral Ca⁺⁺ appears to be a significant step in the vascular muscle relaxant action of both K⁺ channel opener drugs, the level of Ca⁺⁺ at peripheral sites was greater in response to pinacidil than to cromakalim. The results of this study suggest that in addition to increasing K⁺ conductance, pinacidil and cromakalim cause 1) decreased Ca⁺⁺ activity in central regions of the myocytes, and 2) a shift in Ca⁺⁺ distribution to primarily subsarcolemmal sites. These observations lead us to hypothesize separate control of peripheral and central Ca⁺⁺ activity within a vascular muscle cell, with Ca⁺⁺ redistribution that can be altered by vasorelaxants. We suggest that intracellular Ca⁺⁺ redistribution may contribute the membrane potential-independent part of the vasorelaxant action of the K⁺ channel openers.This study was supported by NIH grants HL38537 and HL38645, and Eli Lilly Co. P.E. was supported by the Swiss Foundation of Cardiology and by the SNF 32-029 975.90     

Acrylamides as potassium channel openers

The acrylamide moiety is widely employed in medicinal chemistry for the design of bioactive molecule and, notably, it has served as a scaffold for a number of potassium channel modulators in recent years. Their synthesis has proven to be an effective approach, especially for the discovery and optimisation of small molecule openers of large-conductance calcium-activated (BK) potassium channels and KCNQ channels. This review provides an overview of the worldwide patent literature surrounding cinnamic amides, with a special focus on their potential medical use and the most salient structural and chemical features involved in the modulation of BK and KCNQ channel activity. Remarkably, such derivatives are among the most potent BK and KCNQ channel openers reported to date.     

Comparative electrophysiological and mechanical actions of ATP-sensitive potassium channel openers in canine Purkinje fibers

• 1.1. Electrophysiological and mechanical effects of ATP-dependent potassium channel openers on canine Purkinje fibers were examined.
• 2.2. Cromakalim and pinacidil (0.3–10 μM) and nicorandil (0.3–1 mM) shortened the action potential duration (APD), and reduced the contractile force, in a concentration-dependent manner. Other action potential parameters were unaffected. The effects were reversible.
• 3.3. Effects of the openers were antagonized by tetraethylammonium (a non-specific potassium channel blocker) and more potently by glibenclamide (a blocker of ATP-sensitive K⁺ channel).
• 4.4. The APD shortening and the negative inotropic effect induced by a switch of stimulation frequency (from 0.5 to 3 Hz) were potentiated by the openers. At high Ca²⁺ (5.4 and 10.8 mM), the effects of the openers on the APD and contractile force were unaffected.
• 5.5. In the spontaneously beating preparations, the openers hyperpolarized the maximum diastolic potential, and an arrest occurred.
• 6.6. Under the calcium overload condition, the K⁺ openers abolished a delayed afterdepolarization, and enhanced the depressed post-rest potentiation.
• 7.7. These results suggest that the K⁺ channel openers increase the K⁺ conductance and might decrease the cellular Ca²⁺ level in calcium overloading cells, and that nicorandil might also act directly ATP-sensitive K⁺ channels.

     

External ATP antagonizes the effect of potassium channel openers in guinea-pig ventricular papillary muscle.

Right ventricular papillary muscles of the guinea-pig heart were electrically stimulated. Cromakalim 10-100 microM and Ro 31-6930 3 microM depressed the contractile force and shortened the duration of action potentials. Glibenclamide 0.3-3 microM, ATP 100 microM and alpha, beta-methylene ATP (alpha, beta-meATP) 30 microM antagonized these effects. Suramin 300 microM failed to reverse the alpha, beta-meATP-evoked antagonism of the action of cromakalim. It is concluded that both intra- and extracellular ATP may interfere with potassium channel openers and that extracellular ATP does not act via the known P2-purinoceptor subtypes.     

Recent developments on KCNQ potassium channel openers

During the past five years, several members of the KCNQ potassium channel gene family have been identified with a high degree of CNS specificity. Within the KCNQ family, the combination of the KCNQ2/KCNQ3 proteins, and the KCNQ5/KCNQ3 arrangement has been identified as the molecular correlates of the different M-currents. Several lines of evidence are emerging demonstrating the importance of these channels in regulating neuronal excitability; for example, determination of the excitability threshold, firing properties, and responsiveness of neurons to synaptic inputs. Recent studies have shown that KCNQ openers have potential for the treatment of several CNS disorders characterized by neuronal hyperexcitability, such as migraine, epilepsy and neuropathic pain. This article reviews the recent developments of KCNQ potassium channel openers.     

Current trends in the study of potassium channel openers

Some eight years ago it was found that certain smooth muscle relaxants exert their effect by opening a specific K⁺ channel resulting in cell membrane hyperpolarization. The use of K⁺channel openers (cromakalim, pinacidil and RP-52891) and compounds which antagonize their actions (glibenclamide, phentolamine and alinidine) has enabled a great deal of research to be performed into the role of this K⁺ channel, not only in smooth muscle, but also in cardiac and skeletal muscle as well as neural and endocrine organ function. Much of the attention has centred on the smooth muscle relaxant actions of the K⁺ channel openers, since they have potential therapeutic use in disorders involving smooth muscle over-reactivity such as hypertension and asthma. More recently the cardiac actions of the K⁺ channel openers have become the focus of interest. Although there appear to be good theoretical reasons why K⁺ channel openers may be of use in some arrhythmias and in ischaemic heart disease there are major hurdles to overcome. In particular, given that the effect of these compounds on vascular smooth muscle occurs at a concentration 20- to 100-fold lower than that required to produce cardiac effects, it is likely their therapeutic usefulness will be limited until a breakthrough in cardiac/vascular selectivity is made. There is also growing interest in endogenous K⁺ channel openers and the physiological role of the K⁺ channel which they open. Opening of K⁺ channels, either spontaneously or by endogenous regulators, could possibly be an important hypotensive mechanism both under normal conditions and in a number of pathological conditions. Research into these areas will need to continue if the true therapeutic potential of K⁺ channel openers and their antagonists are to be realized.     

Peripheral channelopathies as targets for potassium channel openers

Potassium channel openers (KCOs) are important tools that are often used to gain a greater understanding of K⁺ channels. Agents that can induce or maintain the opening of K⁺ channels also offer a therapeutic approach to controlling of cell excitability and offer a means of producing stability in biological systems. The pathogenesis of a broad range of peripheral disorders (e.g., LQT syndrome, hypokalemic periodic paralysis, hyperinsulinism in infancy and erectile dysfunction) are associated with dysfunctional K⁺ channels due to mutations in genes encoding channel proteins. The therapeutic potential of KCOs in peripheral K⁺ channelopathies is discussed. The identification of K⁺ channel subtype-specific openers offers discrete modulation of cellular systems creating a realistic therapeutic advance in the treatment of K⁺ channelopathies.     

ATP-sensitive potassium channel openers and 2,3-dimethyl-2-butylamine derivatives

ATP-sensitive potassium (K(ATP)) channels have important functions through their coupling of cellular energetic networks and their ability to decode metabolic signals, and they are implicated in diseases of many organs. K(ATP) channels are formed by the physical association between the inwardly rectifier potassium channels (Kir6.x) and the regulatory sulfonylurea receptor subunit (SUR), which form a hetero-octameric complex. Different subtypes of K(ATP) channels exist in various tissues. K(ATP) channel openers (KCOs) are classified into nine chemical families according to their molecular structures: (1) benzopyrans, (2) cyanoguanidines, (3) thioformamides, (4) pyrimidine derivatives, (5) pyridine derivatives, (6) benzothiadiazines, (7) dihydropyridines, (8) nicotinamide derivatives, and (9) aliphatic amines. Although the model also predicts that KCOs have four co-binding areas, it was hypothesized that the main contribution lies in the binding domain of hydrophobicity of the side chain. A series of compounds containing the skeleton of the aliphatic secondary amines as a side chain was designed. It was found that N-isopropyl 2,3-dimethyl-2-butylamine (iptakalim, 91) is a novel KCOs. Iptakalim regulates the pore selectively of the inwardly rectifier potassium channel and dilates smaller arteries, but has little effect on vasodilatation of the aorta. Iptakalim administered p.o. has selective and long-lasting antihypertensive effects in hypertensive animals and does not induce tolerance, but has little effect on blood pressure in normotensive animals. Meanwhile, it also reverses cardiovascular remodeling and protects the brain and kidney against damage caused by hypertension in animal models. Iptakalim is in phase II clinical trials now and has a promising future as a treatment for hypertension.     

钾通道开放剂对培养兔主动脉平滑肌细胞增殖的抑制作用

钾通道开放剂对培养兔主动脉平滑肌细胞增殖的抑制作用１史道华２，郭兆贵（湖南医科大学药理研究室，长沙４１００７８，中国）关键词血管平滑肌；胸主动脉；培养的细胞；细胞分裂；苯福林；钾通道目的：探讨吡那地尔（Ｐｉｎ），尼可地尔（Ｎｉｃ），ＲＰ４９３５６（Ｒ...     

钾通道开放剂的抗过敏作用及其机制

用多种抗变态反应实验方法,研究钾通道开放剂米诺地尔(Min)与二氮嗪(Dia)的抗过敏作用,并探讨其作用机制。结果表明,Min能抑制大鼠同种被动皮肤过敏反应,拮抗5-HT引起的大鼠皮肤血管通透性增高。Dia和Min均能抑制豚鼠离体回肠平滑肌的过敏性收缩,Dia并能抑制A₂₃₁₈₇和化合物48/80诱发的肥大细胞释放组胺。因此钾通道开放剂Min与Dia具有抗过敏作用,作用的主要机理是抑制肥大细胞外Ca²⁺内流和细胞内贮存钙的释放,从而抑制组胺的释放,此可能与药物开放钾通道的作用有关。     

New insights into the development of ATP-sensitive potassium channel openers

ATP-sensitive potassium channel openers are expected to be valuable drugs for the treatment of a wide variety of pathologies. However, their clinical interest depends mainly on their tissue selectivity, which is linked to their affinity for a specific ATP-sensitive potassium (K_ATP) channel subtype. Hypertension was the first indication for K_ATP channel openers currently on the market. Recent progress in the synthesis of tissue-selective compounds led to the discovery of potential candidates for the management (prevention and/or treatment) of several pathological states such as Type 1 or Type 2 diabetes, obesity, urinary incontinence and congestive heart failure. The present report focuses on patent applications introduced between 2001 – 2004, describing original K_ATP channel openers.     

Effects of rubidium on responses to potassium channel openers in rat isolated aorta.

1. In a physiological salt solution (PSS) in which potassium (K) was replaced by rubidium (Rb), segments of rat aorta precontracted with 20 mM RbCl were fully relaxed by K-channel openers with an order of potency levcromakalim > cromakalim > aprikalim > RP 49356. These relaxations were inhibited by glibenclamide. 2. Segments of rat aorta bathed in normal PSS and precontracted with 20 mM KCl were also relaxed by these K-channel openers with an order of potency levcromakalim > cromakalim > aprikalim > RP 49356. These relaxations were glibenclamide-sensitive. However, the absolute potencies of the K-channel openers were approximately four times greater in normal PSS than in RbPSS. 3. In RbPSS, minoxidil sulphate relaxed segments of aorta precontracted with 20 mM RbCl by approximately 20% whereas in normal PSS it fully relaxed those contracted with 20 mM KCl. 4. In RbPSS, levcromakalim-induced relaxation of aortic segments precontracted with 20 mM RbCl was initially well-maintained but then faded by approximately 60% of the initial relaxation to a new, stable level. Subsequent exposure to RP 49356 or to higher concentrations of levcromakalim was without further relaxant effect. Similar changes were observed when RP 49356 was the initial relaxant and tissues were exposed to either RP 49356 or levcromakalim. In normal PSS, levcromakalim- or RP 49356-induced relaxation of contractions produced by 20 mM KCl was well-maintained. 5. In RbPSS, minoxidil sulphate-induced relaxation of aortic segments precontracted with 20 mM RbCl was well-maintained. Subsequent exposure to either levcromakalim or to RP 49356 produced further tissue relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of potassium channel openers and their antagonists on rat locus coeruleus neurones.

1. Intracellular recordings were obtained from a pontine slice preparation of the rat brain containing the locus coeruleus (LC). Two openers of ATP-sensitive potassium (K(ATP)) channels, RO 31-6930 (10 microM) and cromakalim (100 microM) decreased the spontaneous discharge of action potentials without altering their amplitude or duration. Neither compound changed the resting membrane potential. 2. Of two K(ATP) channel blockers, tolbutamide (300 microM) increased the firing rate, while glibenclamide (3 microM) only tended to do so. In addition, both compounds antagonized the effect of RO 31-6930 (10 microM). Neither glibenclamide (3 microM) nor tolbutamide (300 microM) altered the resting membrane potential. 3. Tetrodotoxin (0.5 microM) depressed the firing, but did not influence the inhibitory action of RO 31-6930 (10 microM). The excitatory amino acid antagonist, kynurenic acid (500 microM), did not change the spontaneous discharge of action potentials. 4. Small shifts (2-4 mV) of the membrane potential by hyper- or depolarizing current injections markedly decreased and increased the firing rate, respectively. 5. Noradrenaline (100 microM) hyperpolarized the cells and decreased their input resistance. This effect was not antagonized by glibenclamide (3 microM) or tolbutamide (300 microM). Ba2+ (2 mM), a blocker of both ATP-sensitive and inwardly rectifying potassium channels, abolished the effects of RO 31-6930 (10 microM) and noradrenaline (100 microM). 6. These data suggest that K(ATP) channels are present on the noradrenergic LC neurones, but are not coupled to alpha 2-adrenoceptors.     

钾通道阻滞剂对大鼠支气管平滑肌细胞增殖的影响

目的探讨电压依赖性延迟整流钾通道(K_V)、Ca²⁺激活钾通道(K_Ca)和ATP敏感性钾通道(K_ATP)对大鼠支气管平滑肌细胞(BSMC)增殖的影响。 方法应用免疫细胞化学、MTT微量比色分析法及流式细胞术,观察K_V,K_Ca和K_ATP对培养中大鼠BSMC增殖的影响。结果K_V阻断剂4-氨基吡啶(4-AP)显著促进大鼠BSMC增殖细胞核抗原的表达,提高BSMC吸光度值,使S+G₂M期细胞数显著增多,并显著提高基础状态下BSMC内Ca²⁺浓度,而K_Ca阻断剂四乙铵(TEA)和K_ATP阻断剂格列本脲(Glib)均无此效应。 结论大鼠BSMC K_V活性的抑制,可提高细胞内Ca²⁺浓度,促进细胞的增殖,而K_Ca和K_ATP对BSMC的增殖均无明显影响。