Pharmacogenetics of new analgesics

Patient phenotypes in pharmacological pain treatment varies between individuals, which could be partly assigned to their genotypes regarding the targets of classical analgesics (OPRM1, PTGS2) or associated signalling pathways (KCNJ6). Translational and genetic research have identified new targets, for which new analgesics are being developed. This addresses voltage-gated sodium, calcium and potassium channels, for which SCN9A, CACNA1B, KCNQ2 and KCNQ3, respectively, are primary gene candidates because they code for the subunits of the respective channels targeted by analgesics currently in clinical development. Mutations in voltage gated transient receptor potential (TRPV) channels are known from genetic pain research and may modulate the effects of analgesics under development targeting TRPV1 or TRPV3. To this add ligand-gated ion channels including nicotinic acetylcholine receptors, ionotropic glutamate-gated receptors and ATP-gated purinergic P2X receptors with most important subunits coded by CHRNA4, GRIN2B and P2RX7. Among G protein coupled receptors, δ-opioid receptors (coded by OPRD1), cannabinoid receptors (CNR1 and CNR2), metabotropic glutamate receptors (mGluR5 coded by GRM5), bradykinin B(1) (BDKRB1) and 5-HT(1A) (HTR1A) receptors are targeted by new analgesic substances. Finally, nerve growth factor (NGFB), its tyrosine kinase receptor (NTRK1) and the fatty acid amide hydrolase (FAAH) have become targets of interest. For most of these genes, functional variants have been associated with neuro-psychiatric disorders and not yet with analgesia. However, research on the genetic modulation of pain has already identified variants in these genes, relative to pain, which may facilitate the pharmacogenetic assessments of new analgesics. The increased number of candidate pharmacogenetic modulators of analgesic actions may open opportunities for the broader clinical implementation of genotyping information.     

Concomitant facilitation of GABAA receptors and KV7 channels by the non-opioid analgesic flupirtine

BACKGROUND AND PURPOSE

Flupirtine is a non-opioid analgesic that has been in clinical use for more than 20 years. It is characterized as a selective neuronal potassium channel opener (SNEPCO). Nevertheless, its mechanisms of action remain controversial and are the purpose of this study.

EXPERIMENTAL APPROACH

Effects of flupirtine on native and recombinant voltage- and ligand-gated ion channels were explored in patch-clamp experiments using the following experimental systems: recombinant K_IR3 and K_V7 channels and α3β4 nicotinic acetylcholine receptors expressed in tsA 201 cells; native voltage-gated Na⁺, Ca²⁺, inward rectifier K⁺, K_V7 K⁺, and TRPV1 channels, as well as GABA_A, glycine, and ionotropic glutamate receptors expressed in rat dorsal root ganglion, dorsal horn and hippocampal neurons.

KEY RESULTS

Therapeutic flupirtine concentrations (≤10 µM) did not affect voltage-gated Na⁺ or Ca²⁺ channels, inward rectifier K⁺ channels, nicotinic acetylcholine receptors, glycine or ionotropic glutamate receptors. Flupirtine shifted the gating of K_V7 K⁺ channels to more negative potentials and the gating of GABA_A receptors to lower GABA concentrations. These latter effects were more pronounced in dorsal root ganglion and dorsal horn neurons than in hippocampal neurons. In dorsal root ganglion and dorsal horn neurons, the facilitatory effect of therapeutic flupirtine concentrations on K_V7 channels and GABA_A receptors was comparable, whereas in hippocampal neurons the effects on K_V7 channels were more pronounced.

CONCLUSIONS AND IMPLICATIONS

These results indicate that flupirtine exerts its analgesic action by acting on both GABA_A receptors and K_V7 channels.     

非选择性阳离子通道与缺血性脑损伤

非选择性阳离子通道是一类对阳离子的选择性较低的配体门控性离子通道。缺血性脑损伤发生时,一价或二价阳离子进入神经元细胞内,可引发和加重神经元的凋亡和坏死。部分非选择性阳离子通道亦分布于血管内皮细胞,脑缺血发生时可导致血管内皮细胞功能异常和脑水肿。啮齿类动物脑缺血模型亦证明非选择性阳离子通道的非特异性阻断剂,如匹诺卡兰和利莫那班等,能明显减小血管的梗死面积,降低死亡率,发挥神经元保护作用。越来越多的实验证明,在缺血性脑损伤中,非选择性阳离子通道及其非特异性阻断剂与脑缺血密切相关,此通道将成为神经元保护的新靶标。     

A nomenclature for ligand-gated ion channels

The ligand-gated ion channels that participate in fast synaptic transmission comprise the nicotinic acetylcholine, 5-hydroxytryptamine₃ (5-HT₃), γ-aminobutyric acid_A (GABA_A), glycine, ionotropic glutamate and P2X receptor families. A consistent and systematic nomenclature for the individual subunits that comprise these receptors and the receptors that result from their co-assembly is highly desirable. There is also a need to develop criteria that aid in deciding which of the vast number of heteromeric combinations of subunits that can be assembled in heterologous expression systems in vitro, are known, or likely, to exist as functional receptors in vivo. The aim of this short article is to summarize the progress being made by the nomenclature committee of IUPHAR (NC-IUPHAR) in formulating recommendations that attempt to address these issues.     

背根神经节在疼痛机制和治疗中的作用

背根神经节作为痛觉传入的第一级神经元在痛觉的外周机制中起着极为重要的作用。对背根神经节中特有的或者主要在背根神经表达的受体、离子通道的认识,对于阐明疼痛的机制和治疗有重要意义。本文对感觉神经元特有的Nav1.8通道,主要在背根神经节表达的Nav1.7,Nav1.9,TRPV1,TRPA1,TRPM8通道及P2X3受体的分布、生理特点及在疼痛机制和治疗中的作用进行了详细的阐述。     

内源性腺苷和K_(ATP)通道介导缺氧窦房结起搏细胞的电生理效应(英文)

目的:观察腺苷脱氨酶(ADase),8-苯茶碱(8-PT)和格列苯脲(Gli)在豚鼠缺氧窦房结起搏细胞的电生理效应.方法:以充有100％氮和无糖的K-H液灌流豚鼠窦房结20 min引起其缺氧.用玻璃微电极技术记录起搏细胞的MDP,APA,APD_(90),V_(max),RPF和VDD等动作电位参数.结果:缺氧增加起搏细胞APA,MDP和V_(max),但减小VDD和RPF.Adase 10 U·L~(-1),8-PT 0.1 μmol·L~(-1)和Gli10 μmol·L~(-1)明显缓解缺氧引起的电生理效应. 结论:内源性腺苷和KATp通道在缺氧所致窦房结起搏细胞电生理效应中起重要作用.     

Heteromeric canonical transient receptor potential 1 and 4 channels play a critical role in epileptiform burst firing and seizure-induced neurodegeneration

Canonical transient receptor potential channels (TRPCs) are receptor-operated cation channels that are activated in response to phospholipase C signaling. Although TRPC1 is ubiquitously expressed in the brain, TRPC4 expression is the most restrictive, with the highest expression level limited to the lateral septum. The subunit composition of neuronal TRPC channels remains uncertain because of conflicting data from recombinant expression systems. Here we report that the large depolarizing plateau potential that underlies the epileptiform burst firing induced by metabotropic glutamate receptor agonists in lateral septal neurons was completely abolished in TRPC1/4 double-knockout mice, and was abolished in 74% of lateral septal neurons in TRPC1 knockout mice. Furthermore, neuronal cell death in the lateral septum and the cornu ammonis 1 region of hippocampus after pilocarpine-induced severe seizures was significantly ameliorated in TRPC1/4 double-knockout mice. Our data suggest that both TRPC1 and TRPC4 are essential for an intrinsic membrane conductance mediating the plateau potential in lateral septal neurons, possibly as heteromeric channels. Moreover, excitotoxic neuronal cell death, an underlying process for many neurological diseases, is not mediated merely by ionotropic glutamate receptors but also by heteromeric TRPC channels activated by metabotropic glutamate receptors. TRPC channels could be an unsuspected but critical molecular target for clinical intervention for excitotoxicity.     

The Effects of General Anesthetics on Synaptic Transmission

General anesthetics are a class of drugs that target the central nervous system and are widely used for various medical procedures. General anesthetics produce many behavioral changes required for clinical intervention, including amnesia, hypnosis, analgesia, and immobility; while they may also induce side effects like respiration and cardiovascular depressions. Understanding the mechanism of general anesthesia is essential for the development of selective general anesthetics which can preserve wanted pharmacological actions and exclude the side effects and underlying neural toxicities. However, the exact mechanism of how general anesthetics work is still elusive. Various molecular targets have been identified as specific targets for general anesthetics. Among these molecular targets, ion channels are the most principal category, including ligand-gated ionotropic receptors like γ-aminobutyric acid, glutamate and acetylcholine receptors, voltage-gated ion channels like voltage-gated sodium channel, calcium channel and potassium channels, and some second massager coupled channels. For neural functions of the central nervous system, synaptic transmission is the main procedure for which information is transmitted between neurons through brain regions, and intact synaptic function is fundamentally important for almost all the nervous functions, including consciousness, memory, and cognition. Therefore, it is important to understand the effects of general anesthetics on synaptic transmission via modulations of specific ion channels and relevant molecular targets, which can lead to the development of safer general anesthetics with selective actions. The present review will summarize the effects of various general anesthetics on synaptic transmissions and plasticity.     

Changes in intracellular Ca2+ by activation of P2 receptors in submucosal neurons in short-term cultures

Electrophysiological and Ca2+ microfluorimetric techniques were used to characterize the pharmacological profile of the P2 receptors expressed in submucosal neurons and the changes in intracellular Ca2+ associated with activation of these receptors. ATP caused a fast and slow membrane depolarizations during intracellular recordings. ATP induced a rapid inward current during whole-cell experiments. Receptors mediating the inward current and fast depolarization have the same pharmacological profile and these ATP responses were more sensitive to pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid than Basilen BlueE-3G, and potentiated by suramin. The slow depolarization was not blocked by these P2 receptor antagonists, pertussis toxin, or KT5720 (protein kinase A inhibitor). N-ethylmaleimide or protein kinase C inhibitors (staurosporine and calphostin) blocked this depolarization. ATP induced complex multi-phasic Ca2+ transients in most neurons, classified as fast, slow, or mixed fast/slow responses. In conclusion, the fast and slow Ca2+ responses were mediated by respective activation of P2X and P2Y receptors and were associated with fast and slow depolarizations, respectively.     

Transient receptor potential (TRP) channels, vascular tone and autoregulation of cerebral blood flow

Members of the transient receptor potential (TRP) channel superfamily are present in vascular smooth muscle cells and play important roles in the regulation of vascular contractility. The TRPC3 and TRPC6 channels are activated by stimulation of several excitatory receptors in vascular smooth muscle cells. Activation of these channels leads to myocyte depolarization, which stimulates Ca2+ entry via voltage-dependent Ca2+ channels (VDCC), leading to vasoconstriction. The TRPV4 channels in arterial myocytes are activated by epoxyeicosatrienoic acids, and activation of the channels enhances Ca2+ spark and transient Ca2+-sensitive K+ channel activity, thereby hyperpolarizing and relaxing vascular smooth muscle cells. The TRPC6 and TRPM4 channels are activated by mechanical stimulation of cerebral artery myocytes. Subsequent depolarization and activation of VDCC Ca2+ entry is directly linked to the development of myogenic tone in vitro and to autoregulation of cerebral blood flow in vivo. These findings imply a fundamental importance of TRP channels in the regulation of vascular smooth muscle tone and suggest that TRP channels could be important targets for drug therapy under conditions in which vascular contractility is disturbed (e.g. hypertension, stroke, vasospasm).     

Negatively charged 2- and 10-microm particles activate vanilloid receptors,increase cAMP,and induce cytokine release

Exposure to airborne pollutants, such as particulate matter (PM), is associated with increased mortality and morbidity. Indirect evidence suggested that PM-induced responses could be initiated by the activation of proton-gated receptors, including vanilloid receptors (VRs) and acid-sensitive ion channels (e.g. ASICS). We tested this hypothesis by characterizing the effects of 10- and 2-microm polystyrene carboxylate-modified particles (PC(10) and PC(2)) on HEK 293 cells expressing VR1 receptors, rat trigeminal ganglion (TG) neurons, and BEAS-2B airway epithelial cells. Zeta potential measurements revealed that these particles are negatively charged, meaning that when they adhere to a membrane they can lower the surface pH and activate proton-gated receptors. Both types of PCs induced currents and/or elevated intracellular Ca(2+) in cells that were capsaicin sensitive (CS). In about 70% of CS neurons, 10 microM capsazepine (CPZ), a VR antagonist, blocked PC-induced responses. In TG neurons in which VRs were blocked or desensitized, PCs induced an amiloride-inhibitable inward current having the characteristics of ASIC-mediated currents. Incubation of TG neurons with either capsaicin or PCs produced a CPZ-sensitive increase in cyclic AMP and cytokine (IL-6) release. In summary, we provide unequivocal evidence demonstrating that negatively charged PCs could activate VR1 and other proton-gated receptors. These data suggest that pharmacological manipulation of such receptors could prevent the physiological actions of PMs.     

Waixenicin A,a marine-derived TRPM7 inhibitor: a promising CNS drug lead

Ion channels are the third largest class of targets for therapeutic drugs. The pharmacology of ion channels is an important research area for identifying new treatment options for human diseases. The past decade or so has seen increasing interest in an ion channel protein belonging to the transient receptor potential (TRP) family, namely the melastatin subfamily member 7 (TRPM7), as an emerging drug target. TRPM7 is a bifunctional protein with a magnesium and calcium-conducting divalent ion channel fused with an active kinase domain. TRPM7 is ubiquitously expressed in human tissues, including the brain, and regulates various cell biology processes such as magnesium and calcium homeostasis, cell growth and proliferation, and embryonic development. TRPM7 provides a link between cellular metabolic status and intracellular calcium homeostasis in neurons due to TRPM7’s unique sensitivity to fluctuating intracellular Mg·ATP levels. Thus, the protein plays a key role in ischemic and hypoxic neuronal cell death and brain injury, and is one of the key nonglutamate mechanisms in cerebral ischemia and stroke. Currently, the most potent and specific TRPM7 inhibitor is waixenicin A, a xenicane diterpenoid from the Hawaiian soft coral Sarcothelia edmondsoni. Using waixenicin A as a pharmacological tool, we demonstrated that TRPM7 is involved in promoting neurite outgrowth in vitro. Most recently, we found that waixenicin A reduced hypoxic–ischemic brain injury and preserved long-term behavioral outcomes in mouse neonates. We here suggest that TRPM7 is an emerging drug target for CNS diseases and disorders, and waixenicin A is a viable drug lead for these disorders.     

TRPM7 in cerebral ischemia and potential target for drug development in stroke

Searching for effective pharmacological agents for stroke treatment has largely been unsuccessful. Despite initial excitement, antagonists for glutamate receptors, the most studied receptor channels in ischemic stroke, have shown insufficient neuroprotective effects in clinical trials. Outside the traditional glutamate-mediated excitotoxicity, recent evidence suggests few non-glutamate mechanisms, which may also cause ionic imbalance and cell death in cerebral ischemia. Transient receptor potential melastatin 7 (TRPM7) is a Ca(2+) permeable, non-selective cation channel that has recently gained attention as a potential cation influx pathway involved in ischemic events. Compelling new evidence from an in vivo study demonstrated that suppression of TRPM7 channels in adult rat brain in vivo using virally mediated gene silencing approach reduced delayed neuronal cell death and preserved neuronal functions in global cerebral ischemia. In this review, we will discuss the current understanding of the role of TRPM7 channels in physiology and pathophysiology as well as its therapeutic potential in stroke.     

新型抗胆碱药物盐酸戊乙奎醚及其4种光学异构体对N受体离子通道的作用

在非洲爪蟾培养的胚胎神经元和骨骼肌细胞上, 本文采用细胞膜片钳技术, 研究新型抗胆碱能药物盐酸戊乙奎醚(PHC)及其4种光学异构体对骨骼肌细胞N受体离子通道的作用. 结果表明PHC可阻断神经肌肉接头乙酰胆碱传递; 其4种光学异构体与之相比在对抗强度上无明显差别. PHC优先阻断开放时间长、电流强度大的N受体通道. 此外, PHC及其4种光学异构体对钠和钾离子通道也具有一定强度的阻断作用.     

新型抗胆碱药物盐酸戊乙奎醚及其4种光学异构体对N受体离子通道的作用

在非洲爪蟾培养的胚胎神经元和骨骼肌细胞上，本文采用细胞膜片钳技术，研究新型抗胆碱能药物盐酸戊乙奎醚（ＰＨＣ）及其４种光学异构体对骨骼肌细胞Ｎ受体离子通道的作用．结果表明ＰＨＣ可阻断神经肌肉接头乙酰胆碱传递；其４种光学异构体与之相比在对抗强度上无明显差别．ＰＨＣ优先阻断开放时间长、电流强度大的Ｎ受体通道．此外，ＰＨＣ及其４种光学异构体对钠和钾离子通道也具有一定强度的阻断作用     

ATP‐sensitive potassium channels: A promising target for protecting neurovascular unit function in stroke

• 1 Stroke is the second most common cause of death and a major cause of disability worldwide. Despite increasing knowledge of the cellular and molecular mechanisms that occur in stroke, there are still large gaps in our understanding that are impeding therapeutic progress. In addition, there are no drugs yet that can be used effectively in stroke patients.
• 2 In recent years, it has been recognized that stroke is a brain dysfunction that involves multiple cell types and that a purely neurocentric focus or targeting a single point in a single pathway fails to yield sufficient protection. Thus, the concept of the ‘neurovascular unit’ has emerged as a new paradigm for stroke investigation and therapy.
• 3 ATP‐sensitive potassium (K_ATP) channels are unique channel proteins that directly couple the metabolic state of a cell to its electrical activity. These channels are found throughout the brain, being found in neurons, glial cells and in the brain vasculature. It is well documented that K_ATP channels play multifactorial roles in protecting against brain injury induced by hypoxia, ischaemia or metabolic inhibition.
• 4 In the present review, we focus on the function of the neurovascular units in stroke and review current knowledge regarding K_ATP channels, with a focus on their potential role in the remodelling of the neurovascular units.

     

Alcohols and neurotransmitter gated ion channels: past, present and future

The neurotransmitter-gated ion channels form a superfamily of neurotransmitter receptors specialized for recognizing transmitters and rapidly gating ion channels that are contained within the same holoprotein complex. A large body of research indicates that alcohols alter the function of this class of receptor at concentrations relevant to the intoxicating and anesthetic effects of the alcohols. In addition, studies have implicated several types of neurotransmitter-gated channels in intoxicating and anesthetic alcohol actions. All of the neurotransmitter-gated ion channels contain conserved features such as N-terminal ligand binding domains, hydrophobic membrane spanning domains and charged pore-lining domains. However, at least two, and possibly three families of receptors have been identified within the superfamily, including the nicotinic ACh-like receptors, the ionotropic glutamate receptors and the ATP-gated ion channels. This review will begin with a brief overview of the structural features of the different receptors, with an emphasis on comparing and contrasting features of the different families of neurotransmitter-gated channels. The emphasis will be mostly on the nicotinic-like receptors and the iGluRs, since more is known about these receptors than about other types of ligand-activated cation channels. The remainder of the review focuses on the latest studies aimed at determining the mechanism of alcohol actions on this superfamily of receptor-channels as well as the relationship between the molecular structure of these channels and the effects of alcohols on channel function. In addition, emerging directions for future study of these effects of alcohols and possible regions of the protein that may be altered during alcohol exposure are discussed. Received: 21 January / Accepted: 21 May 1997     

Ethanol differentially affects ATP-gated P2X(3) and P2X(4) receptor subtypes expressed in Xenopus oocytes

P2X receptors are cation-selective, ligand-gated ion channels activated by synaptically released, extracellular adenosine 5'-triphosphate (ATP). ATP-gated currents are inhibited by ethanol when tested in dorsal root ganglion and CA1 neurons. Recently, we reported differences in sensitivity to ethanol inhibition between homomeric P2X(2) and P2X(4) receptors expressed in Xenopus oocytes, which suggested that subunit composition of native P2X receptors determines their ethanol sensitivity. The present study extended the investigation to P2X(3) receptors. The effects of ethanol and zinc ions (Zn(2+)) were tested on homomeric P2X(3) and P2X(4) receptors expressed in Xenopus oocytes using two-electrode voltage clamp. Ethanol potentiated ATP-gated P2X(3) receptor currents in a concentration dependent manner. In contrast, ethanol inhibited P2X(4) receptor function. Ethanol did not directly alter receptor function, nor did it alter the Hill coefficient or maximal ATP response (E(max)) in either P2X(3) or P2X(4) receptors. Ethanol increased the maximal response to Zn(2+) ATP-gated currents in P2X3 receptors which suggests that ethanol and Zn(2+) act on different sites. The differences in ethanol response of P2X(3) and P2X(4) receptors set the stage for future investigations that will use chimeric P2X receptors or other molecular manipulations of P2X structure to investigate the molecular sites and mechanisms of action of ethanol.     

P2 receptor modulation and cytotoxic function in cultured CNS neurons

In this study we investigate the presence, modulation and biological function of P2 receptors and extracellular ATP in cultured cerebellar granule neurons. As we demonstrate by RT-PCR and western blotting, both P2X and P2Y receptor subtypes are expressed and furthermore regulated as a function of neuronal maturation. In early primary cultures, mRNA for most of the P2 receptor subtypes, except P2X(6), are found, while in older cultures only P2X(3), P2Y(1) and P2Y(6) mRNA persist. In contrast, P2 receptor proteins are more prominent in mature neurons, with the exception of P2Y(1). We also report that extracellular ATP acts as a cell death mediator for fully differentiated and mature granule neurons, for dissociated striatal primary cells and hippocampal organotypic cultures, inducing both apoptotic and necrotic features of degeneration. ATP causes cell death with EC(50) in the 20-50 microM range within few minutes of exposure and with a time lapse of at most two hours. Additional agonists for P2 receptors induce toxic effects, whereas selected antagonists are protective. Cellular swelling, lactic dehydrogenase release and nuclei fragmentation are among the features of ATP-evoked cell death, which also include direct P2 receptor modulation. Comparably to P2 receptor antagonists previously shown preventing glutamate-toxicity, here we report that competitive and non-competitive NMDA receptor antagonists inhibit the detrimental consequences of extracellular ATP.Due to the massive extracellular release of purine nucleotides and nucleosides often occurring during a toxic insult, our data indicate that extracellular ATP can now be included among the potential causes of CNS neurodegenerative events.     

心房颤动发病机制研究进展

目的介绍近年来国内外对于心房颤动发病机制研究的新进展。方法依据作者所在的研究室多年研究成果及国内外本领域研究进展,对心房颤动发病的机制从离子通道、受体、纤维化相关分子及microRNA等方面研究进行综述。结果多种离子通道、microRNA和信号转导通路参与心房颤动的发生,这些因素的相互作用是心房颤动发生的原因。结论 心房颤动的防治靶点研究可从纠正离子通道、microRNA和相关小分子的平衡失调入手。