胍丁胺中枢作用的复杂性及多样性

1994年 ,人们发现胍丁胺在牛脑中具有可乐定替代物质的特性。随之的研究表明 ,胍丁胺的作用机制复杂 ,作用靶点多样 ,生物学效应广泛。最近的研究显示 ,胍丁胺是中枢的一种新的神经递质 ,胍丁胺可能是咪唑啉受体的内源性配体     

利用RNA干扰技术研究I1咪唑啉受体在阿片依赖中的作用

目的：我们和国外实验室先后研究发现胍丁胺对阿片依赖具有调节作用，其作用可能与I1咪唑啉受体（I1-imidazoline receptor，I1R）有关。但由于没有特异性咪唑啉受体拮抗剂且胍丁胺的作用靶点较多，到目前为止，I1R是否为胍丁胺抗阿片依赖的主要作用靶点尚不能完全确定。因此，本文旨在利用RNA干扰技术确定I1R是否介导胍丁按对阿片依赖的调节作用。方法：利用RNA干扰技术下调细胞内源性I1R的表达，     

胍丁胺调节阿片功能的分子和神经生物学机制

胍丁胺是一种内源性生物活性物质,由左旋精氨酸在左旋精氨酸脱羧酶作用下脱羧生成。我们的前期研究发现不管是内源性还是外源性胍丁胺都具有明确的阿片功能调节作用,表现为镇痛、增强吗啡镇痛、抑制吗啡耐受,躯体依赖和精神依赖。近年来,在这些工作的基础上,我们研究了胍丁胺调节阿片功能的分子和神经生物学机制。提出了胍丁胺调节阿片功能可能和激活咪唑啉受体有关这一假说。为了证明这一假说的客观性,本实验室首次建立了IRAS-Cho细胞稳定表达系统,通过观察IRAS的亚细胞定位、配体结合特性、生物学功能和信号转等证明了由美国科学家克隆的IRAS就是野生型I1咪唑啉受体。在此细胞模型上,我们首次证明胍丁胺调节阿片功能与激活I1咪唑啉受体相关。为了在神经元上,特别是在整体行为学实验模型上证明上述发现的存在,本实验室用RNA干涉技术在大鼠原代培养海马神经元和大鼠摇体实验模型上进一步证明胍丁胺调节阿片功能与激活I1咪唑啉受体相关。     

胍丁胺对炎性疼痛的镇痛作用及对吗啡镇痛作用的影响

目的观察胍丁胺对炎性疼痛的镇痛作用及其对吗啡镇痛作用的影响,研究胍丁胺的镇痛作用是否与激动咪唑啉受体或影响受体前谷氨酸和γ-氨基丁酸(gamma-aminobutyr-icacid,GABA)释放有关。方法应用福尔马林致大鼠炎性疼痛模型,观察胍丁胺镇痛和增强吗啡镇痛的作用。应用高效液相色谱技术测定胍丁胺对脊髓切片孵育液中谷氨酸和GABA基础释放量及对高钾诱发神经元去极化引起神经递质释放的影响。结果单侧足底注射5%福尔马林使大鼠出现明显的双相伤害性行为反应。胍丁胺抑制福尔马林引起的第二相疼痛行为反应及痛觉过敏,并增强吗啡对第二相疼痛的镇痛作用,但在第一相疼痛过程中,无明显镇痛和增强吗啡镇痛的作用。咪唑啉受体拮抗剂咪唑克生不能拮抗胍丁胺镇痛及增强吗啡镇痛的作用。1~1000μmol.L-1胍丁胺对脊髓谷氨酸和GABA的基础释放量和高钾诱发谷氨酸和GABA释放量的升高均没有影响。结论胍丁胺对炎性疼痛具有明确的镇痛作用,并明显增强吗啡的镇痛效果,其镇痛机制可能与咪唑啉受体无关,也不是通过在受体前水平抑制谷氨酸或促进GABA释放来实现的。     

吗啡长期给药后大鼠脑内MAO—B活性及咪唑啉受体的下调

目的:探讨吗啡长期给药处理后大鼠不同脑区MAO-B活性及咪唑啉受体含量的变化。方法:用[~3H]咪唑克生配体结合试验测定咪唑啉受体含量,用高效液相色谱法测定MAO-B活性。结果:咪唑克生和吗啡能剂量依赖性地抑制大鼠脑匀浆MAO-B活性。咪唑啉受体的内源性配体胍丁胺既不影响MAO-B活性,也不影响咪唑克生及吗啡对MAO-B活性的抑制作用。吗啡连续给药16d后大鼠大脑、海马、丘脑、纹状体及小脑内MAO-B活性均显著下调(P<0.01)。纳洛酮及咪唑克生单次给药对吗啡依赖大鼠上述脑区MAO-B活性均没有进一步影响;胍丁胺伴随吗啡给药后能显著抑制吗啡降低MAO-B活性的作用。吗啡连续给药后大鼠皮层和小脑咪唑啉受体数量减少而亲和力上调(P<0.05)或P<0.01)。结论:MAO-B活性与吗啡依赖大鼠发生戒断综合征相关,但与胍丁胺对吗啡镇痛作用的影响无关;胍丁胺对吗啡药理作用的影响与其激活咪唑啉受体有关。     

咪唑啉结合位点及受体与心血管功能

人及大鼠心肌细胞存在 2 型咪唑啉结合位点 ,多种动物的血管平滑肌存在 1 型和 2 型咪唑啉结合位点 ,其中 2 型咪唑啉结合位点可能参与血管平滑肌的增殖。支配心血管系统的交感神经末梢存在突触前咪唑啉受体 ,激动该受体抑制NE的释放。与多数咪唑啉类化合物不同 ,莫索尼定对突触前咪唑啉受体无效 ,而大麻受体拮抗剂SR141716A对该受体具有拮抗作用。已经证实多数咪唑啉类化合物具有抗心律失常作用 ;咪唑啉受体的内源性配基胍丁胺降低动物窦房结起搏细胞的放电频率 ,延长人与动物心肌细胞的动作电位时程 ,对异丙肾上腺素诱发的后除极具有抑制作用。但是 ,咪唑啉类和胍类化合物非竞争性抑制心血管系统的KATP通道 ,可能干扰IK .ATP的心脏保护作用。     

咪唑啉I_2受体研究进展

咪唑啉I2 受体是新近发现的一种咪唑啉受体 ,根据其与阿米洛利的亲和力可进一步将其分为I2A和I2B两个亚型 ,主要分布于肾、脑和肝细胞的线粒体外膜上 ,其内源性配体是胍丁胺。许多证据提示咪唑啉I2 受体与单胺氧化酶 B具有高度同源性 ,但其与配体的结合位点不同于该酶的催化位点。激活咪唑啉I2 受体可能产生神经元保护、抗血管平滑肌增生及调节阿片功能等多种药理作用。咪唑啉I2 受体与抑郁症、帕金森病、亨廷顿病、阿片成瘾及阿尔茨海默病等疾病的发生有关。     

胍丁胺及其主要生物学作用

胍丁胺作为咪唑啉受体的内源性配体，能调节某些神经递质的释放，并有多方面的生物学作用如降糖、利尿、对抗阿片类药物所致耐受和依赖、抗炎等，因此胍丁胺很可能是又一个新的神经递质或调质。     

Analgesic effect of agmatine and its enhancement on morphine analgesia in mice and rats

目的：观察胍丁胺镇痛和对吗啡镇痛的作用．方法：在小鼠热辐射甩尾,醋酸扭体,大鼠４％盐水实验中观察胍丁胺的镇痛作用;在小鼠和大鼠热辐射甩尾实验中观察其对吗啡和可乐定镇痛的作用．结果：胍丁胺不延长小鼠甩尾潜伏期,使小鼠醋酸扭体次数减少,完全抑制大鼠盐水扭体．在小鼠甩尾实验中,胍丁胺剂量依赖性地增强吗啡和可乐定的镇痛,使吗啡和可乐定的镇痛ＥＤ５０减小了７５％．胍丁胺的上述作用可被咪唑克生所对抗．结论：胍丁胺通过激动咪唑啉受体而具有较弱镇痛和加强吗啡及可乐定镇痛作用     

胍丁胺抑制麻醉大鼠颈动脉窦压力反射(英文)

目的:观察胍丁胺对颈动脉窦压力感受器反射的影响。方法:利用灌流左颈动脉窦方法,观察胍丁胺对麻醉大鼠压力反射机能参数的影响。结果:(1)胍丁胺1,5,10mmol/L均使颈动脉窦压力反射机能曲线向右上方移位,曲线最大斜率和反射性平均动脉压下降幅度均减小,提示胍丁胺对压力感受器反射有抑制作用;(2)预先应用咪唑啉受体(IR)和肾上腺素能α_2受体(α_2-AR)拮抗剂咪唑克生(idazox-an,0.1mmol/L),则可完全阻断胍丁胺5mmol/L的效应。预先应用α_2受体拮抗剂育亨宾(yohimbine,15μmol/L),则可部分阻断其效应;(3)预先应用NOS抑制剂L-NAME(500μmol/L),对胍丁胺的抑制作用无影响。结论:胍丁胺对颈动脉窦压力反射有抑制作用,并由咪唑啉受体和α_2受体介导。     

胍丁胺对吗啡长期处理引起的NMDA受体蛋白表达改变的影响

目的:观察胍丁胺对吗啡长期处理引起的NMDA受体蛋白改变的影响。方法:采用吗啡递增给药制备大鼠慢性依赖模型,并观察依赖状态下大鼠海马和伏隔核NMDA受体NR1和NR2B亚基蛋白表达量的变化,以及胍丁胺对吗啡作用的影响。结果:与对照组相比,吗啡慢性处理大鼠在纳洛酮催促下能出现典型的戒断综合征,提示依赖模型建立成功。用免疫印记(Western blotting)技术发现,海马部位的NR2B亚基明显下调,而NR1亚基未见显著性变化;吗啡慢性处理不引起伏隔核NR2B亚基的明显变化,但NR1亚基却显著上调。胍丁胺与吗啡伴随给药能逆转吗啡对两脑区NMDA受体蛋白表达的调节作用。结论:胍丁胺调节阿片依赖可能与其逆转吗啡对NMDA受体亚基数量和构成的调节有关。     

Agmatine: Biological role and therapeutic potentials in morphine analgesia and dependence

Agmatine is an amine that is formed by decarboxylation of L-arginine by the enzyme arginine decarboxylase (ADC) and hydrolyzed by the enzyme agmatinase to putrescine. Agmatine binds to several target receptors in the brain and has been proposed as a novel neuromodulator. In animal studies, agmatine potentiated morphine analgesia and reduced dependence/withdrawal. While the exact mechanism is not clear, the interactions with N-methyl-D-aspartate (NMDA) receptors, alpha2-adrenergic receptors, and intracellular cyclic adenosine monophosphate (cAMP) signaling have been proposed as possible targets. Like other monoamine transmitter molecules, agmatine is rapidly metabolized in the periphery and has poor penetration into the brain, which limits the use of agmatine itself as a therapeutic agent. However, the development of agmatinase inhibitors will offer a useful method to increase endogenous agmatine in the brain as a possible therapeutic approach to potentiate morphine analgesia and reduce dependence/withdrawal. This review provides a succinct discussion of the biological role/therapeutic potential of agmatine during morphine exposure/pain modulation, with an extensive amount of literature cited for further details.     

Acquisition deficit and time-dependent retrograde amnesia for contextual fear conditioning in agmatine-treated rats

Cumulative evidence indicates that the hippocampus plays a time-limited role in contextual learning paradigms. Pharmacological studies have indicated that acquisition of background contextual cues during Pavlovian fear conditioning is dependent upon hippocampal function, whereas early inactivation of the hippocampus after training produces retrograde amnesia. When administered prior to contextual fear conditioning, agmatine (5 and 10 mg/kg, i.p.), an endogenous polyamine and N-methyl-D-aspartate (NMDA) receptor ligand found at excitatory synapses in the hippocampus, impaired the acquisition of contextual fear (measured as defensive freezing 26 hours later) without a reduction in baseline motor activity during training. Furthermore, ascending doses of agmatine were found not to exert analgesic effects on response thresholds to peripheral shock. This negated the possibility that the observed learning deficit resulted from a difference in perceived shock intensity. Post-training agmatine treatment produced a time-dependent impairment of consolidation, with subjects approaching a level of fear equivalent to that of a reference group as the delay of treatment increased (up to 6 hours). Since physiologically high levels of agmatine are able to inhibit NMDA receptor activity, these results suggest that polyamine modulation of NMDA receptors, most likely within the hippocampus, is required for the acquisition and consolidation of contextual fear stimuli.     

Antidepressant-like effect of agmatine and its possible mechanism

In mammalian brain, agmatine is an endogenous neurotransmitter and/or neuromodulator, which is considered as an endogenous ligand for imidazoline receptors. In this study, the antidepressant-like action of agmatine administered p.o. or s.c. was evaluated in three behavioral models in mice or rats. Agmatine at doses 40 and 80 mg/kg (p.o.) reduced immobility time in the tail suspension test and forced swim test in mice or at dose 20 mg/kg (s.c.) in the forced swim test. Agmatine also reduced immobility time at 10 mg/kg (p.o.) or at 1.25, 2.5 and 5 mg/kg (s.c.) in the forced swim test in rats. These results firstly indicated that agmatine possessed an antidepressant-like action. With 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and lactic dehydrogenase (LDH) assay, 1, 10 and 100 microM agmatine or a classical antidepressant, 2.5 and 10 microM desipramine, protected PC12 cells from the lesion induced by 300 microM N-methyl-D-aspartate (NMDA) treatment for 24 h. Using high-performance liquid chromatography with electrochemical detection (HPLC-ECD), it was found that the levels of monoamines including norepinephrine, epinephrine, dopamine or 5-hydroxytryptamine (5-HT) in PC12 cells decreased after the treatment with 200 microM NMDA for 24 h, while in the presence of 1 and 10 microM agmatine or 1 and 5 microM desipramine, the levels of norepinephrine, epinephrine or dopamine were elevated significantly while 5-HT did not change. Moreover, norepinephrine, 5-HT or dopamine had the same cytoprotective effect as agmatine at doses 0.1, 1 and 10 microM. In the fura-2/AM (acetoxymethyl ester) labeling assay, 1 and 10 microM agmatine, 1 and 5 microM desipramine or monoamines norepinephrine, 5-HT at doses 0.1 and 1 microM attenuated the intracellular Ca(2+) overloading induced by 200 microM NMDA treatment for 24 h in PC12 cells. In summary, we firstly demonstrated that agmatine has an antidepressant-like effect in mice and rats. A classical antidepressant, desipramine, as well as agmatine or monoamines protect the PC12 cells from the lesion induced by NMDA treatment. Agmatine reverses the NMDA-induced intracellular Ca(2+) overloading and the decrease of monoamines (including norepinephrine, epinephrine or dopamine) contents in PC12 cells, indicating that agmatine's antidepressant-like action may be related to its modulation of NMDA receptor activity and/or reversal of the decrease of monoamine contents and Ca(2+) overloading induced by NMDA.     

Protection by imidazol(ine) drugs and agmatine of glutamate-induced neurotoxicity in cultured cerebellar granule cells through blockade of NMDA receptor.

This study was designed to assess the potential neuroprotective effect of several imidazol(ine) drugs and agmatine on glutamate-induced necrosis and on apoptosis induced by low extracellular K+ in cultured cerebellar granule cells. Exposure (30 min) of energy deprived cells to L-glutamate (1-100 microM) caused a concentration-dependent neurotoxicity, as determined 24 h later by a decrease in the ability of the cells to metabolize 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) into a reduced formazan product. L-glutamate-induced neurotoxicity (EC50=5 microM) was blocked by the specific NMDA receptor antagonist MK-801 (dizocilpine). Imidazol(ine) drugs and agmatine fully prevented neurotoxicity induced by 20 microM (EC100) L-glutamate with the rank order (EC50 in microM): antazoline (13)>cirazoline (44)>LSL 61122 [2-styryl-2-imidazoline] (54)>LSL 60101 [2-(2-benzofuranyl) imidazole] (75)>idazoxan (90)>LSL 60129 [2-(1,4-benzodioxan-6-yl)-4,5-dihydroimidazole](101)>RX82 1002 (2-methoxy idazoxan) (106)>agmatine (196). No neuroprotective effect of these drugs was observed in a model of apoptotic neuronal cell death (reduction of extracellular K+) which does not involve stimulation of NMDA receptors. Imidazol(ine) drugs and agmatine fully inhibited [3H]-(+)-MK-801 binding to the phencyclidine site of NMDA receptors in rat brain. The profile of drug potency protecting against L-glutamate neurotoxicity correlated well (r=0.90) with the potency of the same compounds competing against [3H]-(+)-MK-801 binding. In HEK-293 cells transfected to express the NR1-1a and NR2C subunits of the NMDA receptor, antazoline and agmatine produced a voltage- and concentration-dependent block of glutamate-induced currents. Analysis of the voltage dependence of the block was consistent with the presence of a binding site for antazoline located within the NMDA channel pore with an IC50 of 10-12 microM at 0 mV. It is concluded that imidazol(ine) drugs and agmatine are neuroprotective against glutamate-induced necrotic neuronal cell death in vitro and that this effect is mediated through NMDA receptor blockade by interacting with a site located within the NMDA channel pore.     

Agmatine : metabolic pathway and spectrum of activity in brain

Agmatine is an endogenous neuromodulator that, based on animal studies, has the potential for new drug development. As an endogenous aminoguanidine compound (1-amino-4-guanidinobutane), it is structurally unique compared with other monoamines. Agmatine was long thought to be synthesised only in lower life forms, until its biosynthetic pathway (decarboxylation of arginine) was described in the mammalian brain in 1994. Human arginine decarboxylase has been cloned and shown to have 48% identity to ornithine decarboxylase. In neurons of the brain and spinal cord, agmatine is packaged into synaptic vesicles and released upon neuronal depolarisation. Other evidence of a neuromodulation role for agmatine is the presence of a specific cellular uptake mechanism and a specific metabolic enzyme (agmatinase; which forms putrescine).Initially, agmatine was conceptualised as an endogenous clonidine-displacing substance of imidazoline receptors; however, it has now been established to have affinity for several transmembrane receptors, such as alpha(2)-adrenergic, imidazoline I(1) and glutamatergic NMDA receptors. In addition to activity at these receptors, agmatine irreversibly inhibits neuronal nitric oxide synthase and downregulates inducible nitric oxide synthase.Endogenous agmatine is induced in response to stress and/or inflammation. Stressful conditions that induce agmatine include hypoxic-ischaemia and cold-restraint stress of ulcerogenic proportion. Induction of agmatine in the brain seems to occur in astrocytes, although neurons also synthesise agmatine. The effects of injected agmatine in animals include anticonvulsant-, antineurotoxic- and antidepressant-like actions. Intraperitoneal or intracerebroventricular injections of agmatine rapidly elicit antidepressant-like behavioural changes in the rodent forced swim test and tail suspension test. Intraperitoneal injections of agmatine into rats and mice also elicit acute anxiolytic-like behavioural changes in the elevated plus-maze stress test. In an animal model of acute stress disorder, intraperitoneal agmatine injections diminish contextual fear learning. Furthermore, intraperitoneal injections of agmatine reduce alcohol and opioid dependence by diminishing behaviour in a rat conditioned place preference paradigm.Based on these findings, agmatine appears to be an endogenous neuromodulator of mental stress. The possible roles and/or beneficial effects of agmatine in stress-related disorders, such as depression, anxiety and post-traumatic stress disorder, merit further investigation.     

Involvement of glial cells in the nociceptive behaviors induced by a high-dose of histamine administered intrathecally

The involvement of spinal glial cells in the nociceptive behaviors induced by 1600 pmol of histamine was determined in mice. Histamine injected intrathecally (i.t.) produced nociceptive behaviors, consisting of scratching, biting and licking. The nociceptive behaviors induced by histamine were significantly suppressed by i.t. pretreatment with the glial cell inhibitor DL-fluorocitric acid or minocycline. In Western blot analysis using lumber spinal cords, i.t. treatment with histamine increased the phosphorylation of the NR1 subunit of N-methyl-D-aspartate (NMDA) receptors. The increased phosphorylation of the NR1 subunit of NMDA receptors by histamine was abolished by i.t. pretreatment with DL-fluorocitric acid or minocycline. We have previously reported that the nociceptive behaviors induced by 1600 pmol of histamine were significantly suppressed by the i.t. co-administration of (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5,10-imine (MK-801), an ion channel blocker of NMDA receptors, or agmatine, an antagonist for the polyamine recognition site on the NR1 subunit of NMDA receptors. In the present study, the increased phosphorylation of the NR1 subunit of NMDA receptors by histamine was also abolished by i.t. co-administration of agmatine or MK-801. The present results suggest that histamine at 1600 pmol elicits nociceptive behaviors by stimulating the polyamine recognition site on the NR1 subunit of NMDA receptors on spinal glial cells.     

Agmatine modulates neuroadaptations of glutamate transmission in the nucleus accumbens of repeated morphine-treated rats

It has been proved that agmatine inhibits opioid dependence, yet the neural mechanism remains unclear. In the present study, the effect of agmatine on the neuroadaptation of glutamate neurotransmission induced by morphine dependence, including changes of the extracellular glutamate level and glutamate receptors in the nucleus accumbens was investigated.We found that agmatine (2.5–20 mg/kg, s.c.) inhibited development of morphine dependence, which was consistent with our previous report. In rats repeatedly treated with morphine, the glutamate level in the nucleus accumbens dialysate was markedly increased after naloxone-precipitated withdrawal. When agmatine (20 mg/kg, s.c.) was co-pretreated with morphine or was applied before naloxone-precipitated withdrawal, this elevation of the extracellular glutamate level was inhibited. In the synaptosome model, repeated morphine treatment and naloxone precipitation induced an increase in glutamate release, while agmatine (20 mg/kg, s.c.) co-pretreated with morphine reversed the increase of glutamate release. However, neither morphine or agmatine treatment alone nor morphine and agmatine co-administration had any influence on [3H]-glutamate uptake. It indicated that the elevation of the glutamate level in the nucleus accumbens might be caused by the increase of glutamate release of synaptosome in the withdrawal conditions of morphine-dependent rat. Furthermore, agmatine concomitant treatment with morphine entirely abolished the up-regulation of the NR1 subunit of N-methyl-d-aspartate (NMDA) receptors in the nucleus accumbens in repeated morphine-treated rats.Taken together, the present study demonstrated that agmatine could modulate the neuroadaptations of glutamate transmission in the nucleus accumbens in the case of morphine dependence, including modulating extracellular glutamate concentration and NMDA receptor expression.     

Agmatine signaling: odds and threads

Agmatine is a metabolite of L-arginine. It is formed by the decarboxylation of L-arginine via arginine decarboxylase in bacteria, plants and mammals. It is becoming clear that it has multiple physiological functions as a potential transmitter. Agmatine binds to alpha2-adrenoceptors and to imidazoline binding sites. It blocks NMDA receptors and other ligand-gated cation channels. It also inhibits nitric oxide synthase, induces release of peptide hormones and antizyme and plays a role during cell proliferation by interacting with the generation and transport of polyamines. Although the precise function of endogenously released agmatine is presently still unclear, this review will summarize several aspects concerning the biological function of agmatine.     

An in vivo evaluation of the antiseizure activity and acute neurotoxicity of agmatine

Agmatine, an endogenous cationic amine, exerts a wide range of biological effects, including modulation of glutamate-activated N-methyl-D-aspartate (NMDA) receptor function in the central nervous system (CNS). Since glutamate and the NMDA receptor have been implicated in the initiation and spread of seizure activity, the capacity of agmatine to inhibit seizure spread was evaluated in vivo. Orally administered agmatine (30 mg/kg) protected against maximal electroshock seizure (MES)-induced seizure spread in rats as rapidly as 15 min and for as long as 6 h after administration. Inhibition of MES-induced seizure spread was also observed when agmatine was administered intraperitoneally. Agmatine's antiseizure activity did not appear to be dose-dependent. An in vivo neurotoxicity screen indicated that agmatine was devoid of any acute neurological toxicity at the doses tested. These preliminary data suggest that agmatine has promising anticonvulsant activity.