Effects of agmatine on tolerance to and substance dependence on morphine in mice

目的：观察胍丁胺对吗啡所致耐受和依赖的作用．方法：分别在小鼠耐受和跳跃实验中观察胍丁胺抑制吗啡所致耐受和物质依赖的作用．结果：胍丁胺０１２５－２５ｍｇ·ｋｇ－１剂量依赖性地阻止小鼠对吗啡耐受．用吗啡预处理小鼠使吗啡镇痛ＥＤ５０（２０１,１４４－２８０ｍｇ·ｋｇ－１）与盐水组相比（６３,５１－７８ｍｇ·ｋｇ－１）增加３倍以上．用胍丁胺和吗啡共同预处理小鼠则使吗啡丧失引起耐受的能力．胍丁胺（２５－１０ｍｇ·ｋｇ－１）剂量依赖性地抑制由纳洛酮引起的吗啡依赖小鼠之戒断跳跃和体重减轻．用胍丁胺和吗啡共同处理小鼠使引起小鼠之戒断跳跃所需纳洛酮ＥＤ５０（２１４,１８４－２４ｍｇ·ｋｇ－１）较吗啡处理组（２５,２１－２８ｍｇ·ｋｇ－１）增大８倍．胍丁胺的上述作用均被咪唑克生所阻断．结论：胍丁胺通过激活咪唑啉受体而阻止小鼠对吗啡耐受和依赖．     

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。     

地尔硫抑制小鼠吗啡戒断反应及血浆和脑匀浆一氧化氮的含量

目的··:观察钙拮抗剂盐酸地尔硫对小鼠吗啡戒断反应及血浆和脑匀浆一氧化氮 (NO)含量的影响。方法··:皮下注射定量吗啡 ,建立小鼠吗啡依赖动物模型 ;腹腔注射纳洛酮催促 ,根据小鼠戒断反应中出现跳跃反应的潜伏期、跳跃次数和体重丢失评定戒断反应的强度 ;用硝酸还原法检测其血浆和脑内NO含量。结果·· :3种不同剂量 (25、50、100mg·kg-1)的盐酸地尔硫对小鼠吗啡戒断反应有明显的抑制作用 ,并呈量效关系;100mg·kg-1的盐酸地尔硫可抑制吗啡戒断反应引起的血浆和脑匀浆NO含量的升高。结论·· :盐酸地尔硫可抑制小鼠吗啡戒断反应 ,并能抑制戒断反应引起的血浆和脑匀浆NO含量的升高。     

褪黑激素抑制小鼠吗啡戒断反应并降低血浆、脑组织中NO含量

目的观察褪黑激素(MT)对小鼠吗啡戒断反应及血浆、脑组织中NO含量的影响。方法定量吗啡sc,建立小鼠吗啡依赖模型;纳洛酮ip催瘾。用褪黑激素连续ig 3 d。根据小鼠戒断反应中出现跳跃反应的潜伏期、跳跃次数、体重丢失评定戒断反应强度。用硝酸还原酶法检测血浆和脑组织中NO含量。结果用褪黑激素ig可明显延长小鼠吗啡戒断跳跃反应的潜伏期、减少跳跃次数, 对体重丢失也有所改善。MT可抑制由戒断反应引起的血浆和脑组织中NO含量的升高。结论褪黑激素可抑制小鼠吗啡戒断反应并降低由戒断反应引起的血浆和脑组织中NO含量的升高。     

谷氨酸钠对小鼠吗啡依赖及戒断时血浆、脑内一氧化氮的影响

目的 :探讨兴奋性氨基酸的钠盐谷氨酸钠( Glu-Na)对小鼠吗啡依赖及吗啡戒断小鼠血浆、脑内一氧化氮的影响。方法 :皮下注射定量吗啡 ,建立小鼠吗啡依赖模型 ,腹腔注射纳洛酮催瘾。在注射吗啡同时每天早 8点用不同剂量 Glu-Na给小鼠灌胃 ( ig) ,观察小鼠戒断反应中出现的跳跃反应的潜伏期、跳跃次数和体重丢失 ,评定戒断反应的强度。用硝酸还原法检测血浆和脑内NO含量。结果 :三种不同剂量 ( 1 0 0、2 0 0、4 0 0 mg/ kg)的Glu-Na对小鼠吗啡戒断有加强作用 ,并呈量效关系。吗啡戒断小鼠脑内和血浆中 NO的含量明显高于正常小鼠 ,中剂量 ( 2 0 0 mg/ kg)的 Glu-Na可进一步提高吗啡戒断小鼠脑内和血浆中 NO的含量。结论 :Glu-Na可加强小鼠吗啡依赖 ,并提高吗啡戒断小鼠血浆和脑内一氧化氮含量     

吗啡依赖对小鼠海马内一氧化氮合酶阳性神经元的影响

目的·· :探讨吗啡依赖对小鼠海马不同亚区一氧化氮合酶(NOS)活性的影响。方法·· :以剂量递增法皮下注射吗啡建立吗啡依赖小鼠模型 ,采用还原型辅酶Ⅱ -黄递酶(NADPH -d)组织化学法显示吗啡依赖组、纳洛酮催促戒断组和正常对照组小鼠海马CA1区、CA3区和齿状回NOS阳性神经元。结果·· :与对照组相比 ,吗啡依赖组和纳洛酮催促戒断组海马CA1区和齿状回NOS阳性神经元的数目均明显减少 (P<0.01) ,纳洛酮催促戒断组更为明显 ,而在CA3区无明显改变。结论·· :吗啡依赖和纳洛酮催促戒断小鼠海马CA1区和齿状回NOS活性降低 ,提示NO合成的能力下降 ,戒断期下降更明显。这些变化可能是吗啡依赖造成学习记忆功能下降的原因之一。     

甲氧氯普胺对吗啡依赖性小鼠戒断症状和脑区cGMP含量的影响

目的 :观察甲氧氯普胺脑室给药对吗啡依赖小鼠戒断症状的影响以及腹腔给药对小鼠不同脑区cGMP含量的影响。方法 :皮下注射 (sc)盐酸吗啡建立吗啡依赖小鼠实验模型 ,在纳洛酮催促戒断前 30min侧脑室微量注射甲氧氯普胺 ,观察其急性给药对吗啡依赖性戒断症状的影响 ;用放射性免疫法观察腹腔注射 (ip)甲氧氯普胺对吗啡依赖小鼠小脑、大脑皮层、海马及丘脑四个脑区cGMP含量的影响。结果 :甲氧氯普胺 (1 0mg·kg- 1 )可有效抑制纳洛酮催促的吗啡依赖小鼠的跳跃反应 (P <0 0 1) ;吗啡依赖小鼠四个脑区中cGMP含量均低于正常鼠 (P <0 0 1) ,甲氧氯普胺急性给药 (2 0mg·kg- 1 ,ip)可使吗啡依赖小鼠cGMP接近正常水平。结论 :中枢神经系统是甲氧氯普胺抑制吗啡依赖小鼠戒断跳跃反应的主要作用部位 ;脑区cGMP水平的恢复作用可能是其抑制吗啡戒断的主要机制之一     

褪黑素抗吗啡戒断反应及其对中脑导水管周围灰质β-内啡肽的影响

目的:观察褪黑素(MEL)抗小鼠吗啡依赖戒断反应与其对脑内中脑导水管周围灰质(PAG)中β-内啡肽(β-EP)影响之间的关系,以探讨MEL抗戒断反应的中枢机制。方法:采用剂量递增法连续皮下注射(sc)吗啡建立小鼠吗啡依赖模型,sc纳洛酮催促戒断,以小鼠体重下降和戒断跳跃次数为指标观察MEL抗小鼠吗啡依赖戒断反应的效应;采用放射免疫分析法,检测小鼠中脑导水管周围灰质β-EP含量。结果:(1)连续8d腹腔注射(ip)10,20,40mg.kg-1的MEL能剂量依赖性地抑制吗啡依赖小鼠的戒断症状;(2)吗啡依赖组小鼠脑内PAG中β-EP含量明显低于生理盐水对照组;(3)连续8dip20mg.kg-1MEL的小鼠,其PAG中β-EP含量明显高于吗啡依赖组小鼠。结论:MEL可减弱小鼠吗啡依赖戒断反应,这种效应可能与其增加脑内PAG中β-EP的含量有关。     

中缝背核参与大鼠吗啡依赖和戒断的形成机制研究

目的观察大鼠脑内神经核団中缝背核(DRN)在一氧化氮(NO)介导的吗啡依赖和戒断形成的作用机制。方法雄性成年SD大鼠随机分为5组:戒断组(腹腔注射吗啡+纳洛酮);依赖组(注射吗啡+生理盐水);生理盐水组(注射生理盐水);纳洛酮组(注射生理盐水+纳洛酮);抑制剂组(注射吗啡加NOS抑制剂+纳洛酮)。戒断组和依赖组,用剂量递增法经腹腔注射吗啡10～100mg.kg-1,每天3次,连续5天,建立吗啡依赖与戒断模型,并进行行为学观测与评分后,用神经元型一氧化氮合酶(nNOS)免疫组织化学标记,计数各组动物相同层面脑片上nNOS标记细胞的表达情况。结果戒断组,戒断症状及总评分较对照组和依赖组均差异显著(P<0.01);NOS抑制剂组,戒断症状评分较戒断组明显降低(P<0.05)。于中缝背核相应区域计数到部分nNOS标记神经元,生理盐水组及纳洛酮组比较无显著性差异;而依赖组与戒断组,其神经元计数明显增加(P<0.05);而NOS抑制剂组,神经元数量较戒断组明显减少(P<0.05)。结论脑内中缝背核可能通过一氧化氮信号通路参与了大鼠吗啡依赖与戒断的形成。     

氯胺酮对大鼠吗啡戒断症状的影响及其作用机理

目的　研究氯胺酮对大鼠吗啡戒断症状的影响及其可能的机理。方法　建立大鼠吗啡依赖模型 ,在用纳洛酮催瘾前 2min给予不同剂量的氯胺酮 ,观察其戒断症状的改变 ;用分光光度法测定戒断时大鼠一氧化氮 (NO)含量、一氧化氮合酶 (NOS)活性 ,用放射免疫法测定环鸟苷酸 (cGMP)含量。结果　 3个剂量的氯胺酮 (5、10和 2 0mg·kg- 1)均可缓解吗啡戒断时探究、扭体、湿狗样抖动、跳跃等运动反应 ,减少活动次数 ,抑制植物神经系统症状。10、2 0mg·kg- 1氯胺酮可显著减轻吗啡戒断所致的体重下降 ,小剂量氯胺酮 (5mg·kg- 1)可抑制吗啡依赖大鼠前额叶皮质、小脑的NOS活性和NO、cGMP含量的增高。结论　氯胺酮可缓解大鼠吗啡戒断症状 ,其作用机理可能与减弱大鼠吗啡戒断时NMDA NO cGMP通路效应有关。     

胍丁胺抑制小鼠吗啡戒断与其抑制一氧化氮合酶的关系

AIM: To study correlation between inhibitions of naloxone-precipitated withdrawal jumps and nitric-oxide synthase (NOS) activity by agmatine. METHODS: NOS activities in mouse brain were measured by determination of concentration of [3H]citrulline, the product of [3H]arginine. RESULTS: Agmatine inhibited NOS activity in naive and morphine-dependent mouse cerebellum, forebrain, and thalamus in substrate-competitive manner in vitro. Naloxone induced withdrawal jumps and an increase in NOS activity in cerebellum, forebrain, and thalamus of abstinent mice. Pretreatment of mice with morphine plus agmatine inhibited the effect of naloxone to precipitate withdrawal jumps and increase in NOS activity. The effect of agmatine was blocked by idazoxan. CONCLUSION: The inhibitory effect of agmatine on naloxone-precipitated withdrawal jumps is related to its inhibition of NOS activity by substrate competitive manner and activation of imidazoline receptors.     

胍丁胺对吗啡所致小鼠耐受和物质依赖的作用

目的 观察胍丁胺对吗啡所致耐受和依赖的作用。方法 分别在小鼠耐受和跳跃实验中观察胍丁胺抑制吗啡所致耐受和物质依赖的作用,结果：胍下胺０．１２５～２．５ｍｇ．ｋｇ＾－１剂量依赖性地阻止小鼠对吗啡耐受,用吗啡预处理小鼠使吗啡镇痛ＥＤ５０（２０．１,１４．４－２８．０ｍｇ．ｋｇ＾－１）与盐水组相比（６．３,５．１－７．８ｍｇ．ｋｇ＾－１）增加３倍以上,用胍丁胺和吗啡共同预处理小鼠则使吗啡丧失引直耐受的能     

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.     

Binding affinity to and dependence on some opioidsin Sf9 insect cells expressing human μ-opioid receptor

AIM: To investigate the receptor binding affinity and naloxone-precipitated cAMP overshoot of dihydroetorphine, fentanyl, heroin, and pethidine in Sf9 insect cells expressing human mu-opioid receptor (Sf9-mu cells). METHODS: Competitive binding assay of [3H]ohmefentanyl was used to reveal the affinity for mu-opioid receptor in Sf9-mu cells. [3H]cAMP RIA was used to determine cAMP level. Antinociceptive activity was evaluated using degree 55 mouse hot plate test. Naloxone-precipitated withdrawal jumping was used to reflect physical dependence in mice. RESULTS: All drugs displayed antinociceptive activity and produced physical dependence in mice. The K(i) values of dihydroetorphine, fentanyl, heroin, and pethidine in competitive binding assay were (0.85+/-0.20) nmol, (59.1+/-11.7) nmol, (0.36+/-0.13) micromol, and (12.2+/-3.8) micromol respectively. The binding affinities of these drugs for mu-opioid receptor in Sf9-mu cells were paralleled to their antinociceptive activities in mice. After chronic pretreatment with these drugs, naloxone induced cAMP withdrawal overshoot in Sf9-mu cells. The dependence index in Sf9-mu cells was calculated as K(i) value in competitive binding assay over EC(50) value in naloxone-precipitated cAMP assay. The physical dependence index in mice was calculated as antinociceptive ED(50)/withdrawal jumping cumulative ED(50). There was a good linear correlation between dependence index in Sf9-mu cells and physical dependence index in mice. CONCLUSION: The Sf9-mu cells could be used as a cell model to evaluate the receptor binding affinity and physical dependent liability of analgesic agents.     

Modification of the expression of naloxone-precipitated withdrawal signs in morphine-dependent mice by diabetes: possible involvement of protein kinase C

The involvement of cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC) in the modulation of naloxone-precipitated withdrawal jumping in morphine-dependent mice by diabetes was examined. Naloxone-precipitated withdrawal jumps were significantly less in morphine-dependent diabetic mice than in morphine-dependent non-diabetic mice. I.c.v. pretreatment with either calphostin C, a PKC inhibitor, or KT-5720, a PKA inhibitor, attenuated naloxone-precipitated withdrawal jumps in morphine-dependent non-diabetic mice. However, naloxone-precipitated withdrawal jumps in morphine-dependent diabetic mice were not attenuated by i.c.v. pretreatment with either calphostin C or KT5720. Moreover, i.c.v. pretreatment with phorbol-12,13-dibutyrate (PDBu), a PKC activator, attenuated naloxone-precipitated withdrawal jumps in morphine-dependent non-diabetic mice, but not in morphine-dependent diabetic mice. The noradrenaline (NA) turnover in the frontal cortex in morphine-dependent non-diabetic mice, but not in morphine-dependent diabetic mice, was significantly increased 5 min after administration of naloxone. Naloxone-induced enhancement of NA turnover in morphine-dependent non-diabetic mice, but not in morphine-dependent diabetic mice, was blocked by i.c.v. pretreatment with either calphostin C or KT5720 1 hr before naloxone challenge and blocked by PDBu 1 hr before the last injection of morphine. These results suggest that the co-activation of PKC and PKA is needed to elicit naloxone-precipitated withdrawal jumps and enhancement of turnover rate of NA in the frontal cortex in morphine-dependent non-diabetic mice. Furthermore, the attenuation of naloxone-precipitated withdrawal jumps in morphine-dependent diabetic mice may be due, in part, to the desensitization of mu-opioid receptors by the activation of PKC.     

Prolonged morphine treatment increases rat brain dihydropyridine binding sites: possible involvement in development of morphine dependence

Regulation of L-type Ca2+ channels by morphine in rat brain was determined by the binding of [3H]nimodipine. Morphine, administered by subcutaneous pellet implantation, increased the density of [3H]nimodipine binding sites in a time- and dose-dependent manner and this effect was reversible upon removal of the pellets. Increases in these dihydropyridine sites were localized to the cortex, hippocampus, hypothalamus and brainstem but not to the cerebellum and striatum. Additional experiments were performed to test the ability of different Ca2+ channel antagonists to affect naloxone-precipitated withdrawal in morphine-dependent mice and rats. These drugs effectively reduced the incidence of naloxone-induced jumping in mice and several of the withdrawal signs in rats. Taken together, our study underscores the plasticity of brain L-type Ca2+ channels and suggests that their upregulation might contribute to morphine dependence.     

Suppression of naloxone-precipitated withdrawal jumps in morphine-dependent mice by stimulation of prostaglandin EP3 receptor.

1. We have shown that intracisternal (i.c.) administration of interleukin-1 beta (IL-1 beta) attenuates naloxone-precipitated withdrawal jumps in morphine-dependent mice, and the effect was partly mediated by the corticotropin-releasing factor. To elucidate further other possible mechanisms involved in the inhibitory effect of IL-1 beta on morphine withdrawal jumping behaviour, in this study, we examined the involvement of the prostaglandin-synthesis pathway, because prostaglandins have been shown to mediate the several central effects of IL-1. Furthermore, we examined the effects of subtype-selective prostaglandin receptor agonists on morphine withdrawal jumping behaviour. 2. Mice were rendered morphine-dependent by subcutaneous implantation of a pellet containing 11.5 +/- 0.3 mg morphine hydrochloride for 48 h. Morphine withdrawal syndromes were precipitated by intraperitoneal (i.p.) injection of naloxone (10 mg kg-1). The degree of physical dependence on morphine was estimated by counting the number of jumps, one of the typical withdrawal signs in mice, for 40 min. 3. The inhibitory effect of IL-1 beta (1 ng/mouse) administered intracisternally 30 min before naloxone (10 mg kg-1, i.p.) was significantly blocked by pretreatment with sodium salicylate (a cyclo-oxygenase inhibitor, 10 ng or 30 ng/mouse) administered intracisternally 15 min before IL-1 beta, while i.c. administration of sodium salicylate alone (3 ng, 10 ng or 30 ng/mouse) followed by i.c. administration of vehicle instead of IL-1 beta did not significantly change the number of jumps precipitated by naloxone. 4. Intracisternal administration of M&B28,767 (an EP3-receptor agonist, 1 fg-30 ng/mouse) and sulprostone (an EP1/EP3-receptor agonist, 10 fg-100 ng/mouse) 30 min before naloxone (10 mg kg,-1 i.p.) attenuated withdrawal jumps with a U-shaped dose-response, reaching a peak at 10 pg/mouse and 100 pg/mouse, respectively. On the other hand, i.c. administration of iloprost (an EP1/IP-receptor agonist, 10 fg-100 ng/mouse), butaprost (an EP2-receptor agonist, 10 fg-100 ng/mouse) or prostaglandin F2 alpha (a FP-receptor agonist, 10 fg-100 ng/mouse) 30 min before naloxone (10 mg kg-1, i.p.) did not significantly change the number of jumps precipitated by naloxone. 5. These results indicate that the prostaglandin-synthesis pathway is, at least in part, involved in the inhibitory effect of IL-1 beta on naloxone-precipitated withdrawal jumps in morphine-dependent mice, and that the prostaglandin synthesized in the brain suppresses the morphine withdrawal jumping behaviour via the EP3-receptor, but not via the EP1-, EP2-, IP- or FP-receptor.     

Polyamines allosterically modulate [3H]nitrendipine binding to the voltage-sensitive calcium channel in rat brain.

The effects of polyamines on radioligand binding to the slow voltage-dependent Ca2+ channel were studied using membranes from the rat cerebral cortex. [3H]Diltiazem binding was inhibited by arcaine (IC50 = 55 microM) and, in decreasing order of potency, by agmatine, spermidine, spermine and putrescine. Under control conditions, only spermidine and spermine allosterically inhibited [3H]nitrendipine binding while arcaine, agmatine and putrescine were inactive. Nevertheless, putrescine antagonized the effect of spermine as well as the allosteric effects of diltiazem and verapamil on the binding of [3H]nitrendipine, in a manner analogous to that shown previously for Ca2+. Thus, polyamines may function as endogenous modulators of the voltage-dependent Ca2+ channel.