Purinoreceptors are involved in the control of acute morphine withdrawal.

The effects exerted by P1 and P2 purinoceptor agonists and antagonists on the acute opiate withdrawal induced by morphine were investigated in vitro. Following a 4 min in vitro exposure to morphine, the guinea-pig isolated ileum exhibited a strong contracture after the addition of naloxone. The P1 purinoceptor agonist, adenosine, was able dose-dependently to reduce morphine withdrawal whereas alpha,beta-methylene ATP (APCPP), a P2 purinoceptor agonist, increased morphine withdrawal. Caffeine, a P1 purinoceptor antagonist, was able significantly and in a concentration dependent manner to increase morphine withdrawal whereas quinidine, a P2 receptor antagonist, reduced it. The results of our experiments indicate that both P1 and P2 purinoceptor agonists and antagonists are able to influence opiate withdrawal in vitro, suggesting an important functional interaction between the purinergic system and opioid withdrawal.     

腺苷A1受体在离体延髓脑片上对节律性呼吸的调制

实验旨在探讨腺苷A1受体在对基本呼吸节律调制中的可能作用。制作新生大鼠离体延髓脑片标本,主要包含面神经后核内侧区(themedial region of the nucleus retrofacialis,mNRF),并保留完整的舌下神经根。以改良Kreb‘s液灌流脑片,记录mNRF吸气神经元的电活动,并同步记录舌下神经根呼吸节律性放电(respiratory rhythmical discharge activity,RRDA)。在灌流液中先分别单独给予腺苷A1受体的特异性拮抗剂8-环戊-1,3-二丙基黄嘌呤(8-cyclopenty 1-1,3-dipropylxanthine,DPCPX)和特异性激动剂R-苯异丙基-腺苷(R-phenylisopropyl-adenosine,R-PIA);再分别先后给予R-PIA和R-PIA DPCPX,观察RRDA和吸气神经元电活动的变化。结果显示,给予腺苷A1受体拮抗剂DPCPX后,呼气时程和呼吸周期明显缩短,吸气神经元中期放电的频率和峰频率显著增大;给予腺苷Al受体激动剂R-PIA后,吸气时程、积分幅度和吸气神经元中期放电的频率和峰频率均显著降低,呼吸周期明显延长,且R-PIA的呼吸抑制作用可部分地被DPCPX逆转。实验结果提示,腺苷A1受体可能通过介导吸气神经元的抑制性突触输入参与节律性呼吸的调制。     

脊髓水平细胞外信号调节激酶激活参与吗啡依赖的形成及戒断反应的表达

在大鼠吗啡依赖和戒断模型上,采用行为学、免疫组织化学和Western blot方法观察吗啡依赖及戒断大鼠脊髓神经元磷酸化细胞外信号调节激酶（phospho-extracellular signal-regulated kinase,pERK）表达的变化,及鞘内注射促分裂原活化蛋白激酶激酶（mitogen-activated protein kinase kinase,MEK）抑制剂U0126或ERK反义寡核苷酸对吗啡依赖大鼠纳洛酮催促戒断反应、触诱发痛及脊髓神经元pERK表达的影响,探讨脊髓水平pERK在介导吗啡依赖和戒断过程中的作用。结果显示：（1）在吗啡依赖形成过程中,大鼠脊髓胞浆与胞核非磷酸化ERK表达没有改变,但pERK表达逐渐增加,纳洛酮催促戒断后,仍有进一步增加的趋势,戒断1h后,其表达量明显下降,但仍高于对照组。（2）鞘内预先注射MEK抑制剂U0126或ERK反义寡核苷酸能明显抑制吗啡戒断反应和戒断引起的痛觉异常;与行为学结果一致,脊髓背角pERK阳性神经元表达与脊髓胞浆和胞核pERK表达也明显降低。上述结果提示,脊髓水平ERK激活和核转位参与吗啡依赖的形成及戒断反应的表达。     

Cocaine attenuates the severity of naloxone-precipitated opioid withdrawal

Cocaine dependence in opioid addicts is associated with less severe naloxone-precipitated opioid withdrawal compared to opioid addicts who are not cocaine dependent, suggesting that cocaine may reduce opioid withdrawal severity. The present study examines this effect further by comparing withdrawal severity in these two groups of opioid addicts. To control for dose, length of exposure, and time of administration of drugs, we also studied this effect in rats. Cocaine reduced the overall severity of opioid withdrawal in both human and rat. This drug-drug interaction may occur through alpha-2 adrenergic neurons in the locus coeruleus, similar to the effect of clonidine on opioid withdrawal.     

Adenosine and opioid receptor-mediated cardioprotection in the rat: evidence for cross-talk between receptors

The relative roles of free-radical production, mitochondrial ATP-sensitive K+ (mitoKATP) channels and possible receptor cross-talk in both opioid and adenosine A1 receptor (A1AR) mediated protection were assessed in a rat model of myocardial infarction. Sprague-Dawley rats were subjected to 30 min of occlusion and 90 min of reperfusion. The untreated rats exhibited an infarct of 58.8 +/- 2.9% [infarct size (IS)/area at risk (AAR), %] at the end of reperfusion. Pretreatment with either the nonselective opioid receptor agonist morphine or the selective A1AR agonist 2-chloro-cyclopentyladenosine (CCPA) dramatically reduced IS/AAR to 41.1 +/- 2.2% and 37.9 +/- 5.5%, respectively (P < 0.05). Protection afforded by either morphine or CCPA was abolished by the reactive oxygen species scavenger N-(2-mercaptopropionyl)glycine or the mitoKATP channel blocker 5-hydroxydecanoate. Both morphine- and CCPA-mediated protection were attenuated by the selective A1AR antagonist 1,3-dipropyl-8-cyclopentylxanthine and the selective delta1-opioid receptor (DOR) antagonist 7-benzylidenealtrexone. Simultaneous administration of morphine and CCPA failed to enhance the infarct-sparing effect of either agonist alone. These data suggest that both DOR and A1AR-mediated cardioprotection are mitoKATP and reactive oxygen species dependent. Furthermore, these data suggest that there are converging pathways and/or receptor cross-talk between A1AR- and DOR-mediated cardioprotection.     

Trans-cranial electrical stimulation attenuates the severity of naloxone-precipitated morphine withdrawal in rats

The expression of morphine withdrawal in rats has been demonstrated as dependent upon the integrity of specific brain regions. Focal intracranial electrical stimulation of some of these sites results in the attenuation of withdrawal severity. The present study demonstrates that electrical auricular stimulation, in a paradigm known to attenuate nociceptive responses of several brain nuclei, attenuates the severity of naloxone-precipitated morphine withdrawal in rats. This simple non-invasive treatment, based on long-standing principles of electroacupuncture, may provide a useful adjunct for therapy of the narcotic withdrawal syndrome.     

Adenosine A2 receptors are involved in the activation of ATP-sensitive K+ currents during metabolic inhibition in guinea pig ventricular myocytes

It has been hypothesized that an interaction among adenosine A(1) receptors, protein kinase C (PKC) activation, and ATP-sensitive potassium channels (K(ATP)) mediates ischemic preconditioning in experiments on different animal species. The purpose of this study was to determine if activation of K(ATP) is functionally coupled to A(1) receptors and (or) PKC activation during metabolic inhibition (MI) in guinea pig ventricular myocytes. Perforated-patch using nystatin and conventional whole-cell recording methods were used to observe the effects of adenosine and adenosine-receptor antagonists on the activation of K(ATP) currents during MI induced by application of 2,4-dinitrophenol (DNP) and 2-deoxyglucose (2DG) without glucose, in the presence or absence of a PKC activator, phorbol 12-myristate 13-acetate (PMA). Adenosine accelerated the time course activation of K(ATP) currents during MI under the intact intracellular condition or dialyzed condition with l mmol/L ATP in the pipette solution. The accelerated effect of adenosine activation of K(ATP) under MI was not reversed by a nonselective Al adenosine receptor antagonist, 8-(p-sulfophenyl)theophylline (SPT), or a specific Al adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). However, the adenosine A(2) receptor antagonist alloxazine reversed the time course activation of the K(ATP) current under MI. An adenylate cyclase activator, forskolin, did not further abbreviate the time course activation of K(ATP) with or without adenosine. Application of a PKC blocker, chelerythrine, reversed the time course activation of K(ATP) by adenosine under MI. In addition, pretreatment with a PKC activator, PMA, had similar effects to adenosine, while adenosine did not further shorten the time required for activation of K(ATP) currents during MI with PMA pretreatment. There is no direct evidence of activation of K(ATP) currents by adenosine A(1) receptor during metabolic inhibition under our experimental condition. However, adenosine A(2) receptor activation is involved in the K(ATP) channel activation in the guinea pig ventricular myocytes, of which effect is not mediated through the increase in intracellular cAMP. Adenosine seems to interact with PKC activation to open K(ATP) during MI, but a possible link between the adenosine A(2) receptor and PKC activation in this process needs further elucidation.     

Adenosine receptors are expressed during differentiation of monocytes to macrophages in vitro. Implications for regulation of phagocytosis

Ingestion by phagocytes is known to be markedly enhanced by physiologic signals such as cytokines and extracellular matrix proteins which may be found in inflammatory sites. Little investigation has been made of mechanisms that may depress this increased rate of phagocytosis during resolution of inflammation. We show that adenosine can act as an inhibitor of phagocytosis by macrophages derived from in vitro culture of human peripheral blood monocytes. Adenosine (Ado) is equally effective at inhibiting IgG Fc and complement-mediated phagocytosis. However, Ado has no effect on phagocytosis by freshly isolated monocytes. Inhibition by Ado begins after 2 days in culture and reaches a plateau by 5 days; these kinetics of induction of inhibition of phagocytosis parallel an increase in specific Ado binding to the macrophage plasma membrane. Ado binds to cultured monocytes with a Kd of 6 microM. This affinity and the observation that 2-chloroadenosine and 5'-N-ethylcarboxamidadenosine are the most potent inhibitors of phagocytosis suggest that the Ado receptors expressed during monocyte differentiation are of the A2 type. The inhibition of phagocytosis may be mediated by cAMP, a second messenger coupled to A2 receptors in several cell types. Thus, plasma membrane expression of A2 receptors dramatically increases during monocyte differentiation in vitro. These data show that a potentially physiologic mediator can have very different effects on the function of monocytes and macrophages. This suggests a mechanism whereby phagocytic function at inflammatory sites can be down-regulated if and only if signals for the recruitment of new phagocytes have subsided.     

Toll-like receptors are temporally involved in host defense

Toll-like receptors (TLRs) are evolutionarily conserved proteins that recognize microbial molecules and initiate host defense. To investigate how TLRs work together to fight infections, we tested the role of TLRs in host defense against the Gram-negative bacterial pathogen, Salmonella. We show that TLR4 is critical for early cytokine production and killing of bacteria by murine macrophages. Interestingly, later on, TLR2, but not TLR4, is required for macrophage responses. Myeloid differentiation factor 88, an adaptor protein directly downstream of TLRs, is required for both early and late responses. TLR4, TLR2, and myeloid differentiation factor 88 are involved in murine host defense against Salmonella in vivo, which correlates with the defects in host defense observed in vitro. We propose a model where the sequential activation of TLRs tailors the immune response to different microbes.     

Mu-opioid receptors are involved in the tolerance to nicotine antinociception

Several studies have shown the participation of the endogenous opioid system on the antinociceptive effects and addictive properties of nicotine. The aim of the present study was to explore the involvement of the mu-opioid receptors in the development of tolerance to nicotine antinociception. Chronic treatment of C57BL/6 mice with nicotine (5 mg/kg s.c., three times daily during 12 days) resulted in tolerance to its antinociceptive responses in the tail-immersion test. We investigated the possible existence of adaptive changes in the expression and/or functional activity of mu-opioid receptors in these tolerant mice by using autoradiography of [(3)H]D-Ala(2)-MePhe(4)-Gly-ol(5) enkephalin ([(3)H]DAMGO) binding and DAMGO-stimulated guanosine [(35)S]5'-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) binding. The density of mu-opioid receptors in the spinal cord was not modified in nicotine-tolerant mice, whereas a decrease was found in the caudate-putamen, as well as in the core and the shell of the nucleus accumbens. However, the functional activity of these receptors was significantly increased in the spinal cord as a consequence of nicotine treatment. To further investigate the role of mu-opioid receptors in the tolerance to nicotine-induced antinociception, we evaluated this response in C57BL/6 mu-opioid receptor knockout mice. Chronic nicotine treatment produced tolerance in both wild-type and knockout animals, but tolerance developed faster in mice lacking mu-opioid receptors. These results indicate that mu-opioid receptors play an important role in the development of tolerance to nicotine antinociceptive effects.     

Renal dopamine receptors are involved in the development of cardiac hypertrophy

The present study examined the effect of fenoldopam, a known dopamine-1 receptor (DA1) agonist in order to understand its involvement in the cardiac hypertrophic process. Male Sprague-Dawley rats underwent abdominal aortic constriction (AB) with placement of a suprarenal ligature while sham operated animals served as controls. The AB groupsshowed an increase in their heart wt, left ventricular (LV) wt, heart wt/body wt and LV wt/body wt ratio. Furthermore, the length of these hearts, as measured from the auriculoventricular border to the apex, LV wall and interventricular (IV) septal thickness were increased from control levels. Treatment with SCH 23390, a DA1 antagonist, on the other hand, was able to partially regress the cardiac hypertrophic changes. All these parameters were also increased in control animals treated with fenoldopam (F). Such changes were more striking in the F+AB group which showed a significant acceleration of the cardiac hypertrophic process on super-imposing the two treatments. Plasma dopamine and renin activity were increased in all the groups as compared to control. These results indicate that dopamine receptors are implicated in the development of cardiac hypertrophy.     

P2 receptors are involved in the mediation of motivation-related behavior

The importance of purinergic signaling in the intact mesolimbic–mesocortical circuit of the brain of freely moving rats is reviewed. In the rat, an endogenous ADP/ATPergic tone reinforces the release of dopamine from the axon terminals in the nucleus accumbens as well as from the somatodendritic region of these neurons in the ventral tegmental area, as well as the release of glutamate, probably via P2Y₁ receptor stimulation. Similar mechanisms may regulate the release of glutamate in both areas of the brain. Dopamine and glutamate determine in concert the activity of the accumbal GABAergic, medium-size spiny neurons thought to act as an interface between the limbic cortex and the extrapyramidal motor system. These neurons project to the pallidal and mesencephalic areas, thereby mediating the behavioral reaction of the animal in response to a motivation-related stimulus. There is evidence that extracellular ADP/ATP promotes goal-directed behavior, e.g., intention and feeding, via dopamine, probably via P2Y₁ receptor stimulation. Accumbal P2 receptor-mediated glutamatergic mechanisms seem to counteract the dopaminergic effects on behavior. Furthermore, adaptive changes of motivation-related behavior, e.g., by chronic succession of starvation and feeding or by repeated amphetamine administration, are accompanied by changes in the expression of the P2Y₁ receptor, thought to modulate the sensitivity of the animal to respond to certain stimuli.     

Effects of harman and harmine on naloxone-precipitated withdrawal syndrome in morphine-dependent rats

The effects of the beta-carbolines, harman and harmine, on naloxone-precipitated withdrawal syndrome in morphine-dependent rats were investigated. Two morphine pellets containing 75 mg morphine base were implanted subcutaneously in the scapular area of adult male Wistar rats (200-250 g) under light ether anesthesia. Rats were then assigned to several groups (n = 12 for each group). Seventy-two hours after morphine implantation, harman (5 and 10 mg/kg), harmine (5 and 10 mg/kg) or saline was injected to rats intraperitoneally (ip). After 45 min, a morphine withdrawal syndrome was precipitated by naloxone (2 mg/kg, ip), and morphine withdrawal signs were observed and evaluated for 15 min. Harmine (5 and 10 mg/kg) attenuated significantly the intensity of all signs of morphine withdrawal except for jumping. While jumping behaviour appearing in morphine withdrawal was intensified by harman (5 and 10 mg/kg) treatment, harmine administration did not produce any significant change in the intensity of this sign. Harman attenuated significantly the intensity of wet dog shakes, writhing, defecation, tremor and ptosis. However, it produced no significant changes in the intensity of teeth chattering and diarrhea. Our results suggest that harman and harmine, beta-carbolines, have some beneficial effects on naloxone-precipitated morphine withdrawal syndrome in rats. Findings from the present study also indicated that harmine was more effective than harman on morphine abstinence syndrome.     

Modulation of naloxone-precipitated morphine withdrawal syndromes in rats by neuropeptide FF analogs

Neuropeptide FF (NPFF) has been reported to be an endogenous anti-opioid peptide that has significant effects on morphine tolerance and dependence. In the present study, we examined the chronic effects of NPFF and its synthetic analogs: the putative agonist, PFRFamide, and the putative antagonists, dansyl-PQRamide and PFR(Tic)amide on naloxone-precipitated morphine withdrawal syndromes in rats. After a 5-day co-administration with morphine [5 mg/kg, intraperitoneally (i.p.), twice per day (b.i.d.)] and the tested peptide [intracerebroventricularly (i.c.v.) or i.p., b.i.d.], naloxone (4 mg/kg, i.p.) was given systemically to evaluate the severity of the morphine withdrawal syndromes. Our results revealed that NPFF significantly potentiated the overall morphine withdrawal syndromes and, on the contrary, dansyl-PQRamide attenuated these syndromes. These results clearly indicate that modulation of the NPFF system in the mammalian central nervous system has significant effects on opiate dependence. In addition, morphine withdrawal syndromes could be practically applied as a valid parameter to functionally characterize the putative NPFF agonists and antagonists.     

脊髓水平nNOS和iNOS在吗啡戒断反应中的作用差异

目的:观察鞘内注射选择性一氧化氮合酶(nNOS)和诱导型一氧化氮合酶(iNOS)抑制剂对吗啡依赖大鼠纳洛酮催促戒断反应、脊髓Fos蛋白表达和脊髓神经元nNOS和iNOS表达的影响,以探讨nNOS和iNOS在吗啡依赖和戒断反应中的作用。方法:在大鼠吗啡依赖和戒断模型上,采用行为学、免疫组织化学和Western blot方法观察鞘内应用nNOS抑制剂7-硝基吲哚(7-Ni)和iNOS抑制剂氨基胍(AG)对吗啡依赖大鼠纳洛酮催促戒断反应、脊髓Fos蛋白表达和脊髓神经元nNOS和iNOS表达的影响。结果:①鞘内注射7-Ni、AG可明显减轻吗啡依赖大鼠戒断症状,戒断组戒断症状评分为28.6±4.89,7-Ni组为16.2±3.99(P<0.01),AG组为22.94±4.0(P<0.05);戒断组TEA评分为13.5±2.55,7-Ni、AG组分别为7.5±2.56、10.5±2.71(P<0.05);②鞘内注射7-Ni、AG可减少脊髓背角Fos阳性神经元的数目,7-Ni、AG组为228.2±49.5、296.8±50.6,低于戒断组(380±71,P<0.05);③7-Ni、AG组nNOS和iNOS阳性神经元的数目分别为169±32、10.2±2.85,均低于戒断组(239±45,16.8±5.1,P<0.05),两给药组脊髓NOS蛋白的表达也显著减少。结论:nNOS和iNOS抑制剂能减轻吗啡依赖及戒断大鼠的戒断症状和在脊髓水平抑制nNOS和iNOS的表达,nNOS起主要作用而iNOS可能起辅助作用。     

Hypothalamic orexin--a neurons are involved in the response of the brain stress system to morphine withdrawal

Both the hypothalamus-pituitary-adrenal (HPA) axis and the extrahypothalamic brain stress system are key elements of the neural circuitry that regulates the negative states during abstinence from chronic drug exposure. Orexins have recently been hypothesized to modulate the extended amygdala and to contribute to the negative emotional state associated with dependence. This study examined the impact of chronic morphine and withdrawal on the lateral hypothalamic (LH) orexin A (OXA) gene expression and activity as well as OXA involvement in the brain stress response to morphine abstinence. Male Wistar rats received chronic morphine followed by naloxone to precipitate withdrawal. The selective OX1R antagonist SB334867 was used to examine whether orexins' activity is related to somatic symptoms of opiate withdrawal and alterations in HPA axis and extended amygdala in rats dependent on morphine. OXA mRNA was induced in the hypothalamus during morphine withdrawal, which was accompanied by activation of OXA neurons in the LH. Importantly, SB334867 attenuated the somatic symptoms of withdrawal, and reduced morphine withdrawal-induced c-Fos expression in the nucleus accumbens (NAc) shell, bed nucleus of stria terminalis, central amygdala and hypothalamic paraventricular nucleus, but did not modify the HPA axis activity. These results highlight a critical role of OXA signalling, via OX1R, in activation of brain stress system to morphine withdrawal and suggest that all orexinergic subpopulations in the lateral hypothalamic area contribute in this response.     

Melanocortin-3 receptors are involved in adaptation to restricted feeding

The central nervous melanocortin system forms a neural network that maintains energy homeostasis. Actions involving neural melanocortin-3 receptors (MC3Rs) regulate the expression rhythms in ingestive behaviors and metabolism anticipating nutrient intake. Here, we characterized the response of Mc3r knockout (Mc3r(-/-)) and wild type (WT) mice to a restricted feeding (RF) schedule where food access was limited to a 4-h period mid light cycle using a mechanical barrier. Mc3r(-/-) mice adapted poorly to the food restriction schedule. Anticipatory activity and the initial bout of intense feeding activity associated with granting food access were attenuated in Mc3r(-/-) mice, resulting in increased weight loss relative to controls. To investigate whether activity in specific hypothalamic nuclei contribute to the Mc3r(-/-) phenotype observed, we assessed hypothalamic FOS-immunoreactivity (FOS-IR) associated with food restriction. Food access markedly increased FOS-IR in the dorsomedial hypothalamus (DMH), but not in the suprachiasmatic or ventromedial hypothalamic nuclei (SCN and VMN, respectively) compared to ad libitum fed mice. Mc3r(-/-) mice displayed a significant reduction in FOS-IR in the DMH during feeding. Analysis of MC3R signaling in vitro indicated dose-dependent stimulation of the extracellular signal-regulated kinase (ERK) pathway by the MC3R agonist d-Trp(8)-γMSH. Treatment of WT mice with d-Trp(8)-γMSH administered intracerebroventricularly increased the number of pERK neurons 1.7-fold in the DMH. These observations provide further support for the involvement of the MC3Rs in regulating adaptation to food restriction. Moreover, MC3Rs may modulate the activity of neurons in the DMH, a region previously linked to the expression of the anticipatory response to RF.