苯肾上腺素诱导乳鼠心肌细胞蛋白质表达变化的蛋白质组分析

α1-肾上腺素受体(α1-Adrenergic receptor,α1-AR)是G蛋白偶联受体(G-protein coupled receptor,GPCR),也是内源性儿茶酚胺、去甲肾上腺素和肾上腺素最重要的靶受体之一.α1-AR广泛分布于机体的各种器官、组织和细胞中,并介导多种生理效应,如血管收缩、蛋白质合成及心脏变力变时作用等[1,2].很多研究已经证实,α1-AR及其信号转导通路与许多心血管疾病存在密切关系[3,4].蛋白质组学可提供一种发现在疾病情况下异常表达蛋白质的方法,为疾病的早期诊断和愈后判断提供指南,并为针对性疾病治疗提供科学依据.本研究以乳鼠心肌细胞为实验模型,利用双向凝胶电泳和飞行时间质谱分析苯肾上腺素诱导乳鼠心肌细胞表达变化的蛋白质.1实验部分1.1试剂苯肾上腺素(Phenylephine,PE)购自Sigma公司;胰蛋白酶和DMEM购自Hyclone公司;IPG预制胶条(pH=5~8,胶条长17 cm),载体两性电解质(B io-Lyte5-8)购于B io-Rad公司;TPCK修饰的测序级胰酶购自Promega公司;其它试剂均为国产分析纯.1.2实验过程(1)乳鼠心肌细胞培养及样...

Study on the GABA gated channels in the neurosecretory cells of MTXO in the eyestalks of Eriocheir sinensis

The responses to rapid application of gamma-aminobutyric acid (GABA) and the GABA receptor characteristics of MTXO neurosecretory cells in the eyestalks of Chinese mitten-handed crab (Eriocheir sinensis) were examined by whole-cell patch clamp. Under current clamp mode, the depolarization and hyperpolarization were evoked from the three types of neurosecretory cells in response to the GABA (0.1 mmol/L) depending on the Nernst Cl- potential. Under voltage clamp mode, the inward Cl- channel currents (I-GABA) were resolved from all three types of neurosecretory cells in response to GABA (0.01similar to5 mmol/L). The GABA currents were activated within 1 200 ms and peaked within 800 ms. No obviously desensitization was observed during GABA application. The dose-response curve showed usual S-shape, with a just-discernible effect at 0.01 mmol/L and near-saturation at 0.5 mmol/L. The GABA currents had reversal potentials that followed Nernst Cl- potentials when [Cl-] was varied. The pharmacological results revealed that the GABA receptor of the crab neurosecretory cells was sensitive to the Cl- channel blockers picrotoxin and niflumic acid (0.5 mmol/L), insensitive to GABA(A) receptor antagonist bicuculline and GABA(C) receptor agonist cis-4-aminocrotonic acid (CACA 1 mmol/L) and trans-4-aminocrotonic (TACA 1 mmol/L).

阿片药物依赖的神经机制---内侧前额叶与药物的心理依赖

Mental dependence, characterized by craving and impulsive seeking behavior, is the matter of intensive study in the field of drug addiction. The mesolimbic dopamine system has been suggested to play an important role in rewarding of drugs and relapse. Although chronic drug use can induce neuroadaptations of the mesolimbic system and changes of drug reinforcement, these mechanisms cannot fully account for the craving and the compulsive drug-using behavior of addicts. Acknowledging the reinforcement effects of drugs, most previous studies have studied the impact of environmental cues and conditioned learning on addiction behavior, often using established classical or operant conditioning model. These studies, however, paid little attention to the role of cognitive control and emotion in addiction. These mental factors that are believed to have an important influence on conditioned learning. The medial prefrontal cortex (mPFC) has close anatomic and functional connections with the mesolimbic dopamine system. A number of the cognitive neurological studies demonstrate that mPFC is involved in motivation, emotional regulation, monitoring of responses and other executive functions. Thus we speculated that the function of abnormality in mPFC following chronic drug use would cause related to the abnormal behavior in addicts including impulse and emotional changes. In the present study of a series of experiments, we used functional magnetic resonance imaging to examine the hemodynamic response of the mPFC and related circuits to various cognitive and emotional stimuli in heroin addicts and to explore the underlying dopamine neuromechnism by microinjection of tool drugs into the mPFC in laboratory animals. In the first experiment, we found that heroin patients, relative to the normal controls, took a much shorter time and committed more errors in completing the more demanding of cognitive regulation in the reverse condition of the task, while the neural activity in anterior cingulate cortex (ACC) was attenuated. In the second experiment, the scores of the heroin patients in self-rating depression scale (SDS) and Self-rating anxiety scale (SAS) were significantly higher than the normal controls and they rated the negative pictures more aversive than the normal controls. Being congruent with the behavioral results, hemodynamic response to negative pictures showed significant difference between the two groups in bilateral ventral mPFC (VMPFC), amygdala, and right thalamus. The VMPFC of patients showed increased activation than normal controls, whereas activation in the amygdala of patients was weaker than that in normal subjects. Our third experiment showed that microinjection of D1 receptor agonist SKF38393 into the mPFC of rats decreased hyperactivity, which was induced by morphine injection, in contrast, D1 receptor antagonist SCH23390 increased the hyperactivity, These findings suggest: (1) The behavior and neural activity in ACC of addicts changed in chronic drug users. Their impulsive behavior might result from the abnormal neural activity in the mPFC especially the ACC. (2) Heroine patients were more depress and anxiety than normal controls. The dysfunction of the mPFC---amygdala circuit of heroine addicts might be related to the abnormal emotion response. (3) Dopamine in the mPFC has an inhibitory effect on morphine induced behavior. The hyperactivity induced by chronic morphine was reduced by dopamine increase with D1 receptor agonist, confirm the first experiment that the neuroadaption of mPFC system induced by chronic morphine administration appears to be the substrate the impulse behavior of drug users.

去甲肾上腺素参与吗啡神经适应性改变的中枢机制

That relapse still exists even after prolonged withdrawal is a difficult issue in the medical cure of drug addiction. Neuro-adaptation induced by prolonged exposure to addictive drugs is the neural mechanisms of both compulsive drug seeking and relapse.Neuro-adaptation caused by addictive drugs increases the individuals’ response to drugs and on the other hand, it reduces the response to natural reward in withdrawn individuals.There must be common neural mechanisms between the co-existing phenomena, and there must also be unique neural mechanisms in the drugs.To reveal the neuro-adaptation arising in the process from random, controllable drug-use to uncontrollable compulsive drug seeking is of great significance both theoretically and practically.Based on the above hypothesis, in order to reveal the function of alpha adrenergic receptor in compulsive drug-seeking motivation during the process of drug addiction, using sensitization of morphine-induced psychomotor activity as behavioral model, through the method of behavioral pharmacology, the neural mechanisms of alpha adrenergic receptor’s involvement in the process of addiction has been studied.The adjustment function caused by alpha receptors in medial prefrontal cortex and nucleus accumbens to morphine-induced psychomotor activity has been compared in the period of first use of drugs and in repetitive-use period. Furthermore, the effect on novelty seeking caused by alpha-receptors in relevant brain areas has also been compared. Major results are as follow: 1 After prolonged morphine exposure, rats’ response to morphine-induced psychomotor activity is strengthened and response to novel object induced reward weakened. 2 Injection of prazosin in medial prefrontal cortex will block morphine-induced psychomotor activity of naïve rats, however, it will not block that of morphine-withdrawn rats, but it will block the novelty seeking behavior of morphine-withdrawn rats. 3 Injection of clonidine in medial prefrontal cortex will block morphine-induced psychomotor effect of both naïve rats and morphine-withdrawn rats, and will block the novelty seeking behavior of morphine-withdrawn rats. 4 Injection of prazosin in nucleus accumbens will not affect the morphine-induced psychomotor effect of either naïve rats or morphine-withdrawn rats, nor will it affect the novelty seeking behavior of morphine-withdrawn rats. 5 Injection of clonidine in nucleus accumbens will block morphine-induced psychomotor effect of naïve rats, however, it will not block that of morphine-withdrawn rats, nor will it affect the novelty seeking behavior of morphine-withdrawn rats. These results show: 1 The weakening of the function of alpha1 receptors in medial prefrontal cortex and alpha2 receptors in nucleus accumbens caused by repetitive exposure to morphine is probably the cause of compulsive drug-seeking activity. 2 Blocking alpha1 receptors in medial prefrontal cortex accelerates the loss of interest in natural reward after morphine withdrawal. 3 Blocking alpha2 receptors in medial prefrontal cortex not only restrains drug-seeking motivation, but also blocks the individual’s seeking motivation for novelty stimulus, which suggests that, while selecting medicine for curing addiction, it should be considered to reduce the influence on natural reward as much as possible and to avoid major side-effect.

鸡胚吗啡用药对学习记忆和成瘾易感性的影响及其受体机制

Prenatal morphine exposure affects neural development of fetus by impairing learning and memory, and increasing susceptibility to morphine abuse. Because nervous systems have different developmental characteristics during different developmental stages, administration of morphine at different stages also has different effects on learning, memory, and susceptibility to morphine. Due to the precise developmental processes of neurotransmitter systems in chick embryo’s brain, and unique superiority of chick embryo model, the purpose of the present studies was to explore critical periods correlated to the memory impairment and the increasing susceptibility to morphine, via one-trial passive avoidance and conditioned place preference as behavior models. Then the possible roles of mu and delta opioid receptors as the possible mechanism were analyzed. Experiment 1 showed that injecting low dose of morphine (1 mg/kg) during the period embryonic 5 to 8 significantly impaired the function of learning and memory, worse than any other periods of the same treatment. Experiment 2 showed that injecting low dose of morphine during the period embryonic 17 to 20 significantly increased the susceptibility to morphine in the new-born chicks. The affected chicks acquired the morphine conditioned place preference more quickly, and maintained it much longer. Experiment 3 showed that during E5-8, injecting delta receptor antagonist naltrindole reversed the learning and memory impairment caused by morphine while delta receptor agonist DPDPE impaired learning and partial memory function. On the other hand, mu opioid receptors had little effect. As for E17-20, given naloxonazine can reverse the increases of susceptibility to morphine, and the mu receptor agonist DAGO cause the increases of susceptibility to morphine. Delta receptors have no effect. The above results demonstrated that prenatal morphine expousure at different developmental periods of chick embryo caused different influences on memory and susceptibility to morphine. That is, E5-8 is the critical period correlate to memory impairment; and E17-20 is the critical period correlate to susceptibility to morphine. Delta receptors were critical in learning and memory impairment while mu receptors in susceptibility.

PLCß介导的钙调腺苷酸环化酶在突触可塑性相关基因转录中的作用和FE65对海马依赖学习及LTP的调节

Gene regulation is required for activity-dependent changes in synaptic plasticity and remodeling. The metabotropic glutamate receptors (mGluRs) contribute to different brain functions, including learning/memory, mental disorders, drug addiction, and persistent pain in the CNS. We found that Gp I mGluRs activate PLCß through Gq and then lead to activation of several calcium-dependent signaling pathways, including ERK, which play an important role in gene transcription. These findings support a calcium-dependent role for Gq in release of Calcium and activation of calcium-stimulated adenylyl cyclases I in activity-dependent transcription in response to application of group I metabotropic glutamate receptors agonist and may provide insights into group I mGluRs-dependent synaptic plasticity through MAP kinases signaling. Moreover, the present study investigated the transcription-dependent changes of Arc in response to the activation of group I mGluRs and suggested the central role of ERK1/2 in group I mGluR-mediated Arc transcription. Further, we selected APP-interaction protein FE65 to investigate the mechanism of transcription-related process in synaptic plasticity. FE65 is expressed predominantly in the brain, and interacts with the C-terminal domain of β-amyloid precursor protein (APP). We examined hippocampus-dependent memory and in vivo long-term potentiation (LTP) at the CA1 synapses with the isoform-specific FE65 knock-out (p97FE65-/-) mice. p97FE65 knock-out mice showed impaired short-term memory for both TDPA and CFC when tested 10min after training, which is transcription-independent. Consistently, at the Schaffer collateral-CA1 synapses, p97FE65 knock-out mice showed defective early phase LTP. These results demonstrate novel roles of FE65 in synaptic plasticity, acquisition, and retention for certain forms of memory formation.

情绪应激对大鼠体液免疫功能的影响及其机制

This study was undertaken to investigate the effect of emotional stress on humoral immunoactivity and to examine whether the sympathetic nervous system was involved in the immunomodulation. In the present study, two types of emotional stressors were used. One was footshock apparatus used to cause the rats which were given footshock before, emotional stressed; the other was an empty water bottle used to cause the rats which were trained to drink water at two set times each day, emotional stressed. The effect of emotional stress on the primary immune function (anti-ovallum antibody level and spleen index), the endocrine response (corticosterone level, epinephrine and norepinephrine level), the behavioral changes (freezing, defecation, grooming and attacking behavior) were investigated. The main results were: 1. Two types of emotional stress significantly increased the level of plasma corticosterone, norepinephrine and epinephrine, as well as freezing, defecation and attacking behavior. 2. Two types of emotional stress significantly decreased the level of anti-ovallum antibody. A negative correlation between catecholamine level (epinephrine and norepinephrine) and antibody level or spleen index was found. 3. β-adrenergic receptor antagonist propranolol could reverse the immunomodulation induced by emotional stress. 4. After two types of emotional stress, c-fos expression was observed in the following brain areas or nucleus; arcuate nucleus, anterior commissure nucleus, diffuse part of dorsalmedial nucleus hypothalamus, lateral dorsal nucleus thalamus, medial nucleus amygdala, solitary nucleus, frontal cortex and cingulum. These brain areas and nucleus are involved in the central modulation of the autonomic nervous system. Taken together, these findings demonstrate that emotional stress can suppress humoral immunity and the activation of the sympathetic nervous system is involved in the humoral immunomodulation induced by emotional stress.

Progettazione e sintesi di nuovi antagonisti CB1 a struttura pirazolica come potenziali radiofarmaci per la PET imaging

Cannabinoid receptors are members of the large family of G-protein coupled receptors. Two types of cannabinoid receptor have been discovered: CB1 and CB2. CB1 receptors are localised predominantly in the brain whereas CB2 receptors are more abundant in peripheral nervous system cells. CB1 receptors have been related with a number of disorders, including depression, anxiety, stress, schizophrenia, chronic pain and obesity. For this reason, several cannabinoid ligands were developed as drug candidates. Among these ligands, a prominent position is occupied by SR141716 (Rimonabant), which is a pyrazole derivative with inverse agonist activity discovered by Sanofi-Synthelabo in 1994. This compound was marketed in Europe as an anti-obesity drug, but subsequently withdrawn due to its side-effects. Since the relationship between the CB1 receptors’ functional modification, density and distribution, and the beginning of a pathological state is still not well understood, the development of radio-ligands suitable for in vivo PET (Positron Emission Tomography) functional imaging of CB1 receptors remains an important area of research in medicine and drug development. To date, a few radiotracers have been synthesised and tested in vivo, but most of them afforded unsatisfactory brain imaging results. A handful of radiolabelled CB1 PET ligands have also been submitted to clinical trials in humans. In this PhD Thesis the design, synthesis and characterization of three new classes of potential high-affinity CB1 ligands as candidate PET tracers is described.

Interazioni neurotrasmettitoriali nel meccanismo d’azione della cocaina: ruolo della serotonina e dell’adenosina

The putative 5-HT6 receptor agonist ST1936 has been shown to increase extracellular dopamine (DA) in the n.accumbens (NAc) Shell and in the medial prefrontal cortex (PFCX). These observations suggest that 5-HT6 receptors modulate DA transmission in mesolimbic and mesocortical terminal DA areas. To investigate the behavioral counterpart of this interaction I studied in rats the effect of 5-HT6 receptor blockade on cocaine stimulated overflow of DA in dialysates from the PFCX and from the NAc Shell and on cocaine i.v. selfadministration. Pretreatment with the 5-HT6 antagonist SB271046 reduced cocaine-induced increase of dialysate DA in the NAc Shell but not in the PFCX and impaired i.v. cocaine selfadministration. These suggest that 5-HT6 receptors play a role in cocaine reinforcement via their facilitatore interaction with DA projections to the NAc Shell. This 5-HT/DA interaction might provide the basis for a new pharmacotherapeutic strategy of cocaine addiction. Caffeine is one of the psychoactive substances most widely used as adulterant in illicit drugs, such as cocaine. Animal studies have demonstrated that caffeine is able to potentiate cocaine actions, although the enhancement of the cocaine reinforcing property by caffeine is less reported, and the results depend on the paradigms and experimental protocols used. In the present study I examined the ability of caffeine to enhance the motivational and rewarding properties of cocaine using the intravenous self-administration paradigm in rats. Additionally, the role of caffeine as a primer cue during extinction was evaluated. To this end, we assessed in naïve rats: 1) the ability of the combination of cocaine (0,125 mg/kg/infusion) and caffeine (0,0625 mg/kg/infusion) to maintain self-administration in fixed ratio (FR) and progressive ratio (PR) schedules of reinforcement compared with cocaine and caffeine alone; 2) the effect of caffeine in the maintenance of responding in the animals exposed to the combination of the drugs during cocaine extinction. Cocaine and the combination of cocaine and caffeine were self-administered on a FR and PR schedules of reinforcement, and the responding for the combination of the drugs was higher than cocaine alone. Caffeine was not reliably self-administered, but was able to maintain a drug-seeking behavior in rats previously exposed to cocaine plus caffeine. These findings suggest that the presence of caffeine enhances the reinforcing effects of cocaine and the motivational value of the drug. Our results highlight the role of active adulterants commonly used in illicit street drugs.

Novel roles for the GABAB receptor in anxiety and cocaine addiction

The GABAB receptor has been postulated as a possible drug target in the treatment of anxiety disorders and cocaine addiction. Indeed, a wealth of preclinical data is emerging that has shown that mice lacking functional GABAB receptors display a highly anxious behaviour across a range of behavioural models of anxiety. Additionally, novel compounds that act by altering the allosteric conformation of the GABAB receptor to a more active state; the GABAB receptor positive modulators, have been repeatedly demonstrated to have anxiolytic effects in animals. In addition to being a putative anxiolytic drug target, the GABAB receptor has been identified as a novel target for antiaddictive therapies. Indeed GABAB receptor positive modulators have been demonstrated to have anti-addictive properties across a broad variety of behavioural paradigms. Despite these findings, several gaps in our knowledge of the role played by the GABAB receptor in both anxiety and drug abuse disorder exist. The aim of this thesis was to use preclinical animal models in an effort to further probe the role played by the GABAB receptor in anxiety and addiction. Our studies initially examined the role played by the GABAB receptor in the neurodevelopmental processes underpinning of anxiety. Our studies demonstrated that treating mouse pups in early life with the GABAB receptor agonist baclofen produced an anxious phenotype in adult life, whereas treatment with the GABAB receptor antagonist CGP52432 produced no effects on adult behaviour. Further to this, we examined whether the anxious behaviour induced by early life blockade of the serotonin reuptake transporter was dependant on alterations in GABAB receptor function. Our studies however revealed no effect of early life selective serotonin reuptake inhibitor treatment on adult life baclofen sensitivity. The next issue addressed in this thesis is the characterization of the effects of a GABAB receptor positive modulator and a GABAB receptor antagonist in a behavioural model of conditioned fear behaviour. These novel classes of GABAB receptor ligands have been considerably less well characterized in this facet of preclinical anxiety behaviour than in terms of innate anxiety behaviour. Our study however revealed that the GABAB receptor positive modulator GS39783 and the GABAB receptor antagonist CGP52432 were without effect on the acquisition, expression or extinction of conditioned fear in our model. The next element of this thesis dealt with the characterization of a novel mouse model, the GABAB(2)- S892A mouse. This mouse has been engineered to express a form of the GABAB(2) receptor subunit wherein the function determining serine phosphorylation site cannot be phosphorylated. We initially tested this mouse in terms of its GABAB receptor function in adult life, followed by testing it in a battery of tests of unconditioned and learned anxiety behaviour. We also examined the behavioural and molecular responses of the GABAB(2)-S892A mouse to cocaine. All of our studies appear to show that the GABAB(2)-S892A mouse is indistinguishable from wildtype controls. The final aim of the thesis was to investigate the behavioural and molecular sensitivity of the GABAB(1) subunit isoform null mice, the GABAB(1a) -/- and GABAB(1b) -/- mice to cocaine. Our studies revealed that these mice display differing behavioural responses to cocaine, with the GABAB(1a) -/- mouse displaying a hypersensitivity to the acute locomotor effects of cocaine, while the GABAB(1b) -/- displayed blunted locomotor sensitisation to cocaine.

Immune and stress factors in the pathophysiology of the mdx mouse model of Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy (DMD) is a fatal multi-system neuromuscular disease caused by loss of dystrophin. The loss of dystrophin from membranes of contractile muscle cells and the dysregulation of the DAPC, induces chronic inflammation due to tissue necrosis and eventual replacement with collagen which weakens muscular force and strength. Dystrophin deficiency may cause under-diagnosed features of DMD include mood disorders such as depression and anxiety and dysfunction of the gastrointestinal tract. The first study in the thesis examined mood in the dystrophin-deficient mdx mouse model of DMD and examined the effects of the tri-cyclic antidepressant, amitriptyline on behaviours. Amitriptyline had anti-depressant and anxiolytic effects in the mdx mice possibly through effects on stress factors such as corticotrophin-releasing factor (CRF). This antidepressant also reduced skeletal muscle inflammation and caused a reduction in circulating interleukin (IL)-6 levels. In the second and third studies, we specifically blocked IL-6 signalling and used Urocortin 2, CRFR2 agonist to investigate their potential as therapeutic targets in mdx mice pathophysiology. Isometric and isotonic contractile properties of the diaphragm, were compared in mdx mice treated with anti IL-6 receptor antibodies (anti IL-6R) and/or Urocortin 2. Deficits in force production, work and power detected in mdx mice were improved with treatment. In study three I investigated contractile properties in gastrointestinal smooth muscle. As compared to wild type mice, mdx mice had slower faecal transit times, shorter colons with thickened muscle layers and increased contractile activity in response to recombinant IL-6. Blocking IL-6 signalling resulted in an increase in colon length, normalised faecal output times and a reduction in IL-6-evoked contractile activity. The findings from these studies indicate that for both diaphragm and gastrointestinal function in a dystrophin-deficient model, targeting of IL-6 and CRFR2 signalling has beneficial therapeutic effects.

Polymorphisms in the ACE and ADRB2 genes and risks of aging-associated phenotypes: the case of myocardial infarction.

Multiple functions of the beta2-adrenergic receptor (ADRB2) and angiotensin-converting enzyme (ACE) genes warrant studies of their associations with aging-related phenotypes. We focus on multimarker analyses and analyses of the effects of compound genotypes of two polymorphisms in the ADRB2 gene, rs1042713 and rs1042714, and 11 polymorphisms of the ACE gene, on the risk of such an aging-associated phenotype as myocardial infarction (MI). We used the data from a genotyped sample of the Framingham Heart Study Offspring (FHSO) cohort (n = 1500) followed for about 36 years with six examinations. The ADRB2 rs1042714 (C-->G) polymorphism and two moderately correlated (r(2) = 0.77) ACE polymorphisms, rs4363 (A-->G) and rs12449782 (A-->G), were significantly associated with risks of MI in this aging cohort in multimarker models. Predominantly linked ACE genotypes exhibited opposite effects on MI risks, e.g., the AA (rs12449782) genotype had a detrimental effect, whereas the predominantly linked AA (rs4363) genotype exhibited a protective effect. This trade-off occurs as a result of the opposite effects of rare compound genotypes of the ACE polymorphisms with a single dose of the AG heterozygote. This genetic trade-off is further augmented by the selective modulating effect of the rs1042714 ADRB2 polymorphism. The associations were not altered by adjustment for common MI risk factors. The results suggest that effects of single specific genetic variants of the ADRB2 and ACE genes on MI can be readily altered by gene-gene or/and gene-environmental interactions, especially in large heterogeneous samples. Multimarker genetic analyses should benefit studies of complex aging-associated phenotypes.

Regulation of G protein-coupled receptor signaling by scaffold proteins.

The actions of many hormones and neurotransmitters are mediated through stimulation of G protein-coupled receptors. A primary mechanism by which these receptors exert effects inside the cell is by association with heterotrimeric G proteins, which can activate a wide variety of cellular enzymes and ion channels. G protein-coupled receptors can also interact with a number of cytoplasmic scaffold proteins, which can link the receptors to various signaling intermediates and intracellular effectors. The multicomponent nature of G protein-coupled receptor signaling pathways makes them ideally suited for regulation by scaffold proteins. This review focuses on several specific examples of G protein-coupled receptor-associated scaffolds and the roles they may play in organizing receptor-initiated signaling pathways in the cardiovascular system and other tissues.

Neuropathic pain activates the endogenous kappa opioid system in mouse spinal cord and induces opioid receptor tolerance.

Release of endogenous dynorphin opioids within the spinal cord after partial sciatic nerve ligation (pSNL) is known to contribute to the neuropathic pain processes. Using a phosphoselective antibody [kappa opioid receptor (KOR-P)] able to detect the serine 369 phosphorylated form of the KOR, we determined possible sites of dynorphin action within the spinal cord after pSNL. KOR-P immunoreactivity (IR) was markedly increased in the L4-L5 spinal dorsal horn of wild-type C57BL/6 mice (7-21 d) after lesion, but not in mice pretreated with the KOR antagonist nor-binaltorphimine (norBNI). In addition, knock-out mice lacking prodynorphin, KOR, or G-protein receptor kinase 3 (GRK3) did not show significant increases in KOR-P IR after pSNL. KOR-P IR was colocalized in both GABAergic neurons and GFAP-positive astrocytes in both ipsilateral and contralateral spinal dorsal horn. Consistent with sustained opioid release, KOR knock-out mice developed significantly increased tactile allodynia and thermal hyperalgesia in both the early (first week) and late (third week) interval after lesion. Similarly, mice pretreated with norBNI showed enhanced hyperalgesia and allodynia during the 3 weeks after pSNL. Because sustained activation of opioid receptors might induce tolerance, we measured the antinociceptive effect of the kappa agonist U50,488 using radiant heat applied to the ipsilateral hindpaw, and we found that agonist potency was significantly decreased 7 d after pSNL. In contrast, neither prodynorphin nor GRK3 knock-out mice showed U50,488 tolerance after pSNL. These findings suggest that pSNL induced a sustained release of endogenous prodynorphin-derived opioid peptides that activated an anti-nociceptive KOR system in mouse spinal cord. Thus, endogenous dynorphin had both pronociceptive and antinociceptive actions after nerve injury and induced GRK3-mediated opioid tolerance.

Hybrid transgenic mice reveal in vivo specificity of G protein-coupled receptor kinases in the heart.

G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors, including alpha(1B)-adrenergic receptors (ARs), resulting in desensitization. In vivo analysis of GRK substrate selectivity has been limited. Therefore, we generated hybrid transgenic mice with myocardium-targeted overexpression of 1 of 3 GRKs expressed in the heart (GRK2 [commonly known as the beta-AR kinase 1], GRK3, or GRK5) with concomitant cardiac expression of a constitutively activated mutant (CAM) or wild-type alpha(1B)AR. Transgenic mice with cardiac CAMalpha(1B)AR overexpression had enhanced myocardial alpha(1)AR signaling and elevated heart-to-body weight ratios with ventricular atrial natriuretic factor expression denoting myocardial hypertrophy. Transgenic mouse hearts overexpressing only GRK2, GRK3, or GRK5 had no hypertrophy. In hybrid transgenic mice, enhanced in vivo signaling through CAMalpha(1B)ARs, as measured by myocardial diacylglycerol content, was attenuated by concomitant overexpression of GRK3 but not GRK2 or GRK5. CAMalpha(1B)AR-induced hypertrophy and ventricular atrial natriuretic factor expression were significantly attenuated with either concurrent GRK3 or GRK5 overexpression. Similar GRK selectivity was seen in hybrid transgenic mice with wild-type alpha(1B)AR overexpression concurrently with a GRK. GRK2 overexpression was without effect on any in vivo CAM or wild-type alpha(1B)AR cardiac phenotype, which is in contrast to previously reported in vitro findings. Furthermore, endogenous myocardial alpha(1)AR mitogen-activated protein kinase signaling in single-GRK transgenic mice also exhibited selectivity, as GRK3 and GRK5 desensitized in vivo alpha(1)AR mitogen-activated protein kinase responses that were unaffected by GRK2 overexpression. Thus, these results demonstrate that GRKs differentially interact with alpha(1B)ARs in vivo such that GRK3 desensitizes all alpha(1B)AR signaling, whereas GRK5 has partial effects and, most interestingly, GRK2 has no effect on in vivo alpha(1B)AR signaling in the heart.