P物质增强大鼠鞘内注射D—丝氨酸诱发的热痛过敏

目的：研究脊髓伤害性信息传递中P物质（SP）与N－甲基－D－天冬氨酸（NMDA）受体甘氨酸位点激动剂D－丝氨酸（D-serine)之间的功能联系。方法：在浅麻大鼠,采用行为学方法,测定甩尾反射潜伏期（TFL）并结合鞘内给药途径观察药物作用。结果：鞘内注射D-serine 1000nmol后1．5分钟,TEL明显缩短;在注射D-serine 10nmol前6分钟鞘内施加SP 0.05nmol,明显增强D-serine 10nmol引起的TEL缩短效应;选择性NMDA受体甘氨酸位点拮抗剂7－氯犬尿酸1pmol及非选择性PKC抑制剂H－7 10μmol均可阻断这种增强作用。结论：SP可使D－丝氨酸诱发的热痛过敏明显加强,NMDA受体甘氨酸位点及胞内蛋白激酶系统参与了脊髓SP与NMDA受体的相互作用。     

激动NMDA受体甘氨酸位点可改善皮质酮致海马长时程增强损伤

目的研究N-甲基-D-天冬氨酸(NMDA)受体甘氨酸位点与皮质酮(CORT)致长时程增强(LTP)损伤之间的关系。方法应用雄性BALB/c小鼠,采用皮下注射CORT50 mg·kg~(-1)造模,60 min后进行电生理实验。NMDA受体甘氨酸位点激动剂300 nmol/只D-丝氨酸,300 nmol/只L-丝氨酸,100 nmol/只甘氨酸(侧脑室注射)和100 mg·kg~(-1)D-氨基酸氧化酶抑制剂MPC(腹腔注射)在CORT前30 min注射,1 mg·kg~(-1)NMDA受体甘氨酸位点拮抗剂L-701,324在D-丝氨酸和MPC前30 min腹腔注射。结果与正常小鼠相比,CORT可显著损伤小鼠海马的LTP(P<0.01),D-丝氨酸、L-丝氨酸、甘氨酸和MPC均可显著改善CORT引起的海马LTP损伤(P<0.01),甘氨酸位点拮抗剂L-701,324可阻断D-丝氨酸和MPC对CORT致海马LTP损伤的改善作用(P<0.01)。结论激动NMDA受体甘氨酸位点可改善CORT致海马LTP损伤,提示CORT引起的LTP损伤可能与NMDA受体功能下降相关。     

丝氨酸331是蛋白激酶C诱导血栓素α受体磷酰化和脱敏的主要部位(英文)

目的:检测人类血栓素α受体(TPα)C端丝氨酸/苏氨酸残基的各种两氨酸诱变体作为PKC底物的能力,以确定被PKC磷酸化和脱敏的特异的丝氨酸/苏氨酸残基.方法:为了易于鉴定被磷酰化的细胞内区段,使用甘胱甘肽S-转移酶(GST)-细胞内区段融合蛋白作纯化的PKC底物,然后突变磷酰化蛋白的cDNA,以找出TPα被PKC磷酰化的主要部位,并将有组氨酸尾野生型或诱变型血栓素受体α稳定地转移到人类胚胎肾(HEK)293细胞,以研究该受体的磷酸化和脱敏.结果:仅C-端能充当纯化PKC底物.丝氨酸-331(mP4)被显示强的磷酰化,丝氨酸-324(mP1)则轻微磷酰化,丝氨酸-329(mP3)则微弱磷酰化,而其它丝氨酸/苏氨酸残基没被发现磷酰化.佛被醇-12-肉豆蔻酸酯-13-乙酸盐(PMA)诱导表达野生型TPα的HEK 293细胞磷酰化.然而不能显示触发表达丝氨酸331丙氨酸诱变受体的HEK 293受体磷酸化.用PMA预处理表达野生型受体的HEK 293细胞能抑制I-B0P诱导Ca~(2 )释放,然而用PMA预处理表达诱变受体的细胞不能中止I-BOP抑制Ca~(2 )释放.结论:丝氨酸331是TPαa磷酰化和脱敏的主要和关键部位.     

丝氨酸331是蛋白激酶C诱导血栓素α受体磷酰化

目的：检测人类血栓素α受体（TPα）C端丝氨酸／苏氨酸残基的各种丙氨酸诱变体作为PKC底物的能力,以确定被PKC磷酰化和脱敏的特异的丝氨酸／苏氨酸残基。方法：为了易于鉴定被磷酰化的细胞内区段,使用甘胱甘肽S－转移酶（GST）－细胞内区段融合蛋白作纯作的PKC底物,然后突变磷酰化蛋白的cDNA,以找出TPα被PKC磷酰化的主要部位,并将有组氨酸尾野生型或诱变型血栓素受体α稳定地转移到人类胚胎肾（EK）293细胞,以研究该受体的磷酰化和脱敏。结果：仅C－端能充当纯化PKC底物。丝氨酸－331 （mP4)被显示强的磷酰化,丝氨酸－324（mP1)则轻微磷酰化,丝氨酸－329（mP3)则微弱磷酰化,而其它丝氨酸／苏氨酸残基没被发现磷酰化。佛波醇－12－肉豆蔻酸酯－13－乙酸盐（PMA）诱导表达野生型TPα的HEK 293细胞磷酰化。然而不能显示触发表达丝氨酸331丙氨酸诱变受体的HEK 293受体磷酰化。用PMA预处理表达野生型受体的HEK 293细胞能抑制I－BOP诱导Ca^2 释放。然而用PMA预处理表达诱变受体的细胞不能中止I－BOP抑制Ca^2 释放。结论：丝氨酸331是TPα磷酰化和脱敏的主要和关键部位。     

G蛋白偶联受体激酶活性调控及其在恶性肿瘤中的作用

G蛋白偶联受体(GPCR),是一类重要的细胞表面受体。G蛋白偶联受体激酶(GRK)属于丝氨酸/苏氨酸蛋白激酶家族,其亚型广泛存在与各种组织,能够特异性地使活化的GPCR发生磷酸化及脱敏,从而终止GPCR介导的信号转导通路。新的研究还发现,GRK不仅作用于GPCR,也可以通过使非GPCR磷酸化或通过非磷酸化作用参与信号转导。GRK不仅能够调节GPCR和非GPCR,其自身活性也可受到多种因素的调节。本文结合GRK的多种功能作用和GRK活性调控,对GRK在脑、内分泌、生殖系统、消化系统及黑色素肿瘤中的作用做简要综述。     

多巴胺D1受体激动剂对中枢神经系统疾病的治疗作用及其靶点通路研究进展

DA受体根据其不同信号转导机制分为：D1样受体和D2样受体。D1样受体与Gs蛋白偶联,受刺激后能提高腺苷酸环化酶（AC）活性,升高细胞内环磷腺苷（cAMP）水平,这类受体包括D1和D52种亚型;D2样受体与Gi蛋白偶联,受刺激后能降低AC的活性,降低细胞内cAMP水平,包括D2、D3、D4受体。     

肾脏多巴胺系统

肾内有一个完整的多巴胺系统的所有成份。多巴胺是控制钠排泌的一个旁分泌物质。多巴胺受体叶分为D1-样(D1和D5)受体(兴奋腺苷酰环化酶)和D2-样(D2、D3和D4)受体(抑制腺苷酰环化酶)。所有5个受体亚型均在肾脏表达,多巴胺是在近端小管细胞处神经元外合成,从这些细胞大量输至小管腔内,并和D1-样受体相互作用以抑制Na　-     

β肾上腺素受体激动对人脐静脉内皮细胞一氧化氮合酶磷酸化的影响

目的：观察血管内皮β肾上腺素受体激动对内皮一氧化氮合酶（eNOS）蛋白表达及磷酸化水平的影响,阐明β受体激动后eNOS调节的分子机制。方法：异丙肾上腺素（ISO）1μmol/L与培养的人脐静脉内皮细胞（HUVEC）孵育30min后,裂解细胞并用免疫沉淀法分离eNOS蛋白,运用同位素两步色谱法（L-[^3H]精氨酸转化法）检测eNOS活性;蛋白免疫印迹增强化学发光法检测eNOS表达水平和eNOS蛋白丝氨酸磷酸化水平,并观察高选择性β1或β2肾上腺素受体阻断剂对上述作用的影响。结果：ISO与内皮细胞孵育30min引起eNOS活性增高;不影响eNOS蛋白表达,但丝氨酸磷酸化水平明显增加;选择性β2受体阻断剂ICI 118551可完全阻断ISO的作用,而选择性β1受体阻断剂CGP 20712A无影响。结论：ISO通过提高eNOS丝氨酸磷酸化水平增加eNOS活性,这种作用是通过β2肾上腺素受体介导的。     

多巴胺受体的分子生物学及相关胃肠疾病研究

随着对脑-肠肽作用认识的不断深入,多巴胺(DA)及多巴胺受体(DR)系统已成为研究热点。根据DR与细胞内信号转导藕联的关系,将DA受体分为D1亚型受体和D2亚型受体。1980年,用放射免疫法,根据配基与DR的不同作用特征,提出有4种受体结合位点,而将DA受体分为D1、D2、D3、D4四种亚型。1     

NKG2D 配体在肿瘤免疫中作用新进展

NKG2D系自然杀伤性细胞(NK)和CD8+T淋巴细胞表面的激活受体。NKG2D配体分子可被细胞在应急状态和发生感染的情况下诱导表达。在肿瘤免疫监视和肿瘤免疫治疗,进行了大量的研究。NKG2D与其配体分子的结合能够激活NK细胞,并为T细胞提供协同刺激,继而激活机体体液免疫和细胞免疫机制,诱导机体的免疫杀伤作用。活化的NK细胞通过IL-15加强来自供体或受体的抗原递呈细胞(APCs)对T细胞的反应,导致机体对移植物的排斥反应。从另一面来说,经NKG2D受体激活NK细胞后,T调节细胞的数量可减小,这种调节T细胞免疫的能力下降。增加NKG2D与其配体分子的结合,可为寻找新的肿瘤免疫治疗提供研究思路。加强对这方面的研究为提高肿瘤存活期具有重要的临床价值。     

D-serine treatment induces oxidative stress in rat brain

D-serine plays a significant role in neuronal activity, including learning, memory, neuronal migration at developmental stages, and cell-death signaling. It has been also suggested that D-serine can potantiate the neurotoxicity induced by N-methyl-D-aspartate (NMDA) receptor activation due to its coagonist function. However, little is known about the role of D-serine in oxidative stress mechanisms. The aim of this study was to determine the possible neurotoxic or oxidative effects of the dose- (50-200 mg/kg) and time-dependent (2 or 6 hours) D-serine administration on lipid, protein, DNA, mitochondrial integrity (i.e., function), levels of antioxidant enzyme activities (e.g., catalase, glutathione peroxidase, and superoxide dismutase), and glutathione (GSH) in the rat brain. Our results showed that D-serine significantly increases the levels of lipid peroxidation, protein carbonyls, and DNA damage. In addition, D-serine treatment changes cellular antioxidant status due to the decreased levels of antioxidant enzymes, GSH, and mitochondrial function. Therefore, it is concluded that the regulation of D-serine levels in the brain may be an important target for the development of neuroprotective strategies against neurodegenerative processes where excitotoxicity is involved.     

Effect of D-serine on AQP4 knockout-induced changes in behavior alteration to cocaine

OBJECTIVE AQP4 is widely expressed in brain astrocytes,and its function is mainly to maintain the steady-state of brain water.D-serine as an important neurotransmitter secreted by glial cells is an endogenous co-agonist of NMDA receptor,and is essential for the induction of long-term potentiation.The present study is to explore whether D-serine is mediated the change of synaptic plasticity and cocaine-induced addictive behavior induced by AQP4 knockout.METHODS The effects of D-serine on NAc LTD and behavior of AQP4 knockout mice were investigated by electrophysiological recording and behavioral tests.RESULTS 10 μmol·L-1 D-serine significantly reversed NMDA receptor-dependent LTD in AQP4 KO mice NAc slices.D-serine concentration-dependently antagonized the effects of AQP4 knockout on cocaine addiction behavior.500 mg·kg-1 D-serine significantly increased cocaine-induced locomotor activity in AQP4 KO mice.D-serine(500 mg·kg-1) significantly restored cocaine-induced conditioned place preferences in AQP4 KO mice.CONCLUSION AQP4 knockout changed the behavior of cocaine addiction through affecting the release of D-serine in glial cells.     

Acute D-serine treatment produces antidepressant-like effects in rodents

Research suggests that dysfunctional glutamatergic signalling may contribute to depression, a debilitating mood disorder affecting millions of individuals worldwide. Ketamine, a N-methyl-D-aspartate (NMDA) receptor antagonist, exerts rapid antidepressant effects in approximately 70% of patients. Glutamate evokes the release of D-serine from astrocytes and neurons, which then acts as a co-agonist and binds at the glycine site on the NR1 subunit of NMDA receptors. Several studies have implicated glial deficits as one of the underlying facets of the neurobiology of depression. The present study tested the hypothesis that D-serine modulates behaviours related to depression. The behavioural effects of a single, acute D-serine administration were examined in several rodent tests of antidepressant-like effects, including the forced swim test (FST), the female urine sniffing test (FUST) following serotonin depletion, and the learned helplessness (LH) paradigm. D-serine significantly reduced immobility in the FST without affecting general motor function. Both D-serine and ketamine significantly rescued sexual reward-seeking deficits caused by serotonin depletion in the FUST. Finally, D-serine reversed LH behaviour, as measured by escape latency, number of escapes, and percentage of mice developing LH. Mice lacking NR1 expression in forebrain excitatory neurons exhibited a depression-like phenotype in the same behavioural tests, and did not respond to D-serine treatment. These findings suggest that D-serine produces antidepressant-like effects and support the notion of complex glutamatergic dysfunction in depression. It is unclear whether D-serine has a convergent influence on downstream synaptic plasticity cascades that may yield a similar therapeutic profile to NMDA antagonists like ketamine.     

Glycine and D-serine act as positive modulators of signal transduction at N-methyl-D-aspartate sensitive glutamate receptors in cultured cerebellar granule cells

In cultures of rat neonatal cerebellar granule cells the signal transduction at ionotropic NMDA-sensitive glutamate receptors (GC1) was measured as an increase of influx of 45Ca2+. This transmitter-mediated influx of Ca2+ was enhanced by glycine and D-serine in a dose-dependent manner. D-Alanine was less active than glycine and D-serine, while L-alanine and L-serine were inactive. These amino acids failed to activate basal influx of Ca2+. Activation of calcium influx at GC2 receptors by kainate was unchanged by the amino acids mentioned above. Glycine and D-serine increased the potency but failed to change the efficacy of GC1 agonists. This action was not changed by strychnine. The enhancement of aspartate signal transduction by glycine and D-serine was inhibited by the noncompetitive GC1 receptor antagonist, phencyclidine, but was even more evident in presence of Mg2+ ions. Hence, glycine and D-serine may function as positive allosteric modulators of signal transduction at NMDA-sensitive (GC1) glutamate receptors.     

D-丝氨酸对N-甲基-D-天冬氨酸诱导大鼠海马cGMP释放作用的影响

The microdialysis technique was utilized to study the effects of exogenous D-serine on the cGMP level in the perfusate of rat hippocampus. Addition of N-methyl-D-aspartate (NMDA) 250 μmol·L^-1 to the perfusate resulted in a striking increase of cGMP in perfusate. The cGMP response elicited by NMDA was prevented by 7-chlorokynurenic acid (7-CKA), a selective antagonist at the glycine site of the NMDA receptor. D-serine completely reversed the antagonism by 7-CKA of the NMDA-evoked cGMP response. The NMDA-evoked cGMP response was enhanced by D-serine. D-serine alone was inactive. The data show that D-serine, administered to freely-moving rats, behaves as a selective agonist at the glycine site of hippocampus NMDA receptor. Furthermore, these results suggest that the glycine modulatory site may not be fully saturated in vivo, and thus can serve to regulate NMDA receptor function.     

Effect of 7-chloro kynurenic acid on glycine modulation of the N-methyl-D-aspartate response in guinea-pig myenteric plexus

The glycine modulation of the N-methyl-D-aspartate (NMDA) response in guinea-pig myenteric plexus was investigated by using D-serine and 7-chloro kynurenic acid as a glycine agonist and antagonist, respectively. D-serine caused a concentration-dependent enhancement of the NMDA response, an effect which was competitively inhibited by 7-chloro kynurenic acid (pA2 = 6.0). In addition, 7-chloro kynurenic acid induced a concentration-dependent, non-competitive inhibition of the NMDA response per se, even in the absence of added D-serine. This inhibition was fully reversed by exogenous D-serine, suggesting that this effect was also due to the occupancy of the glycine site. These results emphasize the usefulness of the guinea-pig myenteric plexus for studying the function of the NMDA receptor complex.     

D-型丝氨酸、丝氨酸消旋酶及D-型氨基酸氧化酶在小鼠出生后个体发育中的分布变化及相互关系

目的：探讨小鼠个体发育过程中皮层、纹状体、小脑、心脏、肺、肝脏、脾脏、肾脏和骨骼肌中D-型丝氨酸,丝氨酸消旋酶(SR)及D-型氨基酸氧化酶(DAO)的分布特征及相互关系。方法：RT-PCR法检测SR和DAO mRNA水平;Western blot检测SR蛋白水平;比色法检测DAO酶活力;高效液相法检测游离D-型丝氨酸含量．结果：小鼠出生后早期个体发育过程中皮层、纹状体和小脑内游离D-型丝氨酸含量急剧增加并伴有SR平行增加．在检测组织中,仅小脑和肾脏可检测到DAO酶活力,且随着小脑和肾脏内DAO酶活力的增加,D-型丝氨酸含量急剧下降,并在出生后第三周末降至成年微量水平,但此时DAO酶活力却仍不断增加．结论：哺乳动物体内D-型丝氨酸主要由SR催化生成,但在皮层和纹状体等D-型丝氨酸浓度很高,但缺乏DAO的组织中D-型丝氨酸的降解尚有其它机制参与．小脑和肾脏中的DA0除参与D-型氨基酸代谢外,可能还具有其它的生理功能。     

Effects of non-steroidal antiinflammatory drugs on D-serine-induced oxidative stress in vitro

Inflammation is deleterious for organs with reduced capacity of regeneration, such as the brain. Recently, studies have focused on investigating the therapeutic effects of nonsteroidal anti-inflammatory drugs (NSAIDs) in Alzheimer's disease, Parkinson's disease, Huntington's disease, and multiple sclerosis. Excitotoxicity is the pathological process when receptors for the excitatory neurotransmitter glutamate, such as the N-methyl-D-aspartate (NMDA), receptors are overactivated. This process may be involved in neurodegenerative diseases. D-serine is one of the coagonist of NMDA receptors, and increased levels of D-serine are associated with excitotoxicity. In our study, the potential neuroprotective effects of mefenamic acid, acetaminophen, and naproxen sodium were investigated against D-serine-induced oxidative stress in the rat brain in vitro. To show their potential neuroprotective properties, NSAIDs were incubated with D-serine and reactive oxygen species (ROS), malondialdehyde, and protein carbonyl content of the brain after different treatments were measured. Our results demostrate that NSAIDs used in the present study significantly reduced ROS production, lipid peroxidation, and protein oxidation against D-serine treatment.     

Effect of aminooxyacetic acid on extracellular level of D-serine in rat striatum: an in vivo microdialysis study

To elucidate the effect of an inhibitor of pyridoxal phosphate-dependent enzymes, aminooxyacetic acid, on the activity of serine racemase in vivo, we have investigated the effect of aminooxyacetic acid on the extracellular concentration of D-serine in the rat striatum using an in vivo microdialysis technique. The intrastriatal perfusion of aminooxyacetic acid caused a significant decline in the extracellular concentration of D-serine. These data, together with the fact that serine racemase is a pyridoxal phosphate-dependent enzyme, suggest that the aminooxyacetic acid-induced reduction of the extracellular D-serine may be at least in part due to the drug's ability to inhibit serine racemase.     

Sodium benzoate attenuates D-serine induced nephrotoxicity in the rat

D-Serine causes selective necrosis to the straight portion of the rat renal proximal tubules. The onset is rapid, occurring within 3-4 h and accompanied by proteinuria, glucosuria and aminoaciduria. The metabolism of D-serine by D-amino acid oxidase (D-AAO) may be involved in the mechanism of toxicity. D-AAO is localized within the peroxisomes of renal tubular epithelial cells, which is also the location of D-serine reabsorption. To address the role of D-AAO in D-serine-induced nephrotoxicity, we have examined the effect of sodium benzoate (SB) on the renal injury. SB has been shown to be a potent, competitive inhibitor of kidney D-AAO in vitro. Male Alderley Park rats were exposed to D-serine (500 mg/kg i.p.) 1 h after exposure to SB (125, 250, 500 or 750 mg/kg i.p.). Urine was collected for 0-6 h, then terminal plasma samples and kidneys were taken at 6.5 h. A second group of animals was given SB (500 mg/kg) followed by D-serine (500 mg/kg i.p.; 1 h later) and urine was collected after 0-6, 6-24 and 24-48 h. Terminal plasma samples and kidneys were taken at 48 h. 1H NMR spectroscopic analysis of urine, combined with principal component analysis, demonstrated that SB was able to prevent D-serine-induced perturbations to the urinary profile in a dose dependent manner. This was confirmed by measurement of plasma creatinine and urinary glucose and protein and histopathological examination of the kidneys. Assessment 48 h after D-serine administration revealed that nephrotoxicity was observed in animals pre-treated with SB (500 mg/kg) although the extent of injury was less pronounced than following D-serine alone. These results demonstrate that whilst prior exposure to SB prevents the initial onset of D-serine-induced nephrotoxicity, renal injury is still apparent at later time points. D-AAO activity in the kidney was decreased by 50% 1 h after dosing with SB suggesting that inhibition of this enzyme may be responsible for the observed protection.