Differential responses of phosphorylated mitogen-activated protein kinase and phosphorylated cyclic-AMP response element-binding protein immunoreactivity in the rat brain to sub-convulsive pentylenetetrazol

The possible advantage of using multiple phospho-specific antibodies to study changes in brain activity was assessed. For this purpose, rats were injected intraperitoneally with either a control treatment or 15 mg/kg pentylenetetrazol. The sub-convulsive dose of pentylenetetrazol did not induce marked behavioural effects. Ten minutes after treatment, the rats were perfused and the brains were dissected. Adjacent brain sections were immunohistochemically stained for phosphorylated cyclic-AMP response element-binding protein and phosphorylated mitogen-activated protein kinase. Opposite effects of pentylenetetrazol treatment were observed on the immunoreactivity of these two antibodies within the hypothalamic paraventricular nucleus, the supraoptic nucleus and the arcuate nucleus. In these regions, pentylenetetrazol treatment increased phosphorylated mitogen-activated protein kinase immunoreactivity, but decreased phosphorylated cyclic-AMP response element-binding protein immunoreactivity.These findings show that changes in the activity of a brain nucleus can be accompanied by differential changes in the activity of two signal transduction pathways, which can be detected immunohistochemically. Therefore, the use of multiple phospho-specific antibodies may enhance our potential to monitor changes in brain activity.     

Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway leading to cyclic AMP response element-binding protein phosphorylation is required for the long-term facilitation process of aversive olfactory learning in young rats

The mitogen-activated protein kinase/extracellular-signal regulated kinase (MAPK/ERK) cascade is an important contributor to synaptic plasticity that underlies learning and memory. ERK activation by the MAPK/ERK kinase (MEK) leading to cyclic-AMP response element binding protein (CREB) phosphorylation is implicated in the formation of long-term memory. We have demonstrated that CREB phosphorylation in the olfactory bulb (OB) is important for aversive olfactory learning in young rats, yet whether MAPK/ERK functions as an upstream regulator are necessary for this olfactory learning remains to be determined. Therefore, we addressed this issue using behavioral and Western blot analyses. The MEK inhibitor PD98059 was continuously infused into the OB of postnatal day 11 rat pups during a 30-min training session regarding the pairing of citral odor and foot shock. On the following day, the time spent in the part of the apparatus where the odor was present was measured as an index of odor aversion. PD98059 impaired olfactory learning in a dose-dependent manner without affecting memory retention 1 h after training. We further tested whether odor-shock training leads to MAPK/ERK activation in the OB and defines the time course of the activation. Phosphorylated ERKs (P-ERKs) 1 and 2 were significantly increased for 60 min after the training without changes in total ERKs 1 and 2. By contrast, intrabulbar infusion of PD98059 during the training significantly reduced P-ERKs 1 and 2 as well as phosphorylated CREB without any effects on the total ERKs or CREB. Taken together with the previous findings, these results indicate that the MAPK/ERK-CREB pathway is required for the long-term, but not the short-term, facilitation process of aversive olfactory learning in young rats.     

gamma-Hydroxybutyrate induces cyclic AMP-responsive element-binding protein phosphorylation in mouse hippocampus: an involvement of GABA(B) receptors and cAMP-dependent protein kinase activation

gamma-Hydroxybutyrate is a widely used recreational drug. Its abuse has been associated with cognitive impairments and development of tolerance and dependence. However, the neural mechanisms underlying these effects remain unclear. In the present study we investigated the possible cellular signaling mechanisms that might mediate gamma-hydroxybutyrate's action. Acute administration of gamma-hydroxybutyrate (500 mg/kg, i.p.) was found to cause a rapid and long-lasting increase in the phosphorylation level of the cAMP-responsive element-binding protein in mouse (C57/BL6) hippocampus. Pretreatment with the specific GABA(B) receptor antagonist [3-[1-(R)-[(3-cyclohexylmethyl)hydroxyphosphinyl]-2-(S)-hydroxy-propyl]amino]ethyl]-benzoic acid (20 mg/kg, i.p.) prevented the action of gamma-hydroxybutyrate, confirming a GABA(B) receptor-mediated mechanism. In addition, acute gamma-hydroxybutyrate administration induced a significant increase in cytosolic cAMP-dependent protein kinase activity in the hippocampus, and pretreatment with the cAMP-dependent protein kinase inhibitor H-89 could prevent the effect of gamma-hydroxybutyrate on cAMP-responsive element-binding protein phosphorylation, indicating a direct involvement of cAMP-dependent protein kinase in gamma-hydroxybutyrate-induced cAMP-responsive element-binding protein phosphorylation. On the other hand, the increased expression of phosphorylated cAMP-responsive element-binding protein was not observed in the hippocampus of mice subjected to repeated gamma-hydroxybutyrate exposure, suggesting the development of a gamma-hydroxybutyrate-induced desensitization of the signaling pathway leading to cAMP-responsive element-binding protein activation. Since cAMP-responsive element-binding protein activation has been implicated in a variety of neural plasticities, our findings may have revealed a new mechanism underlying gamma-hydroxybutyrate-induced neuroadaptations.     

Cyclic AMP receptor protein and cyclic AMP-dependent protein kinase activity in rabbit peritoneal neutrophils

The cAMP receptor protein and cAMP-dependent protein kinase activity in rabbit peritoneal neutrophils have been identified. The cAMP receptor protein in either the plasma membrane or cytosol fractions, identified by photoaffinity labeling with 8-N3-[32P]cAMP, has an apparent molecular weight of 54,000. The cytosol and membrane receptor proteins have apparent dissociation constants for 8-N3-[32P]cAMP of 0.20 microM and 0.06 microM, respectively. The molecular weight and dissociation constant for 8-N3-[32P]cAMP of this cAMP receptor protein are similar to what has been known for RII, the regulatory subunit of the type II cAMP-dependent protein kinase. Unlike the human neutrophils, no evidence of RI activity was detected. cAMP-dependent protein kinase activity was identified by using histone as a substrate. Subcellular fractionation studies showed that the cAMP receptor protein and the cAMP-dependent protein kinase activity are most enriched in the cytosol fraction.     

磷酸化环磷酸腺苷反应元件结合蛋白在小鼠海马发育中的表达

目的:观察胚龄(E)18 d以及生后日龄(P)为1、3、7、14、21 d和28 d的小鼠海马磷酸化的cAMP反应元件结合蛋白(pCREB)的分布,从而为深入研究海马发育机制提供基础实验依据。方法:取E 18 d、P 1 d、P 3 d、P 7 d、P 14 d、P 21 d和P 28 d的小鼠胎鼠和仔鼠海马组织。利用免疫组织化学、免疫印迹结合图像分析技术对pCREB的表达变化进行系统的定性和定量分析。结果:免疫组织化学检测显示,pCREB主要表达在海马阿蒙角锥体细胞层和齿状回颗粒细胞层神经元的细胞核中。E 18 d,海马pCREB阳性细胞稀疏,染色浅,光密度低;E 18 d~P 7 d,pCREB阳性细胞逐渐密集,染色逐渐加深,光密度逐渐增高,P 7 d达高峰。P 14 d~P 21 d,pCREB阳性细胞密集程度逐渐降低,染色逐渐变浅,光密度逐渐降低;P 28 d表达与P 21 d相近,趋于稳定。免疫印迹检测显示,E 18 d~P 7 d,pCREB表达逐渐增高,P 7 d达高峰;P 14 d~P 21 d,表达逐渐降低,P 28 d表达稳定。结论:pCREB与小鼠海马发育有关,pCREB可能参与了小鼠海马发育中的细胞增殖。     

Neonatal handling and the maternal odor preference in rat pups: Involvement of monoamines and cyclic AMP response element-binding protein pathway in the olfactory bulb

Early-life environmental events, such as the handling procedure, can induce long-lasting alterations upon several behavioral and neuroendocrine systems. However, the changes within the pups that could be causally related to the effects in adulthood are still poorly understood. In the present study, we analyzed the effects of neonatal handling on behavioral (maternal odor preference) and biochemical (cyclic AMP response element-binding protein (CREB) phosphorylation, noradrenaline (NA), and serotonin (5-HT) levels in the olfactory bulb (OB)) parameters in 7-day-old male and female rat pups. Repeated handling (RH) abolished preference for the maternal odor in female pups compared with nonhandled (NH) and the single-handled (SH) ones, while in RH males the preference was not different than NH and SH groups. In both male and female pups, RH decreased NA activity in the OB, but 5-HT activity increased only in males. Since preference for the maternal odor involves the synergic action of NA and 5-HT in the OB, the maintenance of the behavior in RH males could be related to the increased 5-HT activity, in spite of reduction in the NA activity in the OB. RH did not alter CREB phosphorylation in the OB of both male and females compared with NH pups. The repeated handling procedure can affect the behavior of rat pups in response to the maternal odor and biochemical parameters related to the olfactory learning mechanism. Sex differences were already detected in 7-day-old pups. Although the responsiveness of the hypothalamic–pituitary–adrenal axis to stressors is reduced in the neonatal period, environmental interventions may impact behavioral and biochemical mechanisms relevant to the animal at that early age.     

Involvement of cAMP response element-binding protein activation in salivary secretion.

OBJECTIVE: Saliva secretion is mediated by cAMP and the calcium signaling pathway in salivary acinar cells. The PKA signaling pathway plays an important role in protein secretion through the activation of cAMP, in fluid secretion through the elevation of intracellular calcium and in the activation of cAMP response element-binding protein (CREB), which is involved in these signaling cascades. In this study, we investigated whether the activation of CREB plays a part in the salivary secretion in mice. METHODS: We examined CREB activation by assessing phosphorylation at the serine-133 position using Western blotting. RESULTS: Carbachol (a muscarinic acetylcholine agonist) and isoproterenol (a beta-adrenergic agonist) markedly activated CREB in parotid acinar cells. Carbachol and isoproterenol-induced CREB phosphorylation was blocked by atropine (a muscarinic acetylcholine antagonist) and propranolol (a beta-adrenergic antagonist), respectively. The PKA inhibitor H89 inhibited CREB activation, but the PLC inhibitor U73122 did not. Moreover, carbachol- and isoproterenol-stimulated amylase secretion from parotid acinar cells was inhibited by H89 and adenoviral dominant-negative CREB. CONCLUSION: These results indicate that the muscarinic and beta-adrenergic activation of CREB was mediated through the PKA pathway and that CREB is involved in protein secretion from parotid acinar cells.     

Nicotine-induced phosphorylation of phosphorylated cyclic AMP response element-binding protein (pCREB) in hippocampal neurons is potentiated by agrin

The scope of this study was to test whether increased levels of the extracellular matrix molecule (ECM) agrin might enhance nicotine effects on those molecular mechanisms that initiate neuroadaptative processes in the hippocampus, a key brain area for learning and memory. We studied the effects of repetitive applications of neuronal agrin to primary hippocampal cell culture on nicotine-induced phosphorylated cyclic AMP response element-binding protein (pCREB) expression, a marker of neuroadaptation, by using immunofluorescence-based assessment of pCREB-positive neurons. We also tested agrin effects on nicotine-induced expression of a marker of metabolic activation, the immediate early gene c-fos. Agrin was shown to significantly enhance nicotine-induced pCREB, but not c-fos, expression. By using Western blotting analysis, cumulative agrin has been shown to increase nicotine-induced pCREB phosphorylation. These analyses, however, showed that inhibition of the CaMKII pathway blocked general pCREB phosphorylation, whereas inhibition of the MAPK pathway potentiated the synergistic effect of cumulative agrin and nicotine. These findings suggest that increasing the concentration of an ECM molecule, i.e. agrin, may enhance nicotine effects on pCREB and that both MAPK and CaMKII signalling may play a regulatory role.     

The midbrain periaqueductal gray and fear extinction: opioid receptor subtype and roles of cyclic AMP, protein kinase A, and mitogen-activated protein kinase

Four experiments studied the opioid receptor subtype and signal transduction mechanisms mediating fear extinction in the ventrolateral quadrant of the midbrain periaqueductal gray (vlPAG). Microinjection of a mu- but not a delta- or kappa-opioid receptor antagonist into the vlPAG retarded extinction. Extinction was also dose-dependently retarded by vlPAG infusions of a cyclic AMP (cAMP) analog but was unaffected by infusions of a protein kinase A activator or a mitogen-activated protein kinase inhibitor across wide dose ranges. The results show that fear extinction occurs via activation of vlPAG mu-opioid receptors and involves reductions in cAMP. These mechanisms are different from the cellular mechanisms for extinction in the amygdala and from the known cellular mechanisms for opioid analgesia in the vlPAG.     

Perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin suppresses contextual fear conditioning-accompanied activation of cyclic AMP response element-binding protein in the hippocampal CA1 region of male rats

We investigated the effect of in utero and lactational exposures to dioxin on adult offspring with contextual fear conditioning, a sex- and hippocampus-dependent learning paradigm; and we measured the conditioning-accompanied activation of cyclic AMP response element-binding protein (CREB) in the hippocampal CA1 region. Pregnant rats were treated with a low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on gestation day 15. TCDD treatment decreased freezing time in conditioning tests of adult male offspring but not of female offspring. A similar, male-specific decrease was observed in the percentage of phosphorylated CREB-immunoreactive neurons in the CA1 region following conditioning in TCDD-treated rats. These results suggest that perinatal TCDD exposure impairs hippocampus-dependent learning in male offspring by suppressing CREB activation.     

Chronic food restriction increases D-1 dopamine receptor agonist-induced phosphorylation of extracellular signal-regulated kinase 1/2 and cyclic AMP response element-binding protein in caudate-putamen and nucleus accumbens

Results of behavioral and c-fos immunohistochemical studies have suggested that chronic food restriction and maintenance of animals at 75-80% of free-feeding body weight may increase d-1 dopamine (DA) receptor function. The purpose of the present study was to determine whether D-1 DA receptor binding and/or mitogen-activated protein kinase (MAPK) signaling in caudate-putamen (CPu) and nucleus accumbens (NAc) are increased in food-restricted subjects. In the first experiment, saturation binding of the D-1 DA receptor antagonist [3H]SCH-23390 indicated no difference between food-restricted and ad libitum fed rats with regard to density or affinity of d-1 binding sites in CPu or NAc. In the second experiment, activation of extracellular signal-regulated kinases (ERK1/2) and cyclic AMP response element-binding protein (CREB) by i.c.v. injection of the D-1 DA receptor agonist SKF-82958 (20 microg) were markedly greater in food-restricted than ad libitum fed rats. Given a prior finding that SKF-82958 does not differentially stimulate adenylyl cyclase in CPu or NAc of food-restricted versus ad libitum fed subjects, the present results suggest that increased D-1 DA receptor-mediated ERK1/2 MAP kinase signaling may mediate the enhanced downstream activation of CREB, c-fos, and behavioral responses in food-restricted subjects. It is of interest that food restriction also increased the activation of c-Jun N-terminal protein kinase/stress-activated protein kinase, but this effect was no greater in rats injected with SKF-82958 than in those injected with saline vehicle. This represents additional evidence of increased striatal cell signaling in food-restricted subjects, presumably in response to the i.c.v. injection procedure, although the underlying receptor mechanisms remain to be determined. There were no differences between feeding groups in protein levels of the major phosphatases, MKP-2 and PP1. The upregulation of striatal MAP kinase signaling in food-restricted animals may adaptively serve to facilitate associative learning but, at the same time, increase vulnerability to the rewarding and addictive properties of abused drugs.     

Down-regulation of p38 mitogen-activated protein kinase activation and proinflammatory cytokine production by mitogen-activated protein kinase inhibitors in inflammatory bowel disease

Crohn's disease and ulcerative colitis are inflammatory bowel diseases (IBD) characterized by chronic relapsing mucosal inflammation. Tumour necrosis factor (TNF)‐α, a known agonist of the mitogen‐activated protein kinase (MAPK) pathway, is a key cytokine in this process. We aimed first to determine whether p38 MAPK is activated in IBD inflamed mucosa, and then studied the effect of four different p38α inhibitory compounds on MAPK phosphorylation and secretion of proinflammatory cytokines by IBD lamina propria mononuclear cells (LPMCs) and organ culture biopsies. In vivo phospho‐p38α and p38α expression was evaluated by immunoblotting on intestinal biopsies from inflamed areas of patients affected by Crohn's disease and ulcerative colitis, and from normal mucosa of sex‐ and age‐matched control subjects. Both mucosal biopsies and isolated LPMCs were incubated with four different p38α selective inhibitory drugs. TNF‐α, interleukin (IL)‐1β and IL‐6 were measured in the organ and cell culture supernatants by enzyme‐linked immunosorbent assay. We found higher levels of phospho‐p38α in the inflamed mucosa of IBD patients in comparison to controls. All the p38α inhibitory drugs inhibited p38α phosphorylation and secretion of TNF‐α, IL‐1β and IL‐6 from IBD LPMCs and biopsies. Activated p38α MAPK is up‐regulated in the inflamed mucosa of patients with IBD. Additionally, all the p38α selective inhibitory drugs significantly down‐regulated the activation of the MAPK pathway and the secretion of proinflammatory cytokines.     

Adenosine-mediated activation of Akt/protein kinase B in the rat hippocampus in vitro and in vivo

Adenosine is considered an endogenous neuroprotective metabolite that through activation of the A(1) receptor results in reduction of neuronal damage following cerebral ischemia. Protein kinase B, also known as Akt/PKB, is part of an endogenous pathway that exerts effective neuroprotection from both necrotic and apoptotic cell death. Using a rat model of unilateral common carotid artery occlusion coupled with hypoxia, and using in vitro rat hippocampal slices, we examined the ability of adenosine to directly activate Akt/PKB. Western blot analysis revealed that levels of phosphorylated Akt/PKB were elevated in vivo under ischemic conditions in an adenosine A(1)-dependent manner and elevated in hippocampal slices treated with an adenosine A(1) agonist. We conclude from these studies that the activation of an adenosine A(1) receptor-mediated signal transduction pathway, either by endogenous adenosine (in vivo) or by an adenosine A(1) agonist (in vitro), results in the activation of the neurotrophic kinase Akt/PKB.     

Cyclic AMP elicits biphasic current whose activation is mediated through protein phosphorylation in snail neurons.

The involvement of protein phosphorylation in cAMP-induced transmembrane current was tested electrophysiologically and pharmacologically in identified neurons of the Japanese land snail, Euhadra peliomphala. Intracellular injection of cAMP elicited a biphasic transmembrane current (cAMP current) which consisted of inward and outward components. The inward component was blocked with Na(+)-free, Ca2(+)-free saline and the outward component abolished by either application of tetraethylammonium or a long-lasting exposure to caffeine in Ca2(+)-free saline. The cAMP current was completely suppressed by the protein kinase inhibitors, protein kinase inhibitor isolated from rabbit muscle or isoquinoline sulfonamide (H-8). The catalytic subunit of cAMP-dependent protein kinase transiently restored the cAMP current suppressed by H-8 nearly to pre-H-8 level. These findings suggest that protein phosphorylation may be an intermediate step in the activation process of the cAMP current.     

抑郁症与cAMP反应元件结合蛋白基因的关联研究

目的 探讨cAMP反应元件结合蛋白(cyclic adenosine monophosphate response elementbinding protein,CREB1)基因与抑郁症的关联关系.方法 采用聚合酶链反应-限制性片段长度多态性方法检测105个抑郁症核心家系CREB1基因上单核苷酸多态性(single nucleotide polymorphisms,SNP)rs10932201和rs6740584的等位基因与基因型分布情况.进行单位点及单倍型的传递/不平衡检验(transmission disequilibrium test,TDT).结果 CREB1基因上SNP位点rs10932201和rs6740584与抑郁症均无显著性关联,TDT χ2 值分别为2.700(P=0.1004)和0.458(P=0.4986),差异均无统计学意义.单倍型TDT分析结果显示由rs10932201和rs6740584构成的单倍型与抑郁症存在显著性关联,差异有统计学意义(总χ2=23.458,df=3,P=0.00003241).单个单倍型A-C和A-T与抑郁症也均有显著性关联,差异有统计学意义(χ2 值分别为5.405和13.623,P值分别为0.020和0.00022).结论 CREB1基因上SNP位点rs10932201和rs6740584与抑郁症均无显著性关联,但由这2个SNP位点构成的单倍型与抑郁症存在显著性关联,提示CREB1基因rs10932201-rs6740584单倍型可能在抑郁症的遗传学发病机制中具有重要作用.     

Ach对人胃腺癌细胞内蛋白激酶A及cAMP的影响

目的：探讨乙酰胆碱(Ach)对人胃腺癌细胞内蛋白激酶A (PKA)及环一磷酸腺苷(cAMP)水平的影响。 方法： 将Ach作用于体外培养的人胃腺癌细胞，利用酶免疫分析法测定癌细胞内PKA活性和cAMP的浓度。 结果： 10 μmol/L Ach使胃腺癌细胞内cAMP水平和PKA活性显著高于空白对照组，该作用可被阿托品(atropine)阻断。 结论： Ach对胃腺癌细胞的增殖分化具有一定的调控作用，这种作用是通过毒蕈碱受体实现的。