阿尔茨海默病与血管性痴呆的18F-FDDNP脑PET显像

目的探讨1-{6-[（2-^18F-氟乙基）-甲氨基]-2-萘基}-亚乙基丙二氰（^18F-FDDNP）PET脑显像鉴别诊断阿尔茨海默病（AD）与血管性痴呆（VaD）的价值。方法分别对9例AD、6例VaD及6例智能正常老年对照者（NC）进行^18F-FDDNPPET脑显像，受试者分别在药物注射后5、25、45min采集图像。结果AD患者大脑皮层及皮层下灰质核团3个时间段放射性清除情况与其他2组图像有明显的不同。药物注射5-45min后脑内放射性清除率：AD组（39％～45％），较NC组（55％～64％）明显减低（P〈0．05）。除外基底节区，VaD组（47％～59％）与NC组比较无显著性差异（P〉0．05）。结论^18F-FDDNPPET脑显像在AD诊断及与VaD的鉴别诊断中有重要的临床应用价值。     

Age-related loss of cardiac preconditioning: impact of protein kinase A

Helium induces preconditioning (He-PC) by mitochondrial calcium-sensitive potassium (mK_Ca) channel-activation, but this effect is lost in the aged myocardium. Both, the upstream signalling pathway of He-PC and the underlying mechanisms for an age-related loss of preconditioning are unknown. A possible candidate as upstream regulator of mK_Ca channels is protein kinase A (PKA).We investigated whether 1) regulation of PKA is involved in He-PC and 2) regulation of PKA is age-dependent.Young (2-3 months) and aged (22-24 months) Wistar rats were randomised to eight groups (each n = 8). All animals underwent 25 min regional myocardial ischemia and 120 min reperfusion. Control (Con, Age Con) animals were not further treated. Young rats inhaled 70% helium for 3 × 5min (He-PC). The PKA-blocker H-89 (10 μg/kg) was administered with and without helium (He-PC + H-89, H-89). Furthermore, we tested the effect of direct activation of mK_Ca channels with NS1619. The adenylyl cyclase activator forskolin (For) was administered in young (300 μg/kg) and aged animals (300 and 1000 μg/kg).He-PC reduced infarct size from 60 ± 4% (Con) to 37 ± 10% (p < 0.05). Infarct size reduction was completely abolished by H-89 (58 ± 5%; p < 0.05), but H-89 alone had no effect (57 ± 2%). NS1619 reduced infarct size in the same concentration in both, young and aged rats (35 ± 6%; p < 0.05 vs. Con and 34 ± 8%; p < 0.05 vs. Age Con). Forskolin in a concentration of 300 μg/kg reduced infarct size in young (37 ± 6%; p < 0.05) but not in aged rats (48 ± 13%; n.s.). In contrast, 1000 μg/kg Forskolin reduced infarct size also in aged rats (28 ± 3%; p < 0.05).He-PC is mediated by activation of PKA. Alterations in PKA regulation might be an underlying mechanism for the age-dependent loss of preconditioning.     

Negative regulation of glucose metabolism in human myotubes by supraphysiological doses of 17β-estradiol or testosterone

Objective

Exposure of skeletal muscle to high levels of testosterone or estrogen induces insulin resistance, but evidence regarding the direct role of either sex hormone on metabolism is limited. Therefore, the aim of this study was to investigate the direct effect of acute sex hormone exposure on glucose metabolism in skeletal muscle.

Materials/Methods

Differentiated human skeletal myotubes were exposed to either 17β-estradiol or testosterone and metabolic characteristics were assessed. Glucose incorporation into glycogen, glucose oxidation, palmitate oxidation, and phosphorylation of key signaling proteins were determined.

Results

Treatment of myotubes with either 17β-estradiol or testosterone decreased glucose incorporation into glycogen. Exposure of myotubes to 17β-estradiol reduced glucose oxidation under basal and insulin-stimulated conditions. However, testosterone treatment enhanced basal palmitate oxidation and prevented insulin action on glucose and palmitate oxidation. Acute stimulation of myotubes with testosterone reduced phosphorylation of S6K1 and p38 MAPK. Exposure of myotubes to either 17β-estradiol or testosterone augmented phosphorylation GSK3β^Ser9 and PKCδ^Thr505, two negative regulators of glycogen synthesis. Treatment of myotubes with a PKC specific inhibitor (GFX) restored the effect of either sex hormone on glycogen synthesis. PKCδ silencing restored glucose incorporation into glycogen to baseline in response to 17β-estradiol, but not testosterone treatment.

Conclusion

An acute exposure to supraphysiological doses of either 17β-estradiol or testosterone regulates glucose metabolism, possibly via PKC signaling pathways. Furthermore, testosterone treatment elicits additional alterations in serine/threonine kinase signaling, including the ribosomal protein S6K1 and p38 MAPK.     

17β-Estradiol stimulates the translocation of endogenous estrogen receptor α at the plasma membrane of normal anterior pituitary cells

In the present work we aimed at identifying ERα in the plasma membrane of normal anterior pituitary cells and investigated if 17β-estradiol was able to induce their subcellular redistribution. Our results show that about 8% of anterior pituitary cells expressed ERα in the plasma membrane, with the geometrical mean fluorescence intensity being increased after steroid hormone treatment. 17β-Estradiol and the selective ERα agonist PPT induced an increase of ERα expression in the plasma membrane and activated the PKCα/ERK 1/2 pathway in a time-course not compatible with genomic actions, thus supporting the notion of membrane-initiated effects. These findings suggest that 17β-estradiol stimulates the translocation of endogenous ERα to the plasma membrane, consequently modulating this ER pool and leading to cellular biological effects in normal anterior pituitary gland.     

Positron emission tomography imaging of adenoviral-mediated transgene expression in liver cancer patients

BACKGROUND & AIMS: In gene-therapy protocols, imaging of gene expression is needed to evaluate the transduction efficiency of the vector, its tissue distribution, and the duration of transgene expression and to assess the feasibility of repeated vector administration. METHODS: We have used positron emission tomography with a fluorine-18-labeled penciclovir analogue to monitor thymidine kinase gene expression after intratumoral injection of a first-generation recombinant adenovirus in patients with hepatocellular carcinoma. Patients were enrolled in a pilot clinical trial and treated with escalating doses of the vector. Two days after adenovirus inoculation, transgene expression was evaluated during the first hours after administration of the radiotracer both on the treated lesion and on a whole-body basis. RESULTS: Transgene expression in the tumor was dependent on the injected dose of the adenovirus and was detectable in all patients who received > or = 10(12) viral particles. However, when the study was repeated 9 days after vector injection, no expression could be observed. It is interesting to note that no specific expression of the transgene could be detected in distant organs or in the surrounding cirrhotic tissue in any of the cases studied. CONCLUSIONS: Our findings show the real possibility of imaging transgene expression in humans by using viral vectors. We show that hepatocarcinoma is a permissive tumor for adenoviral infection and that the nontumoral cirrhotic liver is spared from transduction when the vector is administered by intratumoral injection. These results show that positron emission tomography imaging may help in the design of gene-therapy strategies and in the clinical assessment of new-generation vectors.     

Chemokine (C-C) motif ligand 20 is regulated by PGF2α-F-prostanoid receptor signalling in endometrial adenocarcinoma and promotes cell proliferation

Prostaglandin F_2α (PGF_2α) is an inflammatory mediator which signals through a G-protein coupled receptor, the F-prostanoid (FP) receptor. We have previously shown elevated FP receptor expression in endometrial adenocarcinoma, a common gynaecological malignancy in Western countries. In this study, the expression of the chemokine CC motif Ligand 20 (CCL20) was determined to be regulated by PGF_2α-FP receptor signalling in endometrial adenocarcinoma explants and cell line, and expression of CCL20 and its receptor CCR6 was elevated in endometrial adenocarcinoma compared to non-malignant endometrium. Both CCL20 and CCR6 were localised to neoplastic endometrial epithelial cells. The induction of CCL20 expression by PGF_2α-FP signalling in an endometrial adenocarcinoma cell line stably expressing the FP receptor (FPS cells) was found to be dependent on the intracellular signalling of Gq, EGFR, ERK, calcineurin and nuclear factor of activated T-cells (NFAT) proteins. The treatment of FPS cells with recombinant CCL20 caused a significant increase in proliferation. Therefore these data demonstrate a role for the FP receptor in regulation of the chemokine CCL20, which can mediate proliferation of endometrial adenocarcinoma epithelial cells.     

Stimulatory effect of insulin on theca-interstitial cell proliferation and cell cycle regulatory proteins through MTORC1 dependent pathway

The present study examined the effect of insulin-mediated activation of the mammalian target of rapamycin complex 1 (MTORC1) signaling network on the proliferation of primary culture of theca-interstitial (T-I) cells. Our results show that insulin treatment increased proliferation of the T-I cells through the MTORC1-dependent signaling pathway by increasing cell cycle regulatory proteins. Inhibition of ERK1/2 signaling caused partial reduction of insulin-induced phosphorylation of RPS6KB1 and RPS6 whereas inhibition of PI3-kinase signaling completely blocked the insulin response. Pharmacological inhibition of MTORC1 with rapamycin abrogated the insulin-induced phosphorylation of EIF4EBP1, RPS6KB1 and its downstream effector, RPS6. These results were further confirmed by demonstrating that knockdown of Mtor using siRNA reduced the insulin-stimulated MTORC1 signaling. Furthermore, insulin-stimulated T-I cell proliferation and the expression of cell cycle regulatory proteins CDK4, CCND3 and PCNA were also blocked by rapamycin. Taken together, the present studies show that insulin stimulates cell proliferation and cell cycle regulatory proteins in T-I cells via activation of the MTORC1 signaling pathway.     

Angiotensin AT2 receptor activates the cyclic-AMP signaling pathway in eel

A unique angiotensin type 2 receptor (AT2) that induces a cAMP signaling pathway was cloned and characterized for the first time in fish, Anguilla japonica. Phylogeny and synteny results showed that the AT2s among fishes and tetrapods share the same origin despite a sub-cluster formation among eel, salmon, and zebrafish. The eel AT2 was expressed abundantly in the spleen and localized at straight arterioles and ellipsoid regions prior to the sinusoid, suggesting a role in the regulation of microcirculation and/or immune response. Various angiotensin (Ang) peptides, including Ang II, Ang III, and Ang IV, were detected in the spleen by a radioimmunoassay coupled with HPLC separation, and these endogenous peptides stimulated a cAMP signaling, which has no crosstalk with cGMP pathway. The common and contrasting features of AT2 between fishes and mammals imply some ancestral characters of AT2, which are important information for receptor binding and evolutionary studies.     

Cannabinoids ameliorate pain and reduce disease pathology in cerulein-induced acute pancreatitis

BACKGROUND & AIMS: The functional involvement of the endocannabinoid system in modulation of pancreatic inflammation, such as acute pancreatitis, has not been studied to date. Moreover, the therapeutic potential of cannabinoids in pancreatitis has not been addressed. METHODS: We quantified endocannabinoid levels and expression of cannabinoid receptors 1 and 2 (CB1 and CB2) in pancreas biopsies from patients and mice with acute pancreatitis. Functional studies were performed in mice using pharmacological interventions. Histological examination, serological, and molecular analyses (lipase, myeloperoxidase, cytokines, and chemokines) were performed to assess disease pathology and inflammation. Pain resulting from pancreatitis was studied as abdominal hypersensitivity to punctate von Frey stimuli. Behavioral analyses in the open-field, light-dark, and catalepsy tests were performed to judge cannabinoid-induced central side effects. RESULTS: Patients with acute pancreatitis showed an up-regulation of cannabinoid receptors and elevated levels of endocannabinoids in the pancreas. HU210, a synthetic agonist at CB1 and CB2, abolished abdominal pain associated with pancreatitis and also reduced inflammation and decreased tissue pathology in mice without producing central, adverse effects. Antagonists at CB1- and CB2-receptors were effective in reversing HU210-induced antinociception, whereas a combination of CB1- and CB2-antagonists was required to block the anti-inflammatory effects of HU210 in pancreatitis. CONCLUSIONS: In humans, acute pancreatitis is associated with up-regulation of ligands as well as receptors of the endocannabinoid system in the pancreas. Furthermore, our results suggest a therapeutic potential for cannabinoids in abolishing pain associated with acute pancreatitis and in partially reducing inflammation and disease pathology in the absence of adverse side effects.     

Reactions of the melatonin metabolite N1-acetyl-5-methoxykynuramine with carbamoyl phosphate and related compounds

Abstract:  N -[2-(6-methoxyquinazolin-4-yl)-ethyl] acetamide (MQA) is a compound formed from the melatonin metabolite N ¹-acetyl-5-methoxykynuramine (AMK). We followed MQA production in reaction systems containing various putative reaction partners, in the absence and presence of hydrogen peroxide and/or copper(II). Although MQA may be formally described as a condensation product of either N ¹-acetyl- N ²-formyl-5-methoxykynuramine (AFMK) with ammonia, or AMK with formamide, none of these combinations led to substantial quantities of MQA. However, MQA formation was observed in mixtures containing AMK, hydrogen peroxide, hydrogen carbonate and ammonia, or AMK, hydrogen peroxide, copper(II) and potentially carbamoylating agents, such as potassium cyanate or, more efficiently, carbamoyl phosphate. In the presence of hydrogen peroxide, copper(II) and carbamoyl phosphate, MQA was the major product obtained from AMK, but the omission of copper(II) mainly led to another metabolite, 3-acetamidomethyl-6-methoxycinnolinone (AMMC). This was caused by nitric oxide (NO) generated under oxidative conditions from carbamoyl phosphate, as shown by an NO spin trap. MQA formation with carbamoyl phosphate was not due to the possible decomposition product, formamide. The reaction of AMK with carbamoyl phosphate under oxidative conditions, in which inorganic phosphate and water are released and which differs from the typical process of carbamoylation via isocyanate, may be considered as a new physiological route of MQA formation.     

Serotonin excites neurons in the human submucous plexus via 5-HT3 receptors

BACKGROUND & AIMS: Serotonin (5-hydroxytryptamine [5-HT]) is a key signaling molecule in the gut. Recently, the neural 5-HT3 receptor received a lot of attention as a possible target in functional bowel diseases. Yet, the 5-HT3 receptor-mediated changes in properties of human enteric neurons is unknown. METHODS: We used a fast imaging technique in combination with the potentiometric dye 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octylamino)-6-naphthyl]vinyl]pyridinium betaine to monitor directly the membrane potential changes in neurons of human submucous plexus from surgical specimens of 21 patients. An Ussing chamber technique was used to study 5-HT3 receptor involvement in chloride secretion. RESULTS: Local microejection of 5-HT directly onto ganglion cells resulted in a transient excitation of enteric neurons characterized by increased spike discharge. This response was mimicked by the 5-HT3 receptor agonist, 2-methyl-5-HT, and blocked by the 5-HT3 receptor antagonist, tropisetron. The proportions of 5-HT-responsive nerve cells per ganglion ranged from 25.5% +/- 18.4% in the duodenum to 54.2% +/- 46.9% in the colon. Interestingly, 2-methyl-5-HT did not evoke chloride secretion in the human intestine but it did in the guinea-pig intestine. Specific 5-HT3A and 5-HT3B receptor subunit immunoreactivity as well as 5-HT3A and 5-HT3B receptor-specific messenger RNA were detected in the tissue samples. Based on co-labeling with the pan-neuronal marker HuC/D we conclude that submucous nerve cells potentially express heteromeric 5-HT3A/B receptors. CONCLUSIONS: We show that 5-HT excited human enteric neurons via 5-HT3 receptors, which may comprise both 5-HT3A and 5-HT3B receptor subunits.     

Inhibition of transient lower esophageal sphincter relaxation and gastroesophageal reflux by metabotropic glutamate receptor ligands

BACKGROUND & AIMS: Transient lower esophageal sphincter relaxation (TLESR) is the major mechanism of gastroesophageal acid reflux. TLESR is mediated via vagal pathways, which may be modulated by metabotropic glutamate receptors (mGluRs). Group I mGluRs (mGluR1 and 5) have excitatory effects on neurons, whereas group II (mGluR2 and 3) and group III (mGluR4, 6, 7, and 8) are inhibitory. This study determined the effect of mGluRs on triggering of TLESR and reflux in an established conscious ferret model. METHODS: Esophageal manometric/pH studies were performed in ferrets with chronic esophagostomies. TLESR were induced by a gastric load of 25 mL glucose (pH 3.5) and 30 mL air. RESULTS: In control treated animals, gastric load induced 3.52 +/- 0.46 TLESRs per 47-minute study, 89.7% of which were associated with reflux episodes (n = 16). The mGluR5 antagonist MPEP inhibited TLESR dose dependently, with maximal 71% +/- 7% inhibition at 35 micromol/kg (n = 9; P < .0001). MPEP also significantly reduced reflux episodes (P < .001) and increased basal lower esophageal sphincter pressure (P < .05). MPEP inhibited swallowing dose dependently, suggesting a common action on trigger mechanisms for swallowing and TLESR. The more selective analogue, MTEP, had more potent effects (90% +/- 6% inhibition TLESR at 40 micromol/kg; n = 8; P < .0001). In contrast, the group I agonist DHPG tended to increase TLESR. The group II agonist (2R, 4R)-APDC was ineffective, whereas the group III agonist L-(AP4 slightly reduced TLESR (33% at 11 micromol/kg; P < .05). The selective mGluR8 agonist (S)-3, 4-DCPG inhibited TLESR by 54% at 15 micromol/kg (P < .01). CONCLUSIONS: mGluR5 antagonists potently inhibit TLESR and reflux in ferrets, implicating mGluR5 in the mechanism of TLESR. mGluR5 antagonists are therefore promising as therapy for patients with GERD.     

C-peptide induces human renal mesangial cell proliferation in vitro, activating Src-kinase, PI-3 kinase and ERK1/2

Background

Elevated levels of C-peptide have been found in patients with insulin resistance and early type 2 diabetes. These patients are at greater risk to develop micro- and macrovascular complications. Since diabetic nephropathy involves glomerular hyperproliferation, the present study evaluates the role of C-peptide on human renal mesangial cell proliferation.

Methods and results

C-peptide induces proliferation of human renal mesangial cells in a concentration-dependent manner with a maximal 2.6 ± 0.4-fold induction at 10 nmol/L (P < 0.05 compared with unstimulated cells; n = 6), as revealed by [3H]-thymidine incorporation experiments. The proliferative effect of C-peptide is prevented by Src-kinase inhibitor-PP2, PI-3 kinase inhibitor-LY294002, and the ERK1/2 inhibitor-U126. Moreover, C-peptide induces phosphorylation of Src, as well as activation of PI-3 kinase and ERK1/2. Furthermore, C-peptide induces cyclin D1 expression as well as phosphorylation of retinoblastoma protein (Rb).

Conclusions

These results demonstrate an active role of C-peptide on the proliferation of human renal mesangial cells in vitro involving PI-3 kinase and MAP kinase signaling pathways, suggesting a possible role of C-peptide in glomerular hyperproliferation in patients with diabetic nephropathy.     

Tangeretin stimulates glucose uptake via regulation of AMPK signaling pathways in C2C12 myotubes and improves glucose tolerance in high-fat diet-induced obese mice

Although the flavonoid tangeretin (5, 6, 7, 8, 4'-pentamethoxyflavone) is known to possess beneficial health effects, the anti-diabetic effects and the mechanism of action have not been elucidated. Treatment with 100 μM tangeretin significantly increased the uptake of 2-NBDG in C2C12 myotubes. We also found that AMPK and AS160 were markedly phosphorylated by tangeretin treatment. In addition, pretreatment with an AMPK inhibitor significantly abrogated tangeretin-stimulated AS160 phosphorylation, glucose uptake, and Glut4 translocation from the cytosol to the plasma membrane. Furthermore, disruption of AMPK using siRNA transfection prevented the glucose uptake stimulated by tangeretin. We also examined the anti-diabetic properties of tangeretin in mice on HFD. Administration of HFD plus 200 mg/kg of tangeretin significantly altered weight gain, glucose tolerance, total cholesterol levels, and the secretion of adipocytokines, such as adiponectin, leptin, resistin, IL-6, and MCP-1. Moreover, AMPK was activated by 200 mg/kg of tangeretin in mouse muscle tissue, as expected from the cell system. These results suggest that tangeretin exerts anti-diabetic effects in both cell culture and mouse models, and these effects are necessary for activating AMPK.     

Using U0126 to dissect the role of the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade in the regulation of gene expression by endothelin-1 in cardiac myocytes

The hypertrophic agonist endothelin-1 rapidly but transiently activates the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade (and other signalling pathways) in cardiac myocytes, but the events linking this to hypertrophy are not understood. Using Affymetrix rat U34A microarrays, we identified the short-term (2-4 h) changes in gene expression induced in neonatal myocytes by endothelin-1 alone or in combination with the ERK1/2 cascade inhibitor, U0126. Expression of 15 genes was significantly changed by U0126 alone, and expression of an additional 78 genes was significantly changed by endothelin-1. Of the genes upregulated by U0126, four are classically induced through the aryl hydrocarbon receptor (AhR) by dioxins suggesting that U0126 activates the xenobiotic response element in cardiac myocytes potentially independently of effects on ERK1/2 signalling. The 78 genes showing altered expression with endothelin-1 formed five clusters: (i) three clusters showing upregulation by endothelin-1 according to time course (4 h > 2 h; 2 h > 4 h; 2 h approximately 4 h) with at least partial inhibition by U0126; (ii) a cluster of 11 genes upregulated by endothelin-1 but unaffected by U0126 suggesting regulation through signalling pathways other than ERK1/2; (iii) a cluster of six genes downregulated by endothelin-1 with attenuation by U0126. Thus, U0126 apparently activates the AhR in cardiac myocytes (which must be taken into account in protracted studies), but careful analysis allows identification of genes potentially regulated acutely via the ERK1/2 cascade. Our data suggest that the majority of changes in gene expression induced by endothelin-1 are mediated by the ERK1/2 cascade.     

Adenosine triggers preconditioning through MEK/ERK1/2 signalling pathway during hypoxia/reoxygenation in neonatal rat cardiomyocytes

Three subtypes of adenosine receptors (A(1), A(2A) and A(3) ARs) are functionally expressed in cardiomyocytes. Adenosine released during ischemia and ischemia/reperfusion plays a major role in cardioprotection. Phosphatidylinositol 3-kinase (PI-3K)/protein kinase B (PKB) and MEK/ERK1/2 pathways are involved in cell survival. Since the role of these pathways in AR-mediated preconditioning is poorly understood, we have investigated whether PI-3K/PKB and/or MEK1/ERK1/2 pathways are involved in AR-induced cardioprotection in neonatal rat cardiomyocytes. Cells were pre-treated (15 min) with adenosine (non-selective), CPA (A(1)), CGS 21680 (A(2A)) or Cl-IB-MECA (A(3)) before 4 h hypoxia (0.5% O(2)) and 18 h reoxygenation (HX4/R). HX4/R-induced increase in LDH release was significantly reduced by adenosine (70%), CPA (59%) and Cl-IB-MECA (46%). The MEK1 inhibitor PD 98059 suppressed the effects of adenosine, CPA, and Cl-IB-MECA on LDH release, whereas the PI-3K inhibitor wortmannin did not reverse this cardioprotection. Western blotting of phosphorylated ERK1/2 and PKB during HX4/R supported the involvement of ERK1/2 and not PKB in A(1) and A(3) agonist-mediated cardioprotection. In addition, adenosine, CPA and Cl-IB-MECA inhibited HX4/R-induced caspase 3 activity by 75%, 70% and 59%, respectively, and this inhibition was abolished by PD 98059. Interestingly, wortmannin inhibited by 66% the anti-apoptotic response triggered by Cl-IB-MECA but had no effect on adenosine or CPA-induced inhibition of caspase 3. CGS 21680 did not modify cell survival or caspase 3 activity. In conclusion, these data show that the preconditioning effect of adenosine requires A(1) and A(3) but not A(2A) ARs and involves an anti-apoptotic effect via MEK1/ERK1/2 pathway in neonatal rat cardiomyocytes. In addition, A(3)AR-induced preconditioning also involves a PI-3K dependent pathway.