Inositol Trisphosphate Receptors and Nuclear Calcium in Atrial Fibrillation.

RATIONALE: The mechanisms underlying atrial fibrillation (AF), the most common clinical arrhythmia, are poorly understood. Nucleoplasmic Ca2+ regulates gene expression, but the nature and significance of nuclear Ca2+-changes in AF are largely unknown. OBJECTIVE: To elucidate mechanisms by which AF alters atrial-cardiomyocyte nuclear Ca2+ ([Ca2+]Nuc) and CaMKII (Ca2+/calmodulin-dependent protein kinase-II)-related signaling. METHODS AND RESULTS: Atrial cardiomyocytes were isolated from control and AF dogs (kept in AF by atrial tachypacing [600 bpm × 1 week]). [Ca2+]Nuc and cytosolic [Ca2+] ([Ca2+]Cyto) were recorded via confocal microscopy. Diastolic [Ca2+]Nuc was greater than [Ca2+]Cyto under control conditions, while resting [Ca2+]Nuc was similar to [Ca2+]Cyto; both diastolic and resting [Ca2+]Nuc increased with AF. IP3R (Inositol-trisphosphate receptor) stimulation produced larger [Ca2+]Nuc increases in AF versus control cardiomyocytes, and IP3R-blockade suppressed the AF-related [Ca2+]Nuc differences. AF upregulated nuclear protein expression of IP3R1 (IP3R-type 1) and of phosphorylated CaMKII (immunohistochemistry and immunoblot) while decreasing the nuclear/cytosolic expression ratio for HDAC4 (histone deacetylase type-4). Isolated atrial cardiomyocytes tachypaced at 3 Hz for 24 hours mimicked AF-type [Ca2+]Nuc changes and L-type calcium current decreases versus 1-Hz-paced cardiomyocytes; these changes were prevented by IP3R knockdown with short-interfering RNA directed against IP3R1. Nuclear/cytosolic HDAC4 expression ratio was decreased by 3-Hz pacing, while nuclear CaMKII phosphorylation was increased. Either CaMKII-inhibition (by autocamtide-2-related peptide) or IP3R-knockdown prevented the CaMKII-hyperphosphorylation and nuclear-to-cytosolic HDAC4 shift caused by 3-Hz pacing. In human atrial cardiomyocytes from AF patients, nuclear IP3R1-expression was significantly increased, with decreased nuclear/nonnuclear HDAC4 ratio. MicroRNA-26a was predicted to target ITPR1 (confirmed by luciferase assay) and was downregulated in AF atrial cardiomyocytes; microRNA-26a silencing reproduced AF-induced IP3R1 upregulation and nuclear diastolic Ca2+-loading. CONCLUSIONS: AF increases atrial-cardiomyocyte nucleoplasmic [Ca2+] by IP3R1-upregulation involving miR-26a, leading to enhanced IP3R1-CaMKII-HDAC4 signaling and L-type calcium current downregulation. Graphic Abstract: A graphic abstract is available for this article.

Protective effect of crocin on bisphenol A - induced spatial learning and memory impairment in adult male rats: Role of oxidative stress and AMPA receptor.

OBJECTIVES: Bisphenol A (BPA), a xenoestrogenic endocrine disrupting agent, is widely used in the production of polycarbonate plastics and has potential adverse effects on the developing nervous system, memory and learning abilities. The protective effect of the crocin, an important active constituent in Crocus sativus L, on memory impairment induced by BPA in rat was determined through evaluation of oxidative stress and the level of NMDA (N-methyl-D-aspartate receptors) and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole-propionicd acid) receptors. MATERIALS AND METHODS: Rats were orally treated with BPA (100 mg/kg) or sesame seed oil in control group for 28 days. Crocin (10, 20, and 40 mg/kg, IP) was administrated in BPA-orally treated groups for 28 days. Memory and learning functions were evaluated by Morris water maze. The level of malondialdehyde (MDA) and glutathione (GSH) contents were determined in rat hippocampus. Additionally, the expression of NMDA and AMPA receptors were analyzed using Western blot method. RESULTS: Administration of BPA significantly reduced memory and learning functions. Crocin significantly protected against learning and memory impairments induced by BPA. BPA administration markedly reduced GSH content and induced lipid peroxidation, while crocin was able to increase GSH content in rat hippocampus. The expression of NMDA receptor did not change in BPA-treated rats, while the significant reduction in AMPA receptor expression was observed. Moreover, crocin (20 mg/kg) significantly elevated the expression of AMPA receptor. CONCLUSION: Crocin recovered spatial learning and memory defects induced by BPA in part through anti-oxidant activity and modulation the expression of AMPA receptor in rat hippocampus.

Alteration of protein profile in cerebral cortex of rats exposed to bisphenol a: a proteomics study.

Bisphenol A (BPA) is one of the most widely used chemicals in plastic industry, which enters the human body through occupational and food contact. We studied the protein changes in rat cerebral cortex to evaluate the neurotoxicity of BPA. Twenty-four adult male rats were randomly selected and divided into four groups and each group respectively received 0, 0.5, 5 and 50 mg/kg of BPA for 4 weeks orally. To determine the oxidative status, reduced glutathione content and the level of malondialdehyde were measured in brain cortical tissue. The proteins of each sample extracted and separated on a two-dimensional acrylamide gel electrophoresis. From the obtained protein map, the 10 most altered protein spots were used for mass spectroscopy analysis. The lipid peroxidation in both doses of 0.5 and 5 mg/kg was significantly higher than the control group, but the glutathione content had no significant difference between the groups. Based on the results of the MS data analysis by the MASCOT database search engine, 10 proteins with altered intensity were identified as pyruvate kinase, alpha-enolase, aconitate hydratase, creatine kinase B-type, phosphatidylethanolamine-binding protein 1, 14-3-3 protein eta, guanine nucleotide-binding protein subunit beta-1, dihydropyrimidinase-related protein 2, glutamine synthetase and the neurofilament light polypeptide. There are several reports suggesting that the increase or decrease in the level and activity of these 10 proteins, similar to those observed in this study, is related to some neurological and psychosocial disorders including neurodegenerative diseases, schizophrenia, depression, epilepsy and some brain tumors.

Proteomics and phosphoproteomics analysis of liver in male rats exposed to bisphenol A: Mechanism of hepatotoxicity and biomarker discovery.

Bisphenol A (BPA), discovered to be an artificial estrogen, has been shown to leach from some containers and mediate oxidative damage to cells and tissues and to be involved in reproductive disorders, obesity, diabetes, and liver dysfunction. In the current study, we investigated the effects of oral chronic exposure to low dose of BPA (0.5 mg kg-1) on the protein and phosphoprotein expression profiles in male Wistar rat liver using a gel-based proteomics approach based on two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ionization mass spectrometry identification. Our results showed that BPA exposure affected the levels of proteins and phosphoproteins involved in diverse biological processes associated with hepatotoxicity, fatty liver, and carcinoma. Moreover, we analyzed the effects of BPA on oxidative stress by assessing levels of malondialdehyde (MDA), a marker of lipid peroxidation, and reduced glutathione (GSH), a non-enzymatic antioxidant agent, in the liver. As expected BPA induced oxidative stress indicated by increased levels of MDA and decreased GSH content in the liver. In conclusion, chronic oral exposure of rats to BPA leads to increased oxidative stress in the liver and major alterations in the liver proteome and phosphoproteome, which may contribute to the pathophysiology of liver diseases.

Protective effect of crocin on BPA-induced liver toxicity in rats through inhibition of oxidative stress and downregulation of MAPK and MAPKAP signaling pathway and miRNA-122 expression.

Bisphenol A (BPA) is an artificial environmental endocrine disrupting chemical and commonly used as a monomer of polycarbonate plastics and epoxy resins. The aim of the present study is to investigate the hepatoprotective effects of crocin, a constituent of saffron, against BPA-induced liver toxicity. We showed that treatment of male Wistar rats with 0.5 mg/kg BPA for 30 days increased the level of 8-isoprostane, decreased the level of reduced glutathione, elevated serum levels of aspartate aminotransferase, lactate dehydrogenase, triglyceride, and glucose, and induced periportal inflammation. Western blot results revealed that BPA increased the phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK1/2), and mitogen-activated protein kinase-activated protein kinase (MAPKAPK), but not p38. BPA also reduced the Akt signaling activation and upregulated microRNA (miR-122) expression. Moreover, we showed here that crocin 20 mg/kg administration ameliorated liver damage and improved elevated levels of TG and liver enzymes of BPA-treated rats possibly though antioxidant activity, downregulation of miR-122 transcript level and lowering the phosphorylation of JNK, ERK1/2, and MAPKAPK and subsequently their activities. Overall, the findings suggest that crocin possesses hepatoprotective effects against BPA-induced liver toxicity by enhancing the antioxidative defense system and regulation of important signaling pathway activities and miR-122 expression.

Effects of berberine on acquisition and reinstatement of morphine-induced conditioned place preference in mice.

OBJECTIVE: It has been shown that berberine, a major component of Berberis vulgaris extract, modulates the activity of several neurotransmitter systems including dopamine (Da) and N-methyl-D-aspartate (NMDA) contributing to rewarding and reinforcing effects of morphine. Drug craving and relapsing even after a long time of abstinence therapy are the most important problems of addiction. In the present study, we investigated the alleviating effects of berberine on the acquisition and reinstatement of morphine-induced conditioned place preference (CPP) in mice. MATERIALS AND METHODS: In male NMRI mice, the acquisition of CPP was established by 40 mg/kg of morphine sulphate injection and extinguished after the extinction training and reinstated by a 10 mg/kg injection of morphine. The effects of different doses of berberine (5, 10, and 20 mg/kg) on the acquisition and reinstatement induced by morphine were evaluated in a conditioned place preference test. RESULTS: The results showed that intraperitoneal administration of berberine (5, 10, and 20 mg/kg) did not induce conditioned appetitive or aversive effects. Injection of berberine (10 and 20 mg/kg) 2 h before the morphine administration reduced acquisition of morphine-induced CPP. In addition, same doses of berberine significantly prevented the reinstatement of morphine-induced CPP. CONCLUSION: These results suggest that berberine can reduce the acquisition and reinstatement of morphine-induced conditioned place preference and may be useful in treatment of morphine addiction.

Antidepressant effects of crocin and its effects on transcript and protein levels of CREB, BDNF, and VGF in rat hippocampus.

BACKGROUND: Antidepressants have been shown to affect levels of brain-derived neurotrophic factor (BDNF) and VGF (non-acronymic) whose transcriptions are dependent on cAMP response element binding protein (CREB) in long term treatment. The aim of this study was to verify the subacute antidepressant effects of crocin, an active constituent of saffron (Crocus sativus L.), and its effects on CREB, BDNF, and VGF proteins, transcript levels and amount of active, phosphorylated CREB (P-CREB) protein in rat hippocampus. METHODS: Crocin (12.5, 25, and 50 mg/kg), imipramine (10 mg/kg; positive control) and saline (1 mL/kg; neutral control) were administered intraperitoneally (IP) to male Wistar rats for 21 days. The antidepressant effects were studied using the forced swimming test (FST) on day 21 after injection. Protein expression and transcript levels of genes in the rat hippocampus were evaluated using western blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR), respectively. RESULTS: Crocin significantly reduced the immobility time in the FST. Western blot analysis showed that 25 and 50 mg/kg of crocin increased the levels of CREB and BDNF significantly and dose dependently. All doses of crocin increased the VGF levels in a dose-dependent manner. Levels of p-CREB increased significantly by 50 mg/kg dose of crocin. Only 12.5 mg/kg crocin could significantly increase the transcript levels of BDNF. No changes in CREB and VGF transcript levels were observed in all groups. CONCLUSIONS: These results suggest that crocin has antidepressant-like action by increasing CREB, BDNF and VGF levels in hippocampus.

Evaluation of in vitro toxicity of peptide (N-acetyl-Leu-Gly-Leu-COOH)-substituted-ß-cyclodextrin derivative, a novel drug carrier, in PC-12 cells.

BACKGROUND: Cyclodextrins (CDs) have been shown to improve physicochemical and biopharmaceutical properties of drugs when low solubility and low safety limit their use in the pharmaceutical field. Recently, a new amphiphilic peptide-substituted-ß-CD, hepta-(N-acetyl-Leu-Gly-Leu)-ß-CD (hepta-(N-acetyl-LGL)-ß-CD), is developed which exhibited good solubility, strong inclusion ability and an appropriate average molecular weight. However, there is limited information available about its toxic effects. This study was designed to evaluate cytotoxic effects of the hepta-(N-acetyl-LGL)-ß-CD (50, 200, 400, and 800 µg/ml) on rat pheochromocytoma PC-12 cells. RESULTS: A significant reduction of cell viability with IC50 values of 1115.0 µg/ml, 762.4 µg/ml, and 464.9 µg/ml at 6, 12, and 24 h post-treatment, respectively, as well as increased lipid peroxide levels and DNA damage were observed. CONCLUSIONS: In conclusion, hepta-(N-acetyl-Leu-Gly-Leu)-ß-CD exhibit significant toxic properties at high concentrations, probably through induction of oxidative stress and genotoxicity.