Role of mitochondria in contraction and pacemaking in the mouse uterus

BACKGROUND AND PURPOSE

Uterine spontaneous contraction and pacemaking are poorly understood. This study investigates the role of the mitochondrial Ca²⁺ store in uterine activity.

EXPERIMENTAL APPROACH

We investigated the effects of mitochondrial and sarco-endoplasmic reticulum (SER) inhibitors on contraction, membrane potential (Vm) and cytosolic Ca²⁺ concentration ([Ca²⁺]_c) in longitudinal smooth muscle of the mouse uterus.

KEY RESULTS

The mitochondrial agents rotenone, carbonylcyanide-3-chlorophenylhydrazone (CCCP), 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one ({"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157) and kaempferol decreased the force of contractions. The ATP synthase inhibitor oligomycin had no significant effect. The effects of these agents were compared with those of SER inhibitors cyclopiazonic acid (CPA), 2-amino ethoxyphenylborate (2-APB) and caffeine. All agents, except CPA and oligomycin, decreased contractile force. CPA and CCCP transiently increased contraction frequency, which returned to control levels, whereas rotenone, {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157, kaempferol and 2-APB decreased frequency and caffeine had no significant effect. Application of the mitochondrial agents when CPA functionally inhibited stores did not change contraction frequency but, with the exception of kaempferol, decreased force. CCCP caused depolarization and maintained increase in [Ca²⁺]_c or depolarization/transient hyperpolarization and transient increase in [Ca²⁺]_c for oestrus and di-oestrus tissues respectively. Rotenone caused hyperpolarization and maintained increase in [Ca²⁺]_c. {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 and kaempferol caused hyperpolarization but no measurable change in [Ca²⁺]_c. Application of a range of K⁺ channel blockers indicated a role of Ca²⁺-activated K⁺ (K_Ca) channels in the CCCP- and {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157-induced actions.

CONCLUSIONS AND IMPLICATIONS

Mitochondria have a modulatory role on uterine contractions, with mitochondrial inhibition reducing contraction amplitude and pacemaker frequency by changes in Vm, [Ca²⁺]_c and/or Ca²⁺ influx.     

Role of perinuclear mitochondria in the spatiotemporal dynamics of spontaneous Ca2+ waves in interstitial cells of Cajal-like cells of the rabbit urethra

BACKGROUND AND PURPOSE

Although spontaneous Ca²⁺ waves in interstitial cells of Cajal (ICC)-like cells (ICC-LCs) primarily arise from endoplasmic reticulum (ER) Ca²⁺ release, the interactions among mitochondrial Ca²⁺ buffering, cellular energetics and ER Ca²⁺ release in determining the spatiotemporal dynamics of intracellular Ca²⁺ remain to be elucidated.

EXPERIMENTAL APPROACH

Spontaneous Ca²⁺ transients in freshly isolated ICC-LCs of the rabbit urethra were visualized using fluo-4 Ca²⁺ imaging, while the intracellular distribution of mitochondria was viewed with MitoTracker Red.

KEY RESULTS

Spontaneous Ca²⁺ waves invariably originated from the perinuclear region where clusters of mitochondria surround the nucleus. Perinuclear Ca²⁺ dynamics were characterized by a gradual rise in basal Ca²⁺ that preceded each regenerative Ca²⁺ transient. Caffeine evoked oscillatory Ca²⁺ waves originating from anywhere within ICC-LCs. Ryanodine or cyclopiazonic acid prevented Ca²⁺ wave generation with a rise in basal Ca²⁺, and subsequent caffeine evoked a single rudimentary Ca²⁺ transient. Inhibition of glycolysis with 2-deoxy-glucose or carbonyl cyanide 3-chlorophenylhydrazone, a mitochondrial protonophore, increased basal Ca²⁺ and abolished Ca²⁺ waves. However, caffeine still induced oscillatory Ca²⁺ transients. Mitochondrial Ca²⁺ uptake inhibition with RU360 attenuated Ca²⁺ wave amplitudes, while mitochondrial Ca²⁺ efflux inhibition with {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 suppressed the initial Ca²⁺ rise to reduce Ca²⁺ wave frequency.

CONCLUSIONS AND IMPLICATIONS

Perinuclear mitochondria in ICC-LCs play a dominant role in the spatial regulation of Ca²⁺ wave generation and may regulate ER Ca²⁺ release frequency by buffering Ca²⁺ within microdomains between both organelles. Glycolysis inhibition reduced mitochondrial Ca²⁺ buffering without critically disrupting ER function. Perinuclear mitochondria may function as sensors of intracellular metabolites.     

Lipoic acid inhibits caspase-dependent and -independent cell death pathways and is neuroprotective against hippocampal damage after pilocarpine-induced seizures

Alpha-lipoic acid has some neuroprotective properties, but this action has not been investigated in models of epilepsy. The aim of the present study was to investigate the protective efficacy of α-lipoic acid (lipoic acid) against pilocarpine-induced cell death through the caspase-dependent or -independent mitochondrial apoptotic pathways. Wistar rats were injected intraperitoneally with 0.9% saline (control group), pilocarpine (400 mg/kg, pilocarpine group) alone, or α-lipoic acid (20 mg/kg) in association with pilocarpine (400 mg/kg) 30 min before administration of α-lipoic acid. After the treatments all groups were observed for 24 h. Cell death was reduced in lipoic acid-treated rats. Cytosolic translocation of cytochrome c and subsequent activation of caspase-3 were reduced by lipoic acid treatment. AIF nuclear translocation and subsequent large-scale DNA fragmentation were also decreased in lipoic acid-treated rats. Our study suggests that lipoic acid inhibits both caspase-dependent and -independent apoptotic pathways and may be neuroprotective against hippocampal damage during pilocarpine-induced seizures.     

Synthesis of New Lipoic Acid Conjugates and Evaluation of Their Free Radical Scavenging and Neuroprotective Activities

A series of new lipoic acid derivatives were designed and synthesized as multitarget ligands against Alzheimer's disease. In particular, analogues combining both lipoic acid and cysteine core structures were synthesized. The antioxidant properties of these compounds were evaluated by 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH), 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid (ABTS^?+) radical cation scavenging assays and ferrous ion chelation. The antioxidant potential of the synthesized compounds was also evaluated in a cellular context and compared to α‐lipoic acid and its reduced form, dihydrolipoic acid. The antioxidant effects observed for these compounds in vitro confirmed the importance of free thiol functions for effective antioxidant capacities. However, these promising in vitro results were not mirrored by the antioxidant activity in T67 cell line. This suggests that multiple factors are at stake and warrant further investigations.     

CGP37157 modulates mitochondrial Ca homeostasis in cultured rat dorsal root ganglion neurons

The effects of 7-chloro-3,5-dihydro-5-phenyl-1H-4,1-benzothiazepine-2-on (CGP37157), an inhibitor of mitochondrial Na⁺/Ca²⁺ exchange, on depolarization-induced intracellular free Ca²⁺ concentration ([Ca²⁺]_i) transients were studied in cultured rat dorsal root ganglion neurons with indo-1-based microfluorimetry. A characteristic plateau in the recovery phase of the [Ca²⁺]_i transient resulted from mitochondrion-mediated [Ca²⁺]_i buffering. It was blocked by metabolic poisons and was not dependent on extracellular Ca²⁺. CGP37157 produced a concentration-dependent decrease in the amplitude of the mitochondrion-mediated plateau phase (IC₅₀=4±1 μM). This decrease in [Ca²⁺]_i was followed by an increase in [Ca²⁺]_i upon removal of the drug, suggesting that Ca²⁺ trapped in the matrix was released when the CGP37157 was removed from the bath. CGP37157 also inhibited depolarization-induced Ca²⁺ influx at the concentrations required to see effects on [Ca²⁺]_i buffering. Thus, CGP37157 inhibits mitochondrial Na⁺/Ca²⁺ exchange and directly inhibits voltage-gated Ca²⁺ channels, suggesting caution in its use to study [Ca²⁺]_i regulation in intact cells.     

Acanthopanax senticosus exerts neuroprotective effects through HO-1 signaling in hippocampal and microglial cells

Extracts of Acanthopanax senticosus, a traditional herb commonly found in Northeastern Asia, are used for treating neurodegenerative disorders such as ischemia and depression. However, the mechanisms of its neuroinflammatory and cytoprotective effects have not been investigated. We examined the mechanism of A. senticosus activity in anti-neuroinflammatory and neuroprotective processes. HO-1 is an inducible enzyme present in most cell lines. ASE increased HO-1 expression, which reduced LPS-induced nitric oxide/ROS production in BV2 cells. Moreover, the induction of HO-1 expression protected cells against glutamate-induced neuronal cell death. Activation of the p38-CREB pathway and translocation of Nrf2 are strongly involved in ASE-induced HO-1 expression. Our results showed that ASE-induced HO-1 expression through the p38-CREB pathway plays an important role in the generation of anti-neuroinflammatory and neuroprotective responses. ASE also increases the translocation of Nrf2 to regulate HO-1 expression. Furthermore, our results indicate that ASE serves as a potential therapeutic agent for neuronal disorders.     

The GABAB receptor antagonists CGP35348 and CGP52432 inhibit glycine exocytosis: Study with GABAB1- and GABAB2-deficient mice

GABA_B receptors mediate inhibition of neurotransmitter exocytosis from nerve endings. Unexpectedly, the well known GABA_B receptor antagonist CGP35348 and, in part, the compound CGP52432, are now found to inhibit on their own the K⁺-evoked exocytosis of glycine when added at low micromolar concentrations to superfused mouse glycinergic nerve endings prelabelled with [³H]glycine through GLYT2 transporters. CGP35348 inhibited [³H]glycine release both in spinal cord and in hippocampus, but was also able to prevent the inhibitory effect of (?)-baclofen; CGP52432 exhibited intrinsic activity only in the hippocampus; in spinal cord, it behaved exclusively as a silent orthosteric antagonist by blocking the release inhibition brought about by (?)-baclofen. The intrinsic activity of CGP35348 in spinal cord was not prevented by CGP52432, indicating that CGP35348 is not a partial GABA_B agonist in this experimental system. CGP54626, an extremely potent antagonist, exhibited only a minimal intrinsic activity. SCH50911, a GABA_B antagonist belonging to a different chemical class, was devoid of significant activity, while phaclofen was effective only at 100–300 μM. In synaptosomes purified from the spinal cord or the hippocampus of mice lacking either the GABA_B1 (GABA_B1?/? mice) or the GABA_B2 (GABA_B2?/? mice) subunit, the evoked exocytosis of [³H]glycine was no longer inhibited by (?)-baclofen, whereas the intrinsic activity of CGP35348 and CGP52432 was not decreased. Activation of unknown sites on glycinergic terminals is likely to be involved. These unexpected effects should not be ignored when interpreting results obtained with the above GABA_B receptor antagonists.     

Participation of nuclear factor (erythroid 2-related), factor 2 in ameliorating lithocholic acid-induced cholestatic liver injury in mice

BACKGROUND AND PURPOSE

Lithocholic acid (LCA), the most toxic bile acid, induces cholestatic liver injury in rodents. We previously showed that LCA activates the oxidative stress-responsive nuclear factor (erythroid-2 like), factor 2 (Nrf2) in cultured liver cells, triggering adaptive responses that reduce cell injury. In this study, we determined whether Nrf2 protects the liver against LCA-induced toxicity in vivo.

EXPERIMENTAL APPROACH

Nrf2 disrupted (Nrf2^−/−) and wild-type mice were treated with LCA (125 mg·kg⁻¹ body weight) to induce liver injury. Levels of mRNA, protein and function of important Nrf2 target genes coupled with liver histology and injury biomarkers of mice were examined.

KEY RESULTS

In 4 day LCA treatments, we observed a significantly higher hepatic induction of Nrf2 target, cytoprotective genes including thioredoxin reductase 1, glutamate cysteine ligase subunits, glutathione S-transferases, haeme oxygenase-1 and multidrug resistance-associated proteins 3 and 4 in the wild type as compared with the Nrf2^−/− mice. Moreover, basal and LCA-induced hepatic glutathione and activities of glutathione S-transferases and thioredoxin reductases were higher in wild-type than in Nrf2^−/− mice. This reduced production of cytoprotective genes against LCA toxicity rendered Nrf2^−/− mice more susceptible to severe liver damage with the presence of multifocal liver necrosis, inflamed bile ducts and elevation of lipid peroxidation and liver injury biomarkers, such as alanine aminotransferase and alkaline phosphatase.

CONCLUSIONS AND IMPLICATIONS

Nrf2 plays a crucial cytoprotective role against LCA-induced liver injury by orchestrating adaptive responses. The pharmacological potential of targeting liver Nrf2 in the management of cholestatic liver diseases is proposed.     

Ibuprofen and Lipoic Acid Diamides as Potential Codrugs with Neuroprotective Activity

Current evidences support the hypothesis that non‐steroidal anti‐inflammatory drugs (NSAIDs) and antioxidant therapy might protect against the development of Alzheimer's disease (AD). In the present work, our attention was focused on ibuprofen (IBU) used in clinical trails to prevent Alzheimer's disease, and (R)‐α‐lipoic acid (LA) considered as a potential neuroprotective agent in AD therapy. In particular, we investigated a series of lipophilic molecular combinations obtained by joining (R)‐α‐lipoic acid and ibuprofen via an amide bond. These new entities might allow targeted delivery of the parent drugs to neurons, where cellular oxidative stress and inflammation seem related to Alzheimer's disease. Our study included the synthesis of conjugates 1–3 and the evaluation of their physicochemical and in‐vitro antioxidant properties. The new compounds are extremely stable in aqueous buffer solutions (pH = 1.3 and 7.4), and in rat and human plasma they showed a slow bioconversion to ibuprofen and (R)‐α‐lipoic acid. Codrugs 1–3 displayed in vitro free radical scavenging activity and were hydrolyzed more rapidly in brain tissue than in rat serum indicating that these new entities might allow targeted delivery of the parent drugs to neurons. The immunohistochemical analysis of Aβ (1‐40) protein showed that Aβ‐injected cerebral cortices treated with ibuprofen or compound 1 showed few plaques within capillary vessels and, in particular, Aβ (1‐40) protein was less expressed in codrug‐ 1 ‐treated than in ibuprofen‐treated cerebral cortex.     

Arsenic intoxication-induced reduction of glutathione level and of the activity of related enzymes in rat brain regions: reversal by dl-α-lipoic acid

The purpose of this study was to examine the effects of dl--lipoic acid (LA) on arsenic (As) induced alteration of glutathione (GSH) level and of the activity of glutathione-related enzymes—glutathione peroxidase (GSH-Px), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PDH)—in rat brain regions (cortex, hypothalamus, striatum, cerebellum and hippocampus). Male Wistar rats of 150±10 g weight were divided into four groups: control and three experimental groups supplemented with arsenic (sodium arsenite) alone (100 ppm mixed in drinking water), lipoic acid alone (70 mg kg^–1 body weight), arsenic plus lipoic acid (100 ppm arsenic in drinking water plus 70 mg lipoic acid kg^–1 body weight). The arsenic content of brain regions was found to increase with the administration of sodium arsenite. Arsenic exposure elicited a significant decline in glutathione content and in the activity of related enzymes, with the greatest decreases seen in the cortex, striatum, and hippocampus, whereas there were no significant differences between control rats and the group treated with lipoic acid alone. Highly elevated content of the thiobarbituric acid-reactive substance malondialdehyde (MDA) in the brain regions of arsenic-exposed rats reflected extensive lipid peroxidation (LPO) processes. Simultaneous lipoic acid treatment was effective in reducing brain regional arsenic levels and lipid peroxidation and in increasing the glutathione content and the activity of its related enzymes. Lipoic acid, by acting as an alternative sulfhydryl nucleophile to glutathione, prevents its oxidation to glutathione disulfide in detoxifying reactions against reactive oxygen species and consequently increases the activity of glutathione-related enzymes.     

The competitive NMDA receptor antagonists CGP 37849 and CGP 39551 are potent,orally-active anticonvulsants in rodents

Summary Anticonvulsant properties of CGP 37849 and CGP 39551, two novel phosphono-amino acids which are competitive NMDA receptor antagonists, were examined in rodents. At optimal pretreatment times CGP 37849 suppressed electroshock-induced seizures in mice and rats with ED₅₀ ^s ranging from 8 to 22 mg/kg after oral administration, and 0.4 to 2.4 mg/kg after i. v. and i. p. injection. Relative to CGP 37849, CGP 39551 was more potent after p. o. (ED₅₀ 3.7–8.1 mg/kg), and less potent after i.v. or i.p. treatment (ED₅₀ 2.7–8.7 mg/kg). Following oral treatment, the duration of action of CGP 37849 was about 8 h, while CGP 39551 still showed good activity after 24 h (ED₅₀ 8.7 mg/kg, mouse; 21 mg/kg, rat). Both compounds were anticonvulsant at doses below those at which overt behavioural side effects were apparent. CGP 39551 delayed the development of kindling in rats at doses of 10 mg/kg p. o. and above, and showed weak anticonvulsant activity against pentylenetetrazolevoked seizures. CGP 37849 and CGP 39551 are the first competitive NMDA antagonists to show oral anticonvulsant properties in a therapeutically-useful dose-range, and hence are interesting candidates for novel antiepileptic therapy in man. Send offprint requests to M. Schmutz at the above address     

CGP 25454A,a novel and selective presynaptic dopamine autoreceptor antagonist

N-(diethylamino-ethyl)-4-chloro-5-cyano-2-methoxy-benzamide-hydrochloride (CGP 25454A) is a new benzamide derivative now in clinical trials in patients with major depression. Here we describe some basic neurochemical and behavioural properties in animal experiments. In vitro, CGP 25454A increased the field-stimulated [³H]- and [¹⁴C]-overflow from rat striatal slices preloaded with [³H]dopamine and [¹⁴C] choline, indicating that CGP 25454A was able to enhance the release of both dopamine (DA) and acetylcholine (ACh). However, CGP 25454A was 12.9 times more potent in increasing, by 1/6 of the apparent maximal increase, the release of [³H]DA than that of [¹⁴C]ACh. In vivo, CGP 25454A increased [³H]spiperone binding to receptors of the D₂ family in rat striatum by 90–110% (ED₅₀: 13 mg/kg i.p.). As a similar increase in [³H]spiperone binding was found with a variety of agents which increase the synaptic concentration of endogenous DA, the effect of CGP 25454A most probably reflects an enhanced release of DA under in vivo conditions. At 30–100 mg/kg, CGP 25454A inhibited [³H]spiperone binding in the pituitary of the same animals as a result of a blockade of postsynaptic DA receptors. This dual mode of action was also apparent in terms of behavioral changes. At doses as low as 5–10 mg/kg, CGP 25454A produced a weak stimulation, suggested by a trend of increased spontaneous rearing and corroborated by a significant potentiation of the elevated rearing induced by (+)-amphetamine. By contrast, at doses of 30–100 mg/kg, it exerted clear-cut sedative and neuroleptic-like properties. These data obtained from three different experimental approaches suggest that CGP 25454A selectively blocks presynaptic DA autoreceptors in the lower dose range whereas at higher doses it also blocks the postsynaptic receptors. Correspondence to: S. Bischoff at the above address     

Caloric Restriction-Mediated Induction of Lipid Metabolism Gene Expression in Liver is Enhanced by Keap1-Knockdown

Purpose

CR increases fatty acid oxidation to decrease tissue lipid content. The Nuclear factor E2-related factor 2 (Nrf2)-Kelch like ECH associated Protein 1 (Keap1) pathway is an antioxidant gene regulatory pathway that has been previously investigated in weight gain. However, limited interaction of Nrf2/Keap1 and CR exists. The purpose of this study was to determine how Keap1 knockdown (Keap1-KD), which is known to increase Nrf2 activity, affects the CR response, such as weight loss, hepatic lipid decrease, and induction of fatty acid oxidation gene expression.

Methods

C57BL/6 and Keap1-KD mice were maintained on 40% CR or fed ad libitum for 6 weeks. Hepatic lipid content, lipid metabolic gene, and miRNA expression was quantified.

Results

CR lowered hepatic lipid content, and induced fatty acid oxidation gene expression to a greater degree in Keap1-KD compared to C57BL/6 mice. CR differentially altered miRNA 34a, 370, let-7b* in livers of Keap1-KD compared to C57BL/6 mice.

Conclusions

CR induced induction of fatty acid oxidation gene expression was augmented with Keap1 knockdown, which was associated with differential expression of several miRNAs implicated in fatty acid oxidation and lipid accumulation.     

Thymoquinone induces heme oxygenase-1 expression in HaCaT cells via Nrf2/ARE activation: Akt and AMPKα as upstream targets

Thymoquinone (TQ), an active constituent of Nigella sativa, possesses anti-inflammatory and anticancer properties. Multiple lines of evidence suggest that the induction of heme oxygenase-1 (HO-1) suppresses inflammation and carcinogenesis. In the present study, we examined the effect of TQ on HO-1 expression in human keratinocytes (HaCaT) and elucidated its underlying molecular mechanisms. TQ induced the expression of HO-1 in HaCaT cells in a concentration- and time-dependent manner. Treatment with TQ increased the localization of nuclear factor (NF)-erythroid2-(E2)-related factor-2 (Nrf2) in the nucleus and elevated the antioxidant response element (ARE)-reporter gene activity. Knockdown of Nrf2 abrogated TQ-induced HO-1 expression and the ARE luciferase activity. TQ induced the phosphorylation of extracellular signal-regulated kinase (ERK), Akt and cyclic AMP-activated protein kinase-α (AMPKα). Pharmacological inhibition of Akt or AMPKα, but not that of ERK, abrogated TQ-induced nuclear localization of Nrf2, the ARE-luciferase activity and the expression of HO-1. TQ also generated reactive oxygen species (ROS) and pretreatment with N-acetyl cysteine (NAC) abrogated TQ-induced ROS accumulation, Akt and AMPKα activation, Nrf2 nuclear localization, the ARE-luciferase activity, and HO-1 expression in HaCaT cells. Taken together, TQ induces HO-1 expression in HaCaT cells by activating Nrf2 through ROS-mediated phosphorylation of Akt and AMPKα.