Abstract Epoxide hydrolases comprise a family of enzymes important in detoxification and conversion of lipid signaling molecules, namely epoxyeicosatrienoic acids (EETs), to their supposedly less ...active form, dihydroxyeicosatrienoic acids (DHETs). EETs control cerebral blood flow, exert analgesic, anti-inflammatory and angiogenic effects and protect against ischemia. Although the role of soluble epoxide hydrolase (sEH) in EET metabolism is well established, knowledge on its detailed distribution in rodent brain is rather limited. Here, we analyzed the expression pattern of sEH and of another important member of the EH family, microsomal epoxide hydrolase (mEH), in mouse brain by immunohistochemistry. To investigate the functional relevance of these enzymes in brain, we explored their individual contribution to EET metabolism in acutely isolated brain cells from respective EH −/− mice and wild type littermates by mass spectrometry. We find sEH immunoreactivity almost exclusively in astrocytes throughout the brain, except in the central amygdala, where neurons are also positive for sEH. mEH immunoreactivity is abundant in brain vascular cells (endothelial and smooth muscle cells) and in choroid plexus epithelial cells. In addition, mEH immunoreactivity is present in specific neuronal populations of the hippocampus, striatum, amygdala, and cerebellum, as well as in a fraction of astrocytes. In freshly isolated cells from hippocampus, where both enzymes are expressed, sEH mediates the bulk of EET metabolism. Yet we observe a significant contribution of mEH, pointing to a novel role of this enzyme in the regulation of physiological processes. Furthermore, our findings indicate the presence of additional, hitherto unknown cerebral epoxide hydrolases. Taken together, cerebral EET metabolism is driven by several epoxide hydrolases, a fact important in view of the present targeting of sEH as a potential therapeutic target. Our findings suggest that these different enzymes have individual, possibly quite distinct roles in brain function and cerebral EET metabolism.
The dystrophin glycoprotein complex (DGC) is a multimeric protein assembly associated with either the X-linked cytoskeletal protein dystrophin or its autosomal homologue utrophin. In striated muscle ...cells, the DGC links the extracellular matrix to the actin cytoskeleton and mediates three major functions: structural stability of the plasma membrane, ion homeostasis, and transmembrane signaling. Mutations affecting the DGC underlie major forms of congenital muscle dystrophies. The DGC is prominent also in the central and peripheral nervous system and in tissues with a secretory function or which form barriers between functional compartments, such as the blood-brain barrier, choroid plexus, or kidney. A considerable molecular heterogeneity arises from cell-specific expression of its constituent proteins, notably short C-terminal isoforms of dystrophin. Experimentally, the generation of mice carrying targeted gene deletions affecting the DGC has clarified the interdependence of DGC proteins for assembly of the complex and revealed its importance for brain development and regulation of the 'milieu intérieur. Here, we focus on recent studies of the DGC in brain, blood-brain barrier and choroid plexus, retina, and kidney and discuss the role of dystrophin isoforms and utrophin for assembly of the complex in these tissues.
Multidrug transporters of the ATP-binding cassette family play an important role in regulating drug distribution and efflux in the brain, owing to their selective distribution in microvessels and ...choroid plexus. Their expression pattern and cellular distribution remain controversial, in part due to technical difficulties in localizing these membrane proteins in closely associated cells, such as endothelial cells and astrocytic end-feet at the blood–brain barrier. Here, we used high-resolution immunofluorescence staining with cell-type specific markers to investigate the distribution of major ATP-binding cassette transporters in mouse brain. We report that four ATP-binding cassette transporters, Mdr1, Mrp1, Mrp2 and Mrp5 can be detected in brain endothelial cells, forming three distinct layers, with Mdr1 and Mrp5 being located on the luminal side, Mrp1 on the abluminal (basal) side, and Mrp2 in between. Mrp3 and Mdr3 were undetectable. In choroid plexus, only Mrp1, Mrp2 and Mrp3 were detected, again with a differential distribution. Mrp1 was targeted basolaterally in epithelial cells, Mrp2 was restricted to endothelial cells, and Mrp3 was co-localized with zonula occludens-1 at tight junctions. Analysis of Mdr1a
0/0 and Mrp1
0/0 mice, generated in the FVB strain, revealed no major alteration in expression of the remaining transporters. An unexpected strain difference was unraveled, with wildtype FVB mice selectively lacking Mrp2 protein in brain, but not liver. In conclusion, these results indicate that ATP-binding cassette transporters provide multiple penetration barriers in the blood–brain barrier and choroid plexus, with a selective cellular and subcellular distribution, emphasizing their potential role for drug resistance in neurological disorders, such as epilepsy.
Unilateral intrahippocampal injection of kainic acid in adult mice reproduces most of the morphological characteristics of hippocampal sclerosis (neuronal loss, gliosis, reorganization of ...neurotransmitter receptors, mossy fiber sprouting, granule cell dispersion) observed in patients with temporal lobe epilepsy. Whereas some neuronal loss is observed immediately after the initial status epilepticus induced by kainate treatment, most reorganization processes develop progressively over a period of several weeks. The aim of this study was to characterize the evolution of seizure activity in this model and to assess its pharmacological reactivity to classical antiepileptic drugs.
Intrahippocampal electroencephalographic recordings showed three distinct phases of paroxystic activity following unilateral injection of kainic acid (1 nmol in 50 nl) into the dorsal hippocampus of adult mice: (i) a non-convulsive status epilepticus, (ii) a latent phase lasting approximately 2 weeks, during which no organized activity was recorded, and (iii) a phase of chronic seizure activity with recurrent hippocampal paroxysmal discharges characterized by high amplitude sharp wave onset. These recurrent seizures were first seen about 2 weeks post-injection. They were limited to the injected area and were not observed in the cerebral cortex, contralateral hippocampus or ipsilateral amygdala. Secondary propagation to the contralateral hippocampus and to the cerebral cortex was rare. In addition hippocampal paroxysmal discharges were not responsive to acute carbamazepine, phenytoin, or valproate treatment, but could be suppressed by diazepam.
Our data further validate intrahippocampal injection of kainate in mice as a model of temporal lobe epilepsy and suggest that synaptic reorganization in the lesioned hippocampus is necessary for the development of organized recurrent seizures.
Benzodiazepine tranquilizers are used in the treatment of anxiety disorders. To identify the molecular and neuronal target mediating the anxiolytic action of benzodiazepines, we generated and ...analyzed two mouse lines in which the α2 or α3 GABAA(γ-aminobutyric acid type A) receptors, respectively, were rendered insensitive to diazepam by a knock-in point mutation. The anxiolytic action of diazepam was absent in mice with the α2(H101R) point mutation but present in mice with the α3(H126R) point mutation. These findings indicate that the anxiolytic effect of benzodiazepine drugs is mediated by α2 GABAAreceptors, which are largely expressed in the limbic system, but not by α3 GABAAreceptors, which predominate in the reticular activating system.
A new benzodiazepine pharmacology Möhler, H; Fritschy, J M; Rudolph, U
The Journal of pharmacology and experimental therapeutics
300, Številka:
1
Journal Article
Recenzirano
Classical benzodiazepine drugs are in wide clinical use as anxiolytics, hypnotics, anticonvulsants, and muscle relaxants. They act by enhancing the gamma-aminobutyric acid(A) (GABA(A)) receptor ...function in the central nervous system. The pharmacological relevance of the multitude of structurally diverse GABA(A) receptor subtypes has only recently been identified. Based on an in vivo point mutation strategy, alpha(1)-GABA(A) receptors were found to mediate sedation, anterograde amnesia, and part of the seizure protection, whereas alpha(2)-GABA(A) receptors, but not alpha(3)-receptors, mediate anxiolysis. Rational drug targeting to specific receptor subtypes has now become possible. Only restricted neuronal networks will be modulated by the new subtype-selective drugs. Promising new anxiolytics have already been developed. A new pharmacology of the benzodiazepine site is on the horizon.
Endogenous adenosine in the brain is thought to prevent the development and spread of seizures via a tonic anticonvulsant effect. Brain levels of adenosine are primarily regulated by the activity of ...adenosine kinase. To establish a link between adenosine kinase expression and seizure activity, we analyzed the expression of adenosine kinase in the brain of control mice and in a kainic acid-induced mouse model of mesial temporal lobe epilepsy. Immunohistochemical analysis of brain sections of control mice revealed intense staining for adenosine kinase, mainly in astrocytes, which were more or less evenly distributed throughout the brain, as well as in some neurons, particularly in olfactory bulb, striatum, and brainstem. In contrast, hippocampi lesioned by a unilateral kainic acid injection displayed profound astrogliosis and therefore a significant increase in adenosine kinase immunoreactivity accompanied by a corresponding increase of enzyme activity, which paralleled chronic recurrent seizure activity in this brain region. Accordingly, seizures and interictal spikes were suppressed by the injection of a low dose of the adenosine kinase inhibitor 5-iodotubercidin. We conclude that overexpression of adenosine kinase in discrete parts of the epileptic hippocampus may contribute to the development and progression of seizure activity.
Adenosine is a potent modulator of excitatory neurotransmission, especially in seizure-prone regions such as the hippocampal formation. In adult brain ambient levels of adenosine are controlled by ...adenosine kinase (ADK), the major adenosine-metabolizing enzyme, expressed most strongly in astrocytes. Since ontogeny of the adenosine system is largely unknown, we investigated ADK expression and cellular localization during postnatal development of the mouse brain, using immunofluorescence staining with cell-type specific markers. At early postnatal stages ADK immunoreactivity was prominent in neurons, notably in cerebral cortex and hippocampus. Thereafter, as seen best in hippocampus, ADK gradually disappeared from neurons and appeared in newly developed nestin- and glial fibrillary acidic protein (GFAP)-positive astrocytes. Furthermore, the region-specific downregulation of neuronal ADK coincided with the onset of myelination, as visualized by myelin basic protein staining. After postnatal day 14 (P14), the transition from neuronal to astrocytic ADK expression was complete, except in a subset of neurons that retained ADK until adulthood in specific regions, such as striatum. Moreover, neuronal progenitors in the adult dentate gyrus lacked ADK. Finally, recordings of excitatory field potentials in acute slice preparations revealed a reduced adenosinergic inhibition in P14 hippocampus compared with adult. These findings suggest distinct roles for adenosine in the developing and adult brain. First, ADK expression in young neurons may provide a salvage pathway to utilize adenosine in nucleic acid synthesis, thus supporting differentiation and plasticity and influencing myelination; and second, adult ADK expression in astrocytes may offer a mechanism to regulate adenosine levels as a function of metabolic needs and synaptic activity, thus contributing to the differential resistance of young and adult animals to seizures.
Objectives – Interindividual variability in intestinal absorption and bioavailability might contribute to inadequate control of seizures under treatment with carbamazepine and phenytoin. We ...therefore correlated intestinal expression levels and genetics of CYP3A4, CYP2C9/19, MDR1 and MRP2 with dose requirement and plasma levels of carbamazepine and phenytoin.
Materials and methods – Epileptic patients on carbamazepine (n = 29) or phenytoin (n = 15) were stratified into a ‘high’‐dose (carbamazepine ≥800 mg/day, phenytoin ≥300 mg/day) and a ‘low’‐dose group (carbamazepine ≤600 mg/day, phenytoin ≤200 mg/day). Duodenal biopsies and DNA were obtained for Western blotting and genotyping studies.
Results – Low carbamazepine plasma levels showed a trend towards higher intestinal MDR1 expression (P = 0.06). Furthermore, carbamazepine dose was positively correlated with MRP2 expression (P = 0.1). Moreover, MDR1 expression and carbamazepine and phenytoin dose requirement was influenced by the genotype in position 2677 and 3435 of the MDR1 gene.
Conclusion – Differences in intestinal MDR1 and MRP2 expression may influence carbamazepine and phenytoin disposition and may account for interindividual pharmacokinetic variability.
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