mRNA In Cortex Research Articles

Age-Dependent Decrease and Alternative Splicing of Methionine Synthase mRNA in Human Cerebral Cortex and an Accelerated Decrease in Autism

The folate and vitamin B12-dependent enzyme methionine synthase (MS) is highly sensitive to cellular oxidative status, and lower MS activity increases production of the antioxidant glutathione, while simultaneously decreasing more than 200 methylation reactions, broadly affecting metabolic activity. MS mRNA levels in postmortem human cortex from subjects across the lifespan were measured and a dramatic progressive biphasic decrease of more than 400-fold from 28 weeks of gestation to 84 years was observed. Further analysis revealed alternative splicing of MS mRNA, including deletion of folate-binding domain exons and age-dependent deletion of exons from the cap domain, which protects vitamin B12 (cobalamin) from oxidation. Although three species of MS were evident at the protein level, corresponding to full-length and alternatively spliced mRNA transcripts, decreasing mRNA levels across the lifespan were not associated with significant changes in MS protein or methionine levels. MS mRNA levels were significantly lower in autistic subjects, especially at younger ages, and this decrease was replicated in cultured human neuronal cells by treatment with TNF-α, whose CSF levels are elevated in autism. These novel findings suggest that rather than serving as a housekeeping enzyme, MS has a broad and dynamic role in coordinating metabolism in the brain during development and aging. Factors adversely affecting MS activity, such as oxidative stress, can be a source of risk for neurological disorders across the lifespan via their impact on methylation reactions, including epigenetic regulation of gene expression.

Regulation of Two Renal Chloride Transporters, AE1 and Pendrin, by Electrolytes and Aldosterone

The renal handling of salt and protons and bicarbonate are intricately linked through shared transport mechanisms for sodium, chloride, protons, and bicarbonate. In the collecting duct, the regulated fine-tuning of salt and acid-base homeostasis is achieved by a series of transport proteins located in different cell types, intercalated and principal cells. Intercalated cells are considered to be of less importance for salt handling but recent evidence has suggested that the anion exchanger pendrin may participate in salt reabsorption and blood pressure regulation. Here, we examined the regulated expression of two functionally related but differentially expressed anion exchangers, AE1 and pendrin, by dietary electrolyte intake and aldosterone. Cortical expression of pendrin was regulated on mRNA and protein level. The combination of NaHCO3 and DOCA enhanced pendrin mRNA and protein levels, whereas DOCA or NaHCO3 alone had no effect. NaCl or KHCO3 increased pendrin mRNA, KCl decreased its mRNA abundance. On protein level, NH4Cl, NaCl, and KCl reduced pendrin expression, the other treatments were without effect. In contrast, AE1 mRNA or protein expression in kidney cortex was regulated by none of these treatments. In kidney medulla, NaHCO3/DOCA or NaHCO3 alone enhanced AE1 mRNA levels. AE1 protein abundance was increased by NH4Cl, NaHCO3/DOCA, and NaCl. Immunolocalization showed that during NH4Cl treatment the relative number of AE1 positive cells was increased and pendrin expressing cells reduced. Thus, pendrin and AE1 are differentially regulated with distinct mechanisms that separately affect mRNA and protein levels. Pendrin is regulated by acidosis and chloride intake, whereas AE1 is enhanced by acidosis, NaCl, and the combination of DOCA and NaHCO3.

Tetramethylpyrazine이 LPS의 뇌실주입에 따른 생쥐 뇌조직의 Pro-Inflammatory Cytokines 발현에 미치는 영향

Objectives : Tetramethylpyrazine (TMP) is an active ingredient in Ligusticum wallichii and has a wide range of neuroprotection effects. This study investigated anti-neuroinflammatory effect of TMP on brain regions in intracerebroventricular (i.c.v.) lipopolysaccharide (LPS)-treated C57BL/6 mice. Methods : TMP was administered intraperitoneally at doses of 10, 20, and 30 mg/kg at 1 h prior to LPS (3 mg/kg) i.c.v. injection. mRNA level of pro-inflammatory cytokines, including tumor necrosis factor-<TEX>${\alpha}$</TEX> (TNF-<TEX>${\alpha}$</TEX>), interleukin (IL)-<TEX>$1{\beta}$</TEX> and IL-6, was measured in the cerebral cortex, hippocampus, and hypothalamus tissue using real-time polymerase chain reaction at 24 h after the LPS injection. Cyclooxygenase-2 (COX-2) positive cells in the hypothalamus was also observed using immunohistochemistry at 24 h after the LPS injection. Results : At a dose of 30 mg/kg TMP significantly attenuated up-regulation of TNF-<TEX>${\alpha}$</TEX> and IL-<TEX>$1{\beta}$</TEX> mRNA in the cerebral cortex and IL-<TEX>$1{\beta}$</TEX> mRNA in the hippocampus. In the hypothalamus, doses of 20 mg/kg and 30 mg/kg TMP significantly attenuated up-regulation of TNF-<TEX>${\alpha}$</TEX>, IL-<TEX>$1{\beta}$</TEX>, and IL-6 mRNA induced by the LPS injection. In addition, TMP (30 mg/kg) significantly reduced the number of COX-2 positive cells in the hypothalamus. Conclusion : These results indicate that TMP has an anti-inflammatory effect on neuroinflammation, especially in the hypothalamus, induced by LPS i.c.v. injection and suggest that TMP-containing Ligusticum wallichii may play a modulatory role on the systemic responses following hypothalamic inflammation.

Changes in secretory pathway Ca(2+)-ATPase 2 following focal cerebral ischemia/reperfusion injury.

This study aimed to investigate changes in secretory pathway Ca2+-ATPase 2 expression following cerebral ischemia/reperfusion injury, and to define the role of Ca2+-ATPases in oxidative stress. A rat model of cerebral ischemia/reperfusion injury was established using the unilateral middle cerebral artery occlusion method. Immunohistochemistry and reverse transcription-PCR assay results showed that compared with the control group, the expression of secretory pathway Ca2+-ATPase 2 protein and mRNA in the cerebral cortex and hippocampus of male rats did not significantly change during the ischemic period. However, secretory pathway Ca2+-ATPase 2 protein and mRNA expression reduced gradually at 1, 3, and 24 hours during the reperfusion period. Our experimental findings indicate that levels of secretory pathway Ca2+-ATPase 2 protein and mRNA expression in brain tissue change in response to cerebral ischemia/reperfusion injury.

Reduction in phencyclidine induced sensorimotor gating deficits in the rat following increased system xc⁻ activity in the medial prefrontal cortex.

Aspects of schizophrenia, including deficits in sensorimotor gating, have been linked to glutamate dysfunction and/or oxidative stress in the prefrontal cortex. System xc(-), a cystine-glutamate antiporter, is a poorly understood mechanism that contributes to both cellular antioxidant capacity and glutamate homeostasis. Our goal was to determine whether increased system xc(-) activity within the prefrontal cortex would normalize a rodent measure of sensorimotor gating. In situ hybridization was used to map messenger RNA (mRNA) expression of xCT, the active subunit of system xc(-), in the prefrontal cortex. Prepulse inhibition was used to measure sensorimotor gating; deficits in prepulse inhibition were produced using phencyclidine (0.3-3 mg/kg, sc). N-Acetylcysteine (10-100 μM) and the system xc(-) inhibitor (S)-4-carboxyphenylglycine (CPG, 0.5 μM) were used to increase and decrease system xc(-) activity, respectively. The uptake of (14)C-cystine into tissue punches obtained from the prefrontal cortex was used to assay system xc(-) activity. The expression of xCT mRNA in the prefrontal cortex was most prominent in a lateral band spanning primarily the prelimbic cortex. Although phencyclidine did not alter the uptake of (14)C-cystine in prefrontal cortical tissue punches, intraprefrontal cortical infusion of N-acetylcysteine (10-100 μM) significantly reduced phencyclidine- (1.5 mg/kg, sc) induced deficits in prepulse inhibition. N-Acetylcysteine was without effect when coinfused with CPG (0.5 μM), indicating an involvement of system xc(-). These results indicate that phencyclidine disrupts sensorimotor gating through system xc(-) independent mechanisms, but that increasing cystine-glutamate exchange in the prefrontal cortex is sufficient to reduce behavioral deficits produced by phencyclidine.

Extinction of Aversive Memories Associated with Morphine Withdrawal Requires ERK-Mediated Epigenetic Regulation of Brain-Derived Neurotrophic Factor Transcription in the Rat Ventromedial Prefrontal Cortex

Recent evidence suggests that histone deacetylase (HDAC) inhibitors facilitate extinction of rewarding memory of drug taking. However, little is known about the role of chromatin modification in the extinction of aversive memory of drug withdrawal. In this study, we used conditioned place aversion (CPA), a highly sensitive model for measuring aversive memory of drug withdrawal, to investigate the role of epigenetic regulation of brain-derived neurotrophic factor (BDNF) gene expression in extinction of aversive memory. We found that CPA extinction training induced an increase in recruiting cAMP response element-binding protein (CREB) to and acetylation of histone H3 at the promoters of BDNF exon I transcript and increased BDNF mRNA and protein expression in the ventromedial prefrontal cortex (vmPFC) of acute morphine-dependent rats and that such epigenetic regulation of BDNF gene transcription could be facilitated or diminished by intra-vmPFC infusion of HDAC inhibitor trichostatin A or extracellular signal-regulated kinase (ERK) inhibitor U0126 (1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene) before extinction training. Correspondingly, disruption of the epigenetic regulation of BDNF gene transcription with U0126 or suppression of BDNF signaling with Trk receptor antagonist K252a or BDNF scavenger tyrosine kinase receptor B (TrkB)-Fc blocked extinction of CPA behavior. We also found that extinction training-induced activation of ERK and CREB and extinction of CPA behavior could be potentiated or suppressed by intra-vmPFC infusion of d-cycloserine, a NMDA receptor partial agonist or aminophosphonopentanoic acid, a NMDA receptor antagonist. We conclude that extinction of aversive memory of morphine withdrawal requires epigenetic regulation of BDNF gene transcription in the vmPFC through activation of the ERK-CREB signaling pathway perhaps in a NMDA receptor-dependent manner.

THE EFFECTS AND ITS MECHANISMS OF GINKGO BILOBA RECIPE ON TGF-β1 IN DIABETIC NEPHROPATHY WITH HYPERTENSION RATS

ObjectivesTo observe the effects of Ginkgo Biloba Recipe (GBR) on transforming growth factor-β1 (TGF-β1) in diabetic nephropathy (DN) with hypertension rats and investigate its mechanisms.MethodsDN with hypertension models were made...

Content of mRNA for NMDA Glutamate Receptor Subunits in the Frontal Cortex and Striatum of Rats after Morphine Withdrawal Is Related to the Degree of Abstinence

We studied the expression of mRNA for genes, whose protein products regulate the glutamate/NO/cGMP signal cascade in the frontal cortex, striatum, midbrain, and hippocampus of rats with various degrees of spontaneous morphine withdrawal syndrome. The concentration of Grin2a mRNA (subunit of the NMDA glutamate receptor) in the frontal cortex increased mainly in animals with severe abstinence. By contrast, the expression of mRNA for the Grin2b subunit in the striatum decreased in animals with mild abstinence. Variations in the content of mRNA for other products of the cascade (isoforms of NO-dependent guanylate cyclase and cGMP-dependent protein kinase) after morphine withdrawal were not related to the degree of abstinence. Our results suggest that the region-specific expression of mRNA for certain subunits of the NMDA glutamate receptor after morphine withdrawal can determine the degree of abstinence.

Interrogation of brain miRNA and mRNA expression profiles reveals a molecular regulatory network that is perturbed by mutant huntingtin

Emerging evidence indicates that microRNAs (miRNAs) may play an important role in the pathogenesis of Huntington's disease (HD). To identify the individual miRNAs that are altered in HD and may therefore regulate a gene network underlying mutant huntingtin-induced neuronal dysfunction in HD, we performed miRNA array analysis combined with mRNA profiling in the cerebral cortex from N171-82Q HD mice. Expression profiles of miRNAs as well as mRNAs in HD mouse cerebral cortex were analyzed and confirmed at different stages of disease progression; the most significant changes of miRNAs in the cerebral cortex were also detected in the striatum of HD mice. Our results revealed a significant alteration of miR-200 family members, miR-200a, and miR-200c in the cerebral cortex and the striatum, at the early stage of disease progression in N171-82Q HD mice. We used a coordinated approach to integrate miRNA and mRNA profiling, and applied bioinformatics to predict a target gene network potentially regulated by these significantly altered miRNAs that might be involved in HD disease progression. Interestingly, miR-200a and miR-200c are predicted to target genes regulating synaptic function, neurodevelopment, and neuronal survival. Our results suggest that altered expression of miR-200a and miR-200c may interrupt the production of proteins involved in neuronal plasticity and survival, and further investigation of the involvement of perturbed miRNA expression in HD pathogenesis is warranted, and may lead to reveal novel approaches for HD therapy.

Absence of SHATI/Nat8l reduces social interaction in mice

We previously identified a novel molecule “Shati/Nat8l” from the nucleus accumbens of mice. However, the physiological roles of the SHATI protein are not clear. To investigate the effect of SHATI on the central nervous system and behavior, we studied knockout mice of this protein. We carried out various behavior tests using Shati-knockout mice. Shati-knockout mice did not differ from wild type mice in learning and memory. In the open field test, Shati-knockout mice did not differ from wild-type mice in time of stay in the outer, middle and center areas. On the other hand, Shati-knockout mice showed increases in rearing and grooming time in the open field test, and exploration time of novel objects. These results suggested that knockout of the Shati gene may increase exploration in specific circumstances. Interestingly, the Shati-knockout mice avoided social interaction with unfamiliar mice out of their home cage, although there was no difference in social interaction in their home cage compared with wild type mice. Lack of the Shati gene increased brain-derived neurotrophic factor (BDNF) mRNA in the prefrontal cortex and hippocampus, and decreased glial cell line-derived neurotrophic factor (GDNF) mRNA in the striatum and hippocampus, and lipopolysaccharides-induced TNF-α factor (LITAF) mRNA in the striatum. Since these factors play important roles in behavior, alteration of expression of these factors may be related to the induction of exploration and reduction of social interaction in Shati-knockout mice.

GABAA Receptor α Subunits Differentially Contribute to Diazepam Tolerance after Chronic Treatment

BackgroundWithin the GABAA-receptor field, two important questions are what molecular mechanisms underlie benzodiazepine tolerance, and whether tolerance can be ascribed to certain GABAA-receptor subtypes.MethodsWe investigated tolerance to acute anxiolytic, hypothermic and sedative effects of diazepam in mice exposed for 28-days to non-selective/selective GABAA-receptor positive allosteric modulators: diazepam (non-selective), bretazenil (partial non-selective), zolpidem (α1 selective) and TPA023 (α2/3 selective). In-vivo binding studies with [3H]flumazenil confirmed compounds occupied CNS GABAA receptors.ResultsChronic diazepam treatment resulted in tolerance to diazepam's acute anxiolytic, hypothermic and sedative effects. In mice treated chronically with bretazenil, tolerance to diazepam's anxiolytic and hypothermic, but not sedative, effects was seen. Chronic zolpidem treatment resulted in tolerance to diazepam's hypothermic effect, but partial anxiolytic tolerance and no sedative tolerance. Chronic TPA023 treatment did not result in tolerance to diazepam's hypothermic, anxiolytic or sedative effects.ConclusionsOur data indicate that: (i) GABAA-α2/α3 subtype selective drugs might not induce tolerance; (ii) in rodents quantitative and temporal variations in tolerance development occur dependent on the endpoint assessed, consistent with clinical experience with benzodiazepines (e.g., differential tolerance to antiepileptic and anxiolytic actions); (iii) tolerance to diazepam's sedative actions needs concomitant activation of GABAA-α1/GABAA-α5 receptors. Regarding mechanism, in-situ hybridization studies indicated no gross changes in expression levels of GABAA α1, α2 or α5 subunit mRNA in hippocampus or cortex. Since selective chronic activation of either GABAA α2, or α3 receptors does not engender tolerance development, subtype-selective GABAA drugs might constitute a promising class of novel drugs.

Alteration of POLDIP3 Splicing Associated with Loss of Function of TDP-43 in Tissues Affected with ALS

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease caused by selective loss of motor neurons. In the ALS motor neurons, TAR DNA-binding protein of 43 kDa (TDP-43) is dislocated from the nucleus to cytoplasm and forms inclusions, suggesting that loss of a nuclear function of TDP-43 may underlie the pathogenesis of ALS. TDP-43 functions in RNA metabolism include regulation of transcription, mRNA stability, and alternative splicing of pre-mRNA. However, a function of TDP-43 in tissue affected with ALS has not been elucidated. We sought to identify the molecular indicators reflecting on a TDP-43 function. Using exon array analysis, we observed a remarkable alteration of splicing in the polymerase delta interacting protein 3 (POLDIP3) as a result of the depletion of TDP-43 expression in two types of cultured cells. In the cells treated with TDP-43 siRNA, wild-type POLDIP3 (variant-1) decreased and POLDIP3 lacking exon 3 (variant-2) increased. The RNA binding ability of TDP-43 was necessary for inclusion of POLDIP3 exon 3. Moreover, we found an increment of POLDIP3 variant-2 mRNA in motor cortex, spinal cord and spinal motor neurons collected by laser capture microdissection with ALS. Our results suggest a loss of TDP-43 function in tissues affected with ALS, supporting the hypothesis that a loss of function of TDP-43 underlies the pathogenesis of ALS.

Presenilin 1 and 2 are expressed differentially in the cerebral cortex of mice during development

Presenilin (PS) 1 and PS2 are multi-pass transmembrane proteins involved in vital brain functions. Studies using transgenic or conditional knockout models show that PS1 is implicated in crucial brain developmental processes. Conversely, PS2 knockout mice do not exhibit any abnormality in the brain morphology, suggesting that PS2 may not be involved in brain development. However, there is no holistic information available for endogenous expression of PS during brain development. Therefore, we have examined the distribution and expression profile of PS1 and PS2 mRNA and protein in the cerebral cortex of prenatal, neonatal and postnatal mice. The results revealed that the distribution and expression profile of PS1 and PS2 mRNA varied significantly in the cerebral cortex during development. In prenatal stages, both PS1 and PS2 mRNA showed high expression at embryonic day (E) 12.5 and downregulation at E18.5. Postnatally, PS1 mRNA showed upregulation from postnatal day 0 (P0) to P45 and thereafter reduction at 20weeks, but PS2 mRNA showed no significant alteration. However, they did not exhibit any significant regional variation except at E18.5, when PS2 showed reduction in temporal and medial temporal lobes as compared to frontal and parietal lobes. Furthermore, PS1 showed significant change in protein expression similar to its mRNA profile. However, PS2 protein expression did not correspond to its mRNA; it was highest at E12.5, downregulated up to P20 and then upregulated at P45 and 20weeks. Taken together, our study demonstrates for the first time that the distribution and expression profile of PS2 is different from PS1 in the mouse cerebral cortex during development.

Astragaloside IV, a Novel Antioxidant, Prevents Glucose-Induced Podocyte Apoptosis In Vitro and In Vivo

Glucose-induced reactive oxygen species (ROS) production initiates podocyte apoptosis, which represents a novel early mechanism leading to diabetic nephropathy (DN). Here, we tested the hypothesis that Astragaloside IV(AS-IV) exerts antioxidant and antiapoptotic effects on podocytes under diabetic conditions. Apoptosis, albuminuria, ROS generation, caspase-3 activity and cleavage, as well as Bax and Bcl-2 mRNA and protein expression were measured in vitro and in vivo. Cultured podocytes were exposed to high glucose (HG) with 50, 100 and 200 µg/ml of AS-IV for 24 h. AS-IV significantly attenuated HG-induced podocyte apoptosis and ROS production. This antiapoptotic effect was associated with restoration of Bax and Bcl-2 expression, as well as inhibition of caspase-3 activation and overexpression. In streptozotocin (STZ)-induced diabetic rats, severe hyperglycemia and albuminuria were developed. Increased apoptosis, Bax expression, caspase-3 activity and cleavage while decreased Bcl-2 expression were detected in diabetic rats. However, pretreatment with AS-IV (2.5, 5, 10 mg·kg−1·d−1) for 14 weeks ameliorated podocyte apoptosis, caspase-3 activation, renal histopathology, podocyte foot process effacement, albuminuria and oxidative stress. Expression of Bax and Bcl-2 mRNA and protein in kidney cortex was partially restored by AS-IV pretreatment. These findings suggested AS-IV, a novel antioxidant, to prevent Glucose-Induced podocyte apoptosis partly through restoring the balance of Bax and Bcl-2 expression and inhibiting caspase-3 activation.

Decoupling of Sleepiness from Sleep Time and Intensity during Chronic Sleep Restriction: Evidence for a Role of the Adenosine System

Sleep responses to chronic sleep restriction (CSR) might be very different from those observed after short-term total sleep deprivation. For example, after sleep restriction continues for several consecutive days, animals no longer express compensatory increases in daily sleep time and sleep intensity. However, it is unknown if these allostatic, or adaptive, sleep responses to CSR are paralleled by behavioral and neurochemical measures of sleepiness. This study was designed to investigate CSR-induced changes in (1) sleep time and intensity as a measure of electrophysiological sleepiness, (2) sleep latency as a measure of behavioral sleepiness, and (3) brain adenosine A1 (A1R) and A2a receptor (A2aR) mRNA levels as a putative neurochemical correlate of sleepiness. Male Sprague-Dawley rats A 5-day sleep restriction (SR) protocol consisting of 18-h sleep deprivation and 6-h sleep opportunity each day. Unlike the first SR day, rats did not sleep longer or deeper on days 2 through 5, even though they exhibited significant elevations of behavioral sleepiness throughout all 5 SR days. For all SR days and recovery day 1, A1R mRNA in the basal forebrain was maintained at elevated levels, whereas A2aR mRNA in the frontal cortex was maintained at reduced levels. CSR LEADS TO A DECOUPLING OF SLEEPINESS FROM SLEEP TIME AND SLEEP INTENSITY, SUGGESTING THAT THERE ARE AT LEAST TWO DIFFERENT SLEEP REGULATORY SYSTEMS: one mediating sleepiness (homeostatic) and the other mediating sleep time/intensity (allostatic). The time course of changes observed in adenosine receptor mRNA levels suggests that the basal forebrain and cortical adenosine system might mediate sleepiness rather than sleep time or intensity.

Amelioration of Renal Injury and Oxidative Stress by the nNOS Inhibitor L-VNIO in the Salt-sensitive mRen2.Lewis Congenic Rat

Salt sensitivity is a key risk factor for cardiovascular disease and renal injury. Alterations in renal nitric oxide may contribute to salt-dependent increases in blood pressure and tissue damage. Therefore, we assessed the expression of nitric oxide synthase (NOS) isoforms in the kidney and the effects of nNOS inhibition on renal injury, inflammation, and oxidative stress in the female mRen2.Lewis rat (mRen), a model of salt-sensitive hypertension. We find that a high-salt diet (4% sodium) significantly reduced endothelial NOS mRNA (2.6-fold) and protein (1.5-fold) but increased nNOS mRNA (2.4-fold) and protein (1.9-fold) in the renal cortex of these animals. Immunostaining for nNOS also seemed higher in macula densa and cortical tubules of the rats fed a high-salt diet. Circulating nitrate and nitrite levels were reduced, including the tissue levels of the NOS cofactor tetrahydrobiopterin. Cortical markers of oxidative stress (4HNE, 8-OH-deoxyguanosine) and fibrosis were increased; however, mRNA levels of the NAD(P)H oxidase components NOX4, p22phox, and p47phox were reduced. Chronic treatment with the nNOS inhibitor N-(1-Imino-3-butenyl)-L-ornithine did not influence systolic blood pressure after 4 weeks but significantly attenuated albuminuria, renal fibrosis, inflammation, and indices of oxidative stress. We conclude that an increase in nNOS expression in conjunction with reduced levels of cortical tetrahydrobiopterin may stimulate oxidative stress and renal injury in the salt-sensitive female mRen2.Lewis rat.

Social isolation stress down-regulates cortical early growth response 1 (Egr-1) expression in mice

Social isolation stress induces behavioral disturbances such as aggression, cognitive impairments, and deficits in prepulse inhibition in mice. Social isolation mice have, therefore, been studied as an animal model of neuropsychiatric disorders such as schizophrenia. Recently, the decrease in early growth response (Egr) gene expression levels were reported in the post-mortem brains of schizophrenia patients. In this study, we investigate the effects of social isolation stress on the expression levels of Egr mRNA and protein in the frontal cortex. Social isolation stress exposure significantly down-regulated the expression of Egr-1 protein and Egr-1 gene transcript in nucleus of cortical neurons in a manner dependent on a social isolation period. This stress had no effect on the expression level of Egr-1 in the striatum or the expression levels of other Egr family members (Egr-2, -3, and -4) in the frontal cortex. These results suggest that the decrease in Egr-1 expression in the frontal cortex may be involved in social isolation stress-induced behavioral abnormalities.

Nicotine-induced changes of brain β-endorphin

A consensus has emerged that endogenous opioid peptides and their receptors play an important role in the psychoactive properties of nicotine. Although behavioral studies have shown that β-endorphin contributes to the rewarding and emotional effects of nicotine, whether the drug alters the function of brain endorphinergic neurons is not fully explored. These studies investigated the effect of acute, 1mg/kg, sc, and chronic, daily injection of 1mg/kg, sc, for 14 days, administration of free base nicotine on brain β-endorphin and its precursor proopiomelanocortin (POMC). Acute and chronic treatment with nicotine decreased β-endorphin content in hypothalamus, the principal site of β-endorphin producing neurons in the brain, and in the endorphinergic terminal fields in striatum and hippocampus. The acute effect of nicotine on β-endorphin was reversed by the nicotinic antagonist mecamylamine and the dopamine antagonist haloperidol, indicating pharmacological specificity and involvement of dopamine D2-like receptors. Similar observations were made in prefrontal cortex. POMC mRNA in hypothalamus and prefrontal cortex was unchanged following acute nicotine, but it decreased moderately with chronic treatment. The nicotine treatments had no effect on pituitary and plasma β-endorphin. Taken together, these results could be interpreted to indicate that nicotine alters the synthesis and release of β-endorphin in the limbic brain in vivo. Altered endorphinergic function may contribute to the behavioral effects of acute and chronic nicotine treatment and play a role in nicotine addiction.

Effect of Glucagon-Like Peptide 2 on Hepatic, Renal, and Intestinal Disposition of 1-Chloro-2,4-dinitrobenzene

The ability of the liver, small intestine, and kidney to synthesize and subsequently eliminate dinitrophenyl-S-glutathione (DNP-SG), a substrate for multidrug resistance-associated protein 2 (Mrp2), was assessed in rats treated with glucagon-like peptide 2 (GLP-2, 12 μg/100 g b.wt. s.c. every 12 h for 5 consecutive days). An in vivo perfused jejunum model with simultaneous bile and urine collection was used. A single intravenous dose of 30 μmol/kg b.wt. 1-chloro-2,4-dinitrobenzene (CDNB) was administered, and its conjugate, DNP-SG, and dinitrophenyl cysteinyl glycine (DNP-CG), resulting from the action of γ-glutamyltransferase on DNP-SG, were determined in bile, intestinal perfusate, and urine by high-performance liquid chromatography. Tissue content of DNP-SG was also assessed in liver, intestine, and kidneys. Biliary excretion of DNP-SG+DNP-CG was decreased in GLP-2 rats with respect to controls. In contrast, their intestinal excretion was substantially increased, whereas urinary elimination was not affected. Western blot and real-time polymerase chain reaction studies revealed preserved levels of Mrp2 protein and mRNA in liver and renal cortex and a significant increase in intestine in response to GLP-2 treatment. Tissue content of DNP-SG detected 5 min after CDNB administration was decreased in liver, increased in intestine, and unchanged in kidney in GLP-2 versus control group, consistent with GLP-2-induced down-regulation of expression of glutathione transferase (GST) Mu in liver and up-regulation of GST-Alpha in intestine at both protein and mRNA levels. In conclusion, GLP-2 induced selective changes in hepatic and intestinal disposition of a common GST and Mrp2 substrate administered systemically that could be of pharmacological or toxicological relevance under therapeutic treatment conditions.

Plasma and Urinary Heme Oxygenase-1 in AKI

AKI induces upregulation of heme oxygenase 1 (HO-1), which exerts cytoprotective effects and modulates the renal response to injury, suggesting that a biomarker of intrarenal HO-1 activity may be useful. Because HO-1 largely localizes to the endoplasmic reticulum and has no known secretory pathway, it is unclear whether plasma or urinary levels of HO-1 reflect intrarenal HO-1 expression. We measured plasma and urinary levels of HO-1 by ELISA during the induction and/or maintenance phases of four mouse models of AKI: ischemia/reperfusion, glycerol-induced rhabdomyolysis, cisplatin nephrotoxicity, and bilateral ureteral obstruction. In addition, we measured levels of HO-1 mRNA and protein in the renal cortex. Each AKI model increased renal HO-1 gene expression, which corresponded with release of HO-1 into plasma and urine by 4 hours. Over time, the magnitudes of plasma and urinary HO-1 paralleled renal cortical gene expression. AKI and the associated uremia did not seem to affect extrarenal HO-1 gene activity assessed in the liver, lung, and spleen. In iron-challenged, cultured proximal tubule cells, we observed a positive correlation between HO-1 mRNA level and HO-1 release. In humans, 10 patients with AKI demonstrated markedly higher levels of plasma and urine HO-1 levels than 10 critically ill patients without AKI or 20 patients with CKD or ESRD. In summary, these data suggest that plasma and urinary HO-1 levels may serve as biomarkers of AKI and intrarenal HO-1 gene activity.