Efficacy of Chronic Paroxetine Treatment in Mitigating Amyloid Pathology and Microgliosis in APPSWE/PS1ΔE9 Transgenic Mice.

BACKGROUND: Modulation of serotonergic signaling by treatment with selective serotonin reuptake inhibitors (SSRIs) has been suggested to mitigate amyloid-ß (Aß) pathology in Alzheimer's disease, in addition to exerting an anti-depressant action. OBJECTIVE: To investigate the efficacy of chronic treatment with the SSRI paroxetine, in mitigating Aß pathology and Aß plaque-induced microgliosis in the hippocampus of 18-month-old APPswe/PS1ΔE9 mice. METHODS: Plaque-bearing APPswe/PS1ΔE9 and wildtype mice were treated with paroxetine per os at a dose of 5 mg/kg/day, from 9 to 18 months of age. The per os treatment was monitored by recording of the body weights and serum paroxetine concentrations, and by assessment of the serotonin transporter occupancy by [3H]DASB-binding in wildtype mice. Additionally, 5,7-dihydroxytryptamine was administered to 9-month-old APPswe/PS1ΔE9 mice, to examine the effect of serotonin depletion on Aß pathology. Aß pathology was evaluated by Aß plaque load estimation and the Aß42/Aß40 ratio by ELISA. RESULTS: Paroxetine treatment led to > 80% serotonin transporter occupancy. The treatment increased the body weight of wildtype mice, but not of APPswe/PS1ΔE9 mice. The treatment had no effect on the Aß plaque load (p = 0.39), the number and size of plaques, or the Aß plaque-induced increases in microglial numbers in the dentate gyrus. Three months of serotonin depletion did not significantly impact the Aß plaque load or Aß42/Aß40 ratio in APPswe/PS1ΔE9 mice at 12 months. CONCLUSION: Our results show that chronic treatment with the SSRI paroxetine does not mitigate Aß pathology and Aß plaque-induced microgliosis in the hippocampus of APPswe/PS1ΔE9 mice.

Ethyl Pyruvate Increases Post-Ischemic Levels of Mitochondrial Energy Metabolites: A 13C-Labeled Cerebral Microdialysis Study.

Mitochondrial dysfunction after transient cerebral ischemia can be monitored by cerebral microdialysis (CMD) using changes in the lactate and pyruvate concentrations and ratio. Other metabolites associated with mitochondrial (dys)function are, e.g., tricyclic acid (TCA) and purine metabolites. Ethyl pyruvate (EP) is a putative neuroprotectant, supposedly targeting mitochondrial energy metabolism, but its effect on cerebral energy metabolism has never been described using microdialysis. In this study we monitored the metabolic effects of EP in the endothelin-1 (ET-1) rat model using perfusion with 13C-succinate and analysis of endogenous and 13C-labeled metabolites in the dialysates by liquid chromatography-mass spectrometry (LC-MS). Adult Sprague Dawley rats (n = 27 of which n = 11 were included in the study) were subjected to the microdialysis experiments. Microdialysis probes were perfused with 13C-labeled succinate (1 mM), and striatal dialysates were collected at 30 min intervals before induction of the insult, during intracerebral application of ET-1, and during intravenous treatment with either EP (40 mg/kg) or placebo, which was administered immediately after the insult. The rats were subjected to transient cerebral ischemia by unilateral microinjection of ET-1 in the piriform cortex, causing vasoconstriction of the medial cerebral artery. Monitoring was continued for 5 h after reperfusion, and levels of endogenous and 13C-labeled energy metabolites before and after ischemia-reperfusion were compared in EP-treated and control groups. Infarct volumes were assessed after 24 h. In both the EP-treated and placebo groups, ET-1-induced vasoconstriction resulted in a transient depression of interstitial glucose and elevation of lactate in the ipsilateral striatum. In the reperfusion phase, the concentrations of labeled malate, isocitrate, and lactate as well as endogenous xanthine were significantly higher in the EP-group than in the placebo-group: (mean ± SEM) labeled malate: 39.5% ± 14.9, p = 0.008; labeled isocitrate: 134.8% ± 67.9, p = 0.047; labeled lactate: 61% ± 22.0, p = 0.007; and endogenous xanthine: 93.9% ± 28.3, p = 0.0009. In the placebo group, significantly elevated levels of uridine were observed (mean ± SEM) 32.5% ± 12.7, p = 0.01. Infarct volumes were not significantly different between EP-treated and placebo groups, p = 0.4679. CMD labeled with 13C-succinate enabled detection of ischemic induction and EP treatment effects in the ET-1 rat model of transient focal cerebral ischemia. EP administered as a single intravenous bolus in the reperfusion-phase after transient cerebral ischemia increased de novo synthesis of several key intermediate energy metabolites (13C-malate, 13C-isocitrate, and endogenous xanthine). In summary, mitochondria process 13C-succinate more effectively after EP treatment.

In Vivo Microdialysis of Endogenous and 13C-labeled TCA Metabolites in Rat Brain: Reversible and Persistent Effects of Mitochondrial Inhibition and Transient Cerebral Ischemia.

Cerebral micro-dialysis allows continuous sampling of extracellular metabolites, including glucose, lactate and pyruvate. Transient ischemic events cause a rapid drop in glucose and a rise in lactate levels. Following such events, the lactate/pyruvate (L/P) ratio may remain elevated for a prolonged period of time. In neurointensive care clinics, this ratio is considered a metabolic marker of ischemia and/or mitochondrial dysfunction. Here we propose a novel, sensitive microdialysis liquid chromatography-mass spectrometry (LC-MS) approach to monitor mitochondrial dysfunction in living brain using perfusion with 13C-labeled succinate and analysis of 13C-labeled tricarboxylic acid cycle (TCA) intermediates. This approach was evaluated in rat brain using malonate-perfusion (10-50 mM) and endothelin-1 (ET-1)-induced transient cerebral ischemia. In the malonate model, the expected changes upon inhibition of succinate dehydrogenase (SDH) were observed, i.e., an increase in endogenous succinate and decreases in fumaric acid and malic acid. The inhibition was further elaborated by incorporation of 13C into specific TCA intermediates from 13C-labeled succinate. In the ET-1 model, increases in non-labeled TCA metabolites (reflecting release of intracellular compounds) and decreases in 13C-labeled TCA metabolites (reflecting inhibition of de novo synthesis) were observed. The analysis of 13C incorporation provides further layers of information to identify metabolic disturbances in experimental models and neuro-intensive care patients.

TNF deficiency causes alterations in the spatial organization of neurogenic zones and alters the number of microglia and neurons in the cerebral cortex.

BACKGROUND: Although tumor necrosis factor (TNF) inhibitors are used to treat chronic inflammatory diseases, there is little information about how long-term inhibition of TNF affects the homeostatic functions that TNF maintains in the intact CNS. MATERIALS AND METHODS: To assess whether developmental TNF deficiency causes alterations in the naïve CNS, we estimated the number of proliferating cells, microglia, and neurons in the developing neocortex of E13.5, P7 and adult TNF knock out (TNF-/-) mice and wildtype (WT) littermates. We also measured changes in gene and protein expression and monoamine levels in adult WT and TNF-/- mice. To evaluate long-term effects of TNF inhibitors, we treated healthy adult C57BL/6 mice with either saline, the selective soluble TNF inhibitor XPro1595, or the nonselective TNF inhibitor etanercept. We estimated changes in cell number and protein expression after two months of treatment. We assessed the effects of TNF deficiency on cognition by testing adult WT and TNF-/- mice and mice treated with saline, XPro1595, or etanercept with specific behavioral tasks. RESULTS: TNF deficiency decreased the number of proliferating cells and microglia and increased the number of neurons. At the same time, TNF deficiency decreased the expression of WNT signaling-related proteins, specifically Collagen Triple Helix Repeat Containing 1 (CTHRC1) and Frizzled receptor 6 (FZD6). In contrast to XPro1595, long-term inhibition of TNF with etanercept in adult C57BL/6 mice decreased the number of BrdU+ cells in the granule cell layer of the dentate gyrus. Etanercept, but not XPro1595, also impaired spatial learning and memory in the Barnes maze memory test. CONCLUSION: TNF deficiency impacts the organization of neurogenic zones and alters the cell composition in brain. Long-term inhibition of TNF with the nonselective TNF inhibitor etanercept, but not the soluble TNF inhibitor XPro1595, decreases neurogenesis in the adult mouse hippocampus and impairs learning and memory after two months of treatment.

Cyclosporin A ameliorates cerebral oxidative metabolism and infarct size in the endothelin-1 rat model of transient cerebral ischaemia.

Cerebral microdialysis can be used to detect mitochondrial dysfunction, a potential target of neuroprotective treatment. Cyclosporin A (CsA) is a mitochondrial stabiliser that in a recent clinical stroke trial showed protective potential in patients with successful recanalisation. To investigate specific metabolic effects of CsA during reperfusion, and hypothesising that microdialysis values can be used as a proxy outcome measure, we assessed the temporal patterns of cerebral energy substrates related to oxidative metabolism in a model of transient focal ischaemia. Transient ischaemia was induced by intracerebral microinjection of endothelin-1 (150 pmol/15 µL) through stereotaxically implanted guide cannulas in awake, freely moving rats. This was immediately followed by an intravenous injection of CsA (NeuroSTAT; 15 mg/kg) or placebo solution during continuous microdialysis monitoring. After reperfusion, the lactate/pyruvate ratio (LPR) was significantly lower in the CsA group vs placebo (n = 17, 60.6 ± 24.3%, p = 0.013). Total and striatal infarct volumes (mm3) were reduced in the treatment group (n = 31, 61.8 ± 6.0 vs 80.6 ± 6.7, p = 0.047 and 29.9 ± 3.5 vs 41.5 ± 3.9, p = 0.033). CsA treatment thus ameliorated cerebral reperfusion metabolism and infarct size. Cerebral microdialysis may be useful in evaluating putative neuroprotectants in ischaemic stroke.

Established amyloid-ß pathology is unaffected by chronic treatment with the selective serotonin reuptake inhibitor paroxetine.

INTRODUCTION: Treatment with selective serotonin reuptake inhibitors has been suggested to mitigate amyloid-ß (Aß) pathology in Alzheimer's disease, in addition to an antidepressant mechanism of action. METHODS: We investigated whether chronic treatment with paroxetine, a selective serotonin reuptake inhibitor, mitigates Aß pathology in plaque-bearing double-transgenic amyloid precursor protein (APP)swe/presenilin 1 (PS1)ΔE9 mutants. In addition, we addressed whether serotonin depletion affects Aß pathology. Treatments were assessed by measurement of serotonin transporter occupancy and high-performance liquid chromatography. The effect of paroxetine on Aß pathology was evaluated by stereological plaque load estimation and Aß42/Aß40 ratio by enzyme-linked immunosorbent assay. RESULTS: Contrary to our hypothesis, paroxetine therapy did not mitigate Aß pathology, and depletion of brain serotonin did not exacerbate Aß pathology. However, chronic paroxetine therapy increased mortality in APPswe/PS1ΔE9 transgenic mice. DISCUSSION: Our results question the ability of selective serotonin reuptake inhibitor therapy to ameliorate established Aß pathology. The severe adverse effect of paroxetine may discourage its use for disease-modifying purposes in Alzheimer's disease.

Intermittent, low dose carbon monoxide exposure enhances survival and dopaminergic differentiation of human neural stem cells.

Exploratory studies using human fetal tissue have suggested that intrastriatal transplantation of dopaminergic neurons may become a future treatment for patients with Parkinson's disease. However, the use of human fetal tissue is compromised by ethical, regulatory and practical concerns. Human stem cells constitute an alternative source of cells for transplantation in Parkinson's disease, but efficient protocols for controlled dopaminergic differentiation need to be developed. Short-term, low-level carbon monoxide (CO) exposure has been shown to affect signaling in several tissues, resulting in both protection and generation of reactive oxygen species. The present study investigated the effect of CO produced by a novel CO-releasing molecule on dopaminergic differentiation of human neural stem cells. Short-term exposure to 25 ppm CO at days 0 and 4 significantly increased the relative content of ß-tubulin III-immunoreactive immature neurons and tyrosine hydroxylase expressing catecholaminergic neurons, as assessed 6 days after differentiation. Also the number of microtubule associated protein 2-positive mature neurons had increased significantly. Moreover, the content of apoptotic cells (Caspase3) was reduced, whereas the expression of a cell proliferation marker (Ki67) was left unchanged. Increased expression of hypoxia inducible factor-1α and production of reactive oxygen species (ROS) in cultures exposed to CO may suggest a mechanism involving mitochondrial alterations and generation of ROS. In conclusion, the present procedure using controlled, short-term CO exposure allows efficient dopaminergic differentiation of human neural stem cells at low cost and may as such be useful for derivation of cells for experimental studies and future development of donor cells for transplantation in Parkinson's disease.

Dopamine plasma clearance is increased in piglets compared to neonates during continuous dopamine infusion.

AIM: Piglets models have often been used to study the effects of dopamine infusion on hypotension in neonates. However, piglets need higher doses of dopamine than neonates to increase blood pressure. We investigated whether this difference was due to interspecific difference in dopamine pharmacokinetics. METHODS: Arterial blood samples were drawn from six neonates admitted to the neonatal intensive care unit of Copenhagen University Hospital and 20 newborn piglets during continuous dopamine infusion. Furthermore, to estimate the piglet plasma dopamine half-life, blood samples were drawn at 2.5-minute intervals after the dopamine infusion was discontinued. The plasma dopamine content was analysed by high-performance liquid chromatography with electrochemical detection. RESULTS: The dopamine displayed first-order kinetics in piglets and had a half-life of 2.5 minutes, while the median plasma clearance was 627.9 mL/kg/minute (interquartile range 452.6-1914.4). Both piglets and neonates showed large interindividual variations in plasma clearance, but the median tended to be lower in neonates (384.9, interquartile range 114.2-480.2 mL/kg/minute). CONCLUSION: Our results suggest that pharmacokinetic differences may explain the interspecific difference in required doses of dopamine infusion to increase blood pressure. This is important when translating the results obtained in piglet models to treating neonatal hypotension with dopamine.

Biomarker Research in Parkinson's Disease Using Metabolite Profiling.

Biomarker research in Parkinson's disease (PD) has long been dominated by measuring dopamine metabolites or alpha-synuclein in cerebrospinal fluid. However, these markers do not allow early detection, precise prognosis or monitoring of disease progression. Moreover, PD is now considered a multifactorial disease, which requires a more precise diagnosis and personalized medication to obtain optimal outcome. In recent years, advanced metabolite profiling of body fluids like serum/plasma, CSF or urine, known as "metabolomics", has become a powerful and promising tool to identify novel biomarkers or "metabolic fingerprints" characteristic for PD at various stages of disease. In this review, we discuss metabolite profiling in clinical and experimental PD. We briefly review the use of different analytical platforms and methodologies and discuss the obtained results, the involved metabolic pathways, the potential as a biomarker and the significance of understanding the pathophysiology of PD. Many of the studies report alterations in alanine, branched-chain amino acids and fatty acid metabolism, all pointing to mitochondrial dysfunction in PD. Aromatic amino acids (phenylalanine, tyrosine, tryptophan) and purine metabolism (uric acid) are also altered in most metabolite profiling studies in PD.

Changes in kynurenine pathway metabolism in Parkinson patients with L-DOPA-induced dyskinesia.

L-3,4-Dihydroxyphenylalanine (L-DOPA) is the most effective drug in the symptomatic treatment of Parkinson's disease, but chronic use is associated with L-DOPA-induced dyskinesia in more than half the patients after 10 years of treatment. L-DOPA treatment may affect tryptophan metabolism via the kynurenine pathway. Altered levels of kynurenine metabolites can affect glutamatergic transmission and may play a role in the development of L-DOPA-induced dyskinesia. In this study, we assessed kynurenine metabolites in plasma and cerebrospinal fluid of Parkinson's disease patients and controls. Parkinson patients (n = 26) were clinically assessed for severity of motor symptoms (UPDRS) and L-DOPA-induced dyskinesia (UDysRS). Plasma and cerebrospinal fluid samples were collected after overnight fasting and 1-2 h after intake of L-DOPA or other anti-Parkinson medication. Metabolites were analyzed in plasma and cerebrospinal fluid of controls (n = 14), Parkinson patients receiving no L-DOPA (n = 8), patients treated with L-DOPA without dyskinesia (n = 8), and patients with L-DOPA-induced dyskinesia (n = 10) using liquid chromatography-mass spectrometry. We observed approximately fourfold increase in the 3-hydroxykynurenine/kynurenic acid ratio in plasma of Parkinson's patients with L-DOPA-induced dyskinesia. Anthranilic acid levels were decreased in plasma and cerebrospinal fluid of this patient group. 5-Hydroxytryptophan levels were twofold increased in all L-DOPA-treated Parkinson's patients. We conclude that a higher 3-hydroxykynurenine/kynurenic acid ratio in plasma may serve as a biomarker for L-DOPA-induced dyskinesia. Longitudinal studies including larger patients cohorts are needed to verify whether the changes observed here may serve as a prognostic marker for L-DOPA-induced dyskinesia.

Cerebrospinal fluid levels of catecholamines and its metabolites in Parkinson's disease: effect of l-DOPA treatment and changes in levodopa-induced dyskinesia.

Levodopa (l-DOPA, l-3,4-dihydroxyphenylalanine) is the most effective drug in the symptomatic treatment of Parkinson's disease (PD), but chronic use initiates a maladaptive process leading to l-DOPA-induced dyskinesia (LID). Risk factors for early onset LID include younger age, more severe disease at baseline and higher daily l-DOPA dose, but biomarkers to predict the risk of motor complications are not yet available. Here, we investigated whether CSF levels of catecholamines and its metabolites are altered in PD patients with LID [PD-LID, n = 8)] as compared to non-dyskinetic PD patients receiving l-DOPA (PD-L, n = 6), or not receiving l-DOPA (PD-N, n = 7) as well as non-PD controls (n = 16). PD patients were clinically assessed using the Unified Parkinson's Disease Rating Scale and Unified Dyskinesia Rating Scale and CSF was collected after overnight fasting and 1-2 h after oral intake of l-DOPA or other anti-Parkinson medication. CSF catecholamines and its metabolites were analyzed by HPLC with electrochemical detection. We observed (i) decreased levels of dihydroxyphenylacetic acid (DOPAC) and homovanillic acid in PD patients not receiving l-DOPA (ii) higher dopamine (DA) levels in PD-LID as compared to controls (iii) higher DA/l-DOPA and lower DOPAC/DA ratio's in PD-LID as compared to PD-L and (iv) an age-dependent increase of DA and decrease of DOPAC/DA ratio in controls. These results suggest increased DA release from non-DA cells and deficient DA re-uptake in PD-LID. Monitoring DA and DOPAC in CSF of l-DOPA-treated PD patients may help identify patients at risk of developing LID.

Carbon Monoxide Releasing Molecule-A1 (CORM-A1) Improves Neurogenesis: Increase of Neuronal Differentiation Yield by Preventing Cell Death.

Cerebral ischemia and neurodegenerative diseases lead to impairment or death of neurons in the central nervous system. Stem cell based therapies are promising strategies currently under investigation. Carbon monoxide (CO) is an endogenous product of heme degradation by heme oxygenase (HO) activity. Administration of CO at low concentrations produces several beneficial effects in distinct tissues, namely anti-apoptotic and anti-inflammatory. Herein the CO role on modulation of neuronal differentiation was assessed. Three different models with increasing complexity were used: human neuroblastoma SH-S5Y5 cell line, human teratocarcinoma NT2 cell line and organotypic hippocampal slice cultures (OHSC). Cell lines were differentiated into post-mitotic neurons by treatment with retinoic acid (RA) supplemented with CO-releasing molecule A1 (CORM-A1). CORM-A1 positively modulated neuronal differentiation, since it increased final neuronal production and enhanced the expression of specific neuronal genes: Nestin, Tuj1 and MAP2. Furthermore, during neuronal differentiation process, there was an increase in proliferative cell number (ki67 mRNA expressing cells) and a decrease in cell death (lower propidium iodide (PI) uptake, limitation of caspase-3 activation and higher Bcl-2 expressing cells). CO supplementation did not increase the expression of RA receptors. In the case of SH-S5Y5 model, small amounts of reactive oxygen species (ROS) generation emerges as important signaling molecules during CO-promoted neuronal differentiation. CO's improvement of neuronal differentiation yield was validated using OHSC as ex vivo model. CORM-A1 treatment of OHSC promoted higher levels of cells expressing the neuronal marker Tuj1. Still, CORM-A1 increased cell proliferation assessed by ki67 expression and also prevented cell death, which was followed by increased Bcl-2 expression, decreased levels of active caspase-3 and PI uptake. Likewise, ROS signaling emerged as key factors in CO's increasing number of differentiated neurons in OHSC. In conclusion, CO's increasing number of differentiated neurons is a novel biological role disclosed herein. CO improves neuronal yield due to its capacity to reduce cell death, promoting an increase in proliferative population. However, one cannot disregard a direct CO's effect on specific cellular processes of neuronal differentiation. Further studies are needed to evaluate how CO can potentially modulate cell mechanisms involved in neuronal differentiation. In summary, CO appears as a promising therapeutic molecule to stimulate endogenous neurogenesis or to improve in vitro neuronal production for cell therapy strategies.

The water channel aquaporin-1 contributes to renin cell recruitment during chronic stimulation of renin production.

Both the processing and release of secretory granules involve water movement across granule membranes. It was hypothesized that the water channel aquaporin (AQP)1 directly contributes to the recruitment of renin-positive cells in the afferent arteriole. AQP1(-/-) and AQP1(+/+) mice were fed a low-salt (LS) diet [0.004% (wt/wt) NaCl] for 7 days and given enalapril [angiotensin-converting enzyme inhibitor (ACEI), 0.1 mg/ml] in drinking water for 3 days. There were no differences in plasma renin concentration at baseline. After LS-ACEI, plasma renin concentrations increased markedly in both genotypes but was significantly lower in AQP1(-/-) mice compared with AQP1(+/+) mice. Tissue renin concentrations were higher in AQP1(-/-) mice, and renin mRNA levels were not different between genotypes. Mean arterial blood pressure was not different at baseline and during LS diet but decreased significantly in both genotypes after the addition of ACEI; the response was faster in AQP1(-/-) mice but then stabilized at a similar level. Renin release after 200 µl blood withdrawal was not different. Isoprenaline-stimulated renin release from isolated perfused kidneys did not differ between genotypes. Cortical tissue norepinephrine concentrations were lower after LS-ACEI compared with baseline with no difference between genotypes. Plasma nitrite/nitrate concentrations were unaffected by genotype and LS-ACEI. In AQP1(-/-) mice, the number of afferent arterioles with recruitment was significantly lower compared with AQP1(+/+) mice after LS-ACEI. We conclude that AQP1 is not necessary for acutely stimulated renin secretion in vivo and from isolated perfused kidneys, whereas recruitment of renin-positive cells in response to chronic stimulation is attenuated or delayed in AQP1(-/-) mice.

Genetic KCa3.1-deficiency produces locomotor hyperactivity and alterations in cerebral monoamine levels.

BACKGROUND: The calmodulin/calcium-activated K(+) channel KCa3.1 is expressed in red and white blood cells, epithelia and endothelia, and possibly central and peripheral neurons. However, our knowledge about its contribution to neurological functions and behavior is incomplete. Here, we investigated whether genetic deficiency or pharmacological activation of KCa3.1 change behavior and cerebral monoamine levels in mice. METHODOLOGY/PRINCIPAL FINDINGS: In the open field test, KCa3.1-deficiency increased horizontal activity, as KCa3.1(-/-) mice travelled longer distances (≈145% of KCa3.1(+/+)) and at higher speed (≈1.5-fold of KCa3.1(+/+)). Working memory in the Y-maze was reduced by KCa3.1-deficiency. Motor coordination on the rotarod and neuromuscular functions were unchanged. In KCa3.1(-/-) mice, HPLC analysis revealed that turn-over rates of serotonin were reduced in frontal cortex, striatum and brain stem, while noradrenalin turn-over rates were increased in the frontal cortex. Dopamine turn-over rates were unaltered. Plasma catecholamine and corticosterone levels were unaltered. Intraperitoneal injections of 10 mg/kg of the KCa3.1/KCa2-activator SKA-31 reduced rearing and turning behavior in KCa3.1(+/+) but not in KCa3.1(-/-) mice, while 30 mg/kg SKA-31 caused strong sedation in 50% of the animals of either genotypes. KCa3.1(-/-) mice were hyperactive (≈+60%) in their home cage and SKA-31-administration reduced nocturnal physical activity in KCa3.1(+/+) but not in KCa3.1(-/-) mice. CONCLUSIONS/SIGNIFICANCE: KCa3.1-deficiency causes locomotor hyperactivity and altered monoamine levels in selected brain regions, suggesting a so far unknown functional link of KCa3.1 channels to behavior and monoaminergic neurotransmission in mice. The tranquilizing effects of low-dose SKA-31 raise the possibility to use KCa3.1/KCa2 channels as novel pharmacological targets for the treatment of neuropsychiatric hyperactivity disorders.

Pharmacological activation of KCa3.1/KCa2.3 channels produces endothelial hyperpolarization and lowers blood pressure in conscious dogs.

BACKGROUND AND PURPOSE: In rodents, the endothelial KCa channels, KCa3.1 and KCa2.3, have been shown to play a crucial role in initiating endothelium-derived hyperpolarizing factor (EDHF) vasodilator responses. However, it is not known to what extent these channels are involved in blood pressure regulation in large mammals, which would also allow us to address safety issues. We therefore characterized canine endothelial KCa3.1 and KCa2.3 functions and evaluated the effect of the KCa3.1/KCa2.3 activator SKA-31 on blood pressure and heart rate in dogs. EXPERIMENTAL APPROACH: Canine endothelial KCa3.1/KCa2.3 functions were studied by patch-clamp electrophysiology and wire myography in mesenteric arteries. Systemic cardiovascular actions of acute SKA-31 administration were monitored in conscious, unstressed beagle dogs. KEY RESULTS: Mesenteric endothelial cells expressed functional KCa3.1 and KCa2.3 channels that were strongly activated by SKA-31. SKA-31 hyperpolarized the endothelial membrane and doubled endothelial hyperpolarization-dependent vasodilator responses in mesenteric arteries. SKA-31 (2 mg·kg(-1), i.v.) rapidly decreased the MAP by 28 ± 6 mmHg; this response was transient (8 ± 1 s), and the initial drop was followed by a fast and pronounced increase in HR (+109 ± 7 beats min(-1)) reflecting baroreceptor activation. SKA-31 significantly augmented similar transient depressor responses elicited by ACh (20 ng·kg(-1)) and doubled the magnitude of the response over time. CONCLUSIONS AND IMPLICATIONS: Activation of endothelial KCa3.1 and KCa2.3 lowers arterial blood pressure in dogs by an immediate electrical vasodilator mechanism. The results support the concept that pharmacological activation of these channels may represent a potential unique endothelium-specific antihypertensive therapy.

Identification of nitrotyrosine containing peptides using combined fractional diagonal chromatography (COFRADIC) and off-line nano-LC-MALDI.

Protein nitration take place on tyrosine residues under oxidative stress conditions and may influence a number of processes including enzyme activity, protein-protein interactions and phospho-tyrosine signalling pathways. Nitrated proteins have been identified in a number of diseases, however, the study of these proteins has been compromised by the lack of good methods for identifying nitrated proteins, their nitration sites and the level of nitration. Here, we present a method for identification of nitrated peptides that allows the site specific assignment of nitration, is easy to use and reproducible, and opens up for the possibility to quantify the level of nitration of specific peptides as function of different oxidative conditions, namely combined fractional diagonal chromatography (COFRADIC) in combination with off-line nano-LC-MALDI. We identify six nitrated peptides from in vitro nitrated bovine serum albumin and propose that automated COFRADIC using nano-LC and off-line MALDI-MS might be a possibility for identification of tyrosine nitrated proteins and the nitration sites in complex samples.

Enhanced proliferation and dopaminergic differentiation of ventral mesencephalic precursor cells by synergistic effect of FGF2 and reduced oxygen tension.

Effective numerical expansion of dopaminergic precursors might overcome the limited availability of transplantable cells in replacement strategies for Parkinson's disease. Here we investigated the effect of fibroblast growth factor-2 (FGF2) and FGF8 on expansion and dopaminergic differentiation of rat embryonic ventral mesencephalic neuroblasts cultured at high (20%) and low (3%) oxygen tension. More cells incorporated bromodeoxyuridine in cultures expanded at low as compared to high oxygen tension, and after 6 days of differentiation there were significantly more neuronal cells in low than in high oxygen cultures. Low oxygen during FGF2-mediated expansion resulted also in a significant increase in tyrosine hydroxylase-immunoreactive (TH-ir) dopaminergic neurons as compared to high oxygen tension, but no corresponding effect was observed for dopamine release into the culture medium. However, switching FGF2-expanded cultures from low to high oxygen tension during the last two days of differentiation significantly enhanced dopamine release and intracellular dopamine levels as compared to all other treatment groups. In addition, the short-term exposure to high oxygen enhanced in situ assessed TH enzyme activity, which may explain the elevated dopamine levels. Our findings demonstrate that modulation of oxygen tension is a recognizable factor for in vitro expansion and dopaminergic differentiation of rat embryonic midbrain precursor cells.

MDMA-evoked changes in the binding of dopamine D(2) receptor ligands in striatum of rats with unilateral serotonin depletion.

We earlier reported an anomalous 50% decrease in [(11)C]N-methylspiperone ([(11)C]NMSP) binding to dopamine D(2)-like receptors in living pig striatum after challenge with 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy"), suggesting either (1) a species peculiarity in the vulnerability of butyrophenone binding to competition from dopamine or (2) a novel consequence of synergistic actions of serotonin and dopamine at dopamine receptors. To distinguish these possibilities, we used microPET to test the vulnerability of [(11)C]NMSP binding in striatum of rats with unilateral telencephalic serotonin lesions, later verified by [(125)I]RTI-55 autoradiography. Baseline [(11)C]NMSP microPET recordings were followed by either saline or MDMA-HCl (4 mg/kg) injections (i.v.), and a second [(11)C]NMSP recording, culminating with injection of [(3)H]raclopride for autoradiography ex vivo. Neither MDMA-challenge nor serotonin lesion had any detectable effect on [(11)C]NMSP binding. In contrast, MDMA challenge increased receptor occupancy by [(3)H]raclopride ex vivo (relative to the B(max) in vitro) from 8% to 12%, and doubled the free ligand concentration in cerebral cortex, apparently by blocking hepatic CYP2D6. Assuming a single binding-site model, the increased [(3)H]raclopride binding indicated doubling of the apparent equilibrium dissociation constant in vivo (K(app) (d)), revealing a 2-fold increase in competition from endogenous dopamine at [(3)H]raclopride binding sites. The results favor hypothesis (1) that the remarkable vulnerability of [(11)C]NMSP binding in pig striatum to MDMA challenge does not generalize to the rodent.

Nitration of soluble proteins in organotypic culture models of Parkinson's disease.

Protein nitration due to oxidative and nitrative stress has been linked to the pathogenesis of Parkinson's disease (PD), but its relationship to the loss of dopamine (DA) or tyrosine hydroxylase (TH) activity is not clear. Here we quantified protein-bound 3-nitrotyrosine (3-NT) by a novel gas chromatography/negative chemical ionization tandem mass spectrometry technique and DA and 3,4-dihydroxyphenylalanine (DOPA) by HPLC in tissues or medium of organotypic, mouse mesencephalon cultures after acute or chronic treatments with the peroxynitrite donor 3-morpholino-sydnonimine (SIN-1), the dopaminergic toxin 1-methyl-4-phenylpyridinium (MPP(+)) or the lipophilic complex I inhibitor rotenone. Incubation with SIN-1 (24 h) or MPP(+) treatments (48 h) caused dose-dependent protein nitration reaching a maximum of eightfold increase by 10 mM SIN-1 or twofold by 10 microM MPP(+), but significant DA depletions occurred at much lower concentrations of MPP(+) (1 microM). Chronic MPP(+) or rotenone treatments (3 weeks) caused maximum protein nitration by 1 microM (twofold) or 10nM (fourfold), respectively. Co-treatment with the nitric oxide synthase inhibitor l-NAME (300 microM) prevented protein nitration by MPP(+), but did not protect against MPP(+)-induced DA depletion or inhibition of TH activity. Acute incubation with 100 microM SIN-1 inhibited TH activity, which could be blocked by co-treatment with the tetrahydrobiopterin precursor l-sepiapterin, but tissue DA depletions required higher doses of SIN-1 (>1 mM, 24 h) and longer survival. In conclusion, protein nitration and TH activity or DA depletion are not directly related in these models.

Timing of potential and metabolic brain energy.

The temporal relationship between cerebral electro-physiological activities, higher brain functions and brain energy metabolism is reviewed. The duration of action potentials and transmission through glutamate and GABA are most often less than 5 ms. Subjects may perform complex psycho-physiological tasks within 50 to 200 ms, and perception of conscious experience requires 0.5 to 2 s. Activation of cerebral oxygen consumption starts after at least 100 ms and increases of local blood flow become maximal after about 1 s. Current imaging technologies are unable to detect rapid physiological brain functions. We introduce the concepts of potential and metabolic brain energy to distinguish trans-membrane gradients of ions or neurotransmitters and the capacity to generate energy from intra- or extra-cerebral substrates, respectively. Higher brain functions, such as memory retrieval, speaking, consciousness and self-consciousness are so fast that their execution depends primarily on fast neurotransmission (in the millisecond range) and action-potentials. In other words: brain functioning requires primarily maximal potential energy. Metabolic brain energy is necessary to restore and maintain the potential energy.