Gabriella Zsilla

A possible role of nitric oxide in the regulation of dopamine transporter function in the striatum

Brain microdialysis and high-performance liquid chromatography with electrochemical detection were used to study the effect of the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME) on striatal dopamine (DA) release in the anesthetized rat. Systemic administration of L-NAME (10 mg/kg, i.p.) significantly decreased the resting release of DA. The peak effect (23% decrease) was reached 45 min after injection. The inactive enantiomer D-NAME (10 mg/kg, i.p.) or the vehicle (saline, 5 ml/kg i.p.) had no effect on the striatal DA level. Neither treatment altered significantly the concentration of dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). To investigate the possible involvement of the DA uptake system L-NAME was injected also in the presence of the DA uptake inhibitor nomifensine. Local application of nomifensine (10 microM in the dialysate medium) increased the extracellular concentration of DA to about eight-fold of the basal value and stabilized it at this higher level. Under these conditions L-NAME (10 mg/kg, i.p.) was not able to alter the striatal DA level. Neither nomifensine nor L-NAME caused any change in the level of DOPAC and HVA. Our data suggest that endogenously produced nitric oxide may influence the activity of the DA transporter which effect may have special importance in the regulation of extracellular transmitter concentration in the striatum.

Inhibitory effect of nitric oxide on dopamine transporters: interneuronal communication without receptors.

Previously we observed that Nomega-nitro-L-arginine methyl ester (l-NAME) decreased the striatal dopamine (DA) release in microdialysis experiments and this effect was completely diminished in the presence of the DA uptake inhibitor nomifensine, indicating that the effect was mediated via the DA transporter. The aim of the present work was to study the direct effect of nitrergic compounds on DA uptake. We measured the uptake of [3H]DA in striatal slices and found that the nitric oxide (NO) generator sodium nitroprussid (100 microM) decreased the uptake by 66%. In contrast, the NO synthase inhibitor L-NAME (100 microM) increased the DA uptake by 80%, while the inactive D-NAME had no effect on uptake. Our data indicate that NO exerts an inhibitory effect on DA transporters. Since the production of NO by neuronal NO synthase is closely related to the activation of NMDA receptors, the level of NO around synapses reflects the activity of glutamatergic neurotransmission. The strength of excitatory input, therefore, can be nonsynaptically signaled by NO to the surrounding dopaminergic neurons via the inhibitory tone on transporters. The concomitant elevation of DA concentration around the activated synapse represents the response of dopaminergic system, which can adapt to the changing excitatory activity without receiving glutamatergic input and without expressing glutamate receptors. Thus, the effect of NO on transporters represents a new form of interneuronal communication, a nonsynaptic interaction without receptors.

Isoproterenol inhibits IL-10, TNF-α, and nitric oxide production in RAW 264.7 Macrophages

Abstract In a previous study we have demonstrated in conscious endotoxemic mice that isoproterenol, a nonselective agonist of β-adrenergic receptors, decreased the production of proinflammatory mediators tumor necrosis factor-α (TNF-α) and nitric oxide (NO), and enhanced the formation of the antiinflammatory cytokine interleukin-10 (IL-10). In the present study we investigated the effect of isoproterenol on the bacterial lipopolysaccharide (endotoxin, LPS; 10 μg/ml)-induced inflammatory response in RAW 264.7 macrophages in vitro. Pretreatment of cells with isoproterenol (10–300 μM) resulted in an inhibition of TNF-α, NO (reflected as its stable breakdown product nitrite), as well as IL-10 production that was paralleled with a restoration of the LPS-induced suppression of mitochondrial respiration. In addition, isoproterenol elevated cAMP accumulation in these cells. Finally, isoproterenol (300 μM) did not influence the nuclear translocation of nuclear factor κB. These data demonstrate that isoproterenol potently downregulates the LPS-induced inflammatory response and further support the notion that stimulation of β-adrenoreceptors can be an effective strategy in the treatment of inflammatory diseases.

Uptake and Release of Norepinephrine by Serotonergic Terminals in Norepinephrine Transporter Knock-Out Mice: Implications for the Action of Selective Serotonin Reuptake Inhibitors

Our aim was to investigate the functional properties of the noradrenergic system in genetically modified mice lacking the norepinephrine transporter (NET). We measured the uptake and release of [3H]norepinephrine ([3H]NE) from hippocampal and cortical slices of NET-/- knock-out (KO) and NET+/+ wild-type (WT) mice and investigated the presynaptic α2-adenoceptor-mediated modulation of NE release in vitro and in vivo. The [3H]NE uptake was reduced to 12.6% (hippocampus) and 33.5% (frontal cortex) of WT control in KO mice. The neuronal component of this residual uptake was decreased by 79.4 and 100%, respectively, when a selective serotonin reuptake inhibitor (SSRI) citalopram was present during the loading. The more preserved neuronal release of [3H]NE (hippocampus, 28.1%; frontal cortex, 74.4%; compared with WT) almost completely disappeared in both regions (94.1 and 95.3% decrease compared with KO, respectively) in the presence of citalopram, suggesting that [3H]NE was taken up and released by serotonergic varicosities. This was further supported by the finding that the release of [3H]NE from hippocampal slices of KO mice was not modulated by the α2-adrenoceptor antagonist 7,8-(methylenedioxy)-14-α-hydroxyalloberbane HCl, whereas the endogenous release of NE measured by microdialysis was even more efficiently enhanced by this drug in NET-deficient mice. These experiments indicate that serotonergic varicosities can accumulate and release NE as a result of the heterologous uptake of transmitters. Because the diffusion of NE may be spatially limited by serotonin transporters, the SSRIs, despite their selectivity, might enhance not only serotonergic but also noradrenergic neurotransmission, which might contribute to their antidepressant action.

Subtype-specificity of the presynaptic α2-adrenoceptors modulating hippocampal norepinephrine release in rat

In vivo brain microdialysis and high-performance liquid chromatography with electrochemical detection were used to study the effect of different selective alpha 2-antagonists on hippocampal norepinephrine (NE) release in freely moving awake rat. Systemic administration (0.5 mg/kg i.p.) of either the alpha 2AD-antagonist BRL 44408 or the alpha 2BC-antagonist ARC 239 did not significantly change the basal release of NE. At a higher dose (5 mg/kg i.p.) ARC 239 was still ineffective, whereas BRL 4408 caused a significant increase of the extracellular level of NF. Similar results were obtained from in vitro perfusion experiments. Rat hippocampal slices were loaded with [3H]NE and the electrical stimulation-evoked release of [3H]NE was determined. The alpha 2-antagonists were applied in a concentration range of 10(-8) to 10(-6) M, ARC 239 was ineffective, whereas BRL 44408 significantly increased the electrically induced release of [3H]NE. In agreement with the data of microdialysis and perfusion experiments, BRL 44408 displaced [3H]yohimbine from hippocampal and cortical membranes of rat brain with high affinity whereas ARC 239 was less effective. The pKi values of eight different alpha 2-adrenergic compounds showed a very good correlation (r = 0.98, slope = 1.11 P < 0.0001) in hippocampus and frontal cortex have the alpha 2-adrenoceptors have been characterized as alpha 2d-subtype. Our data indicate that hippocampal NE release in rat is regulated by alpha 2D-adrenoceptors, a species variation of the human alpha 2A-subtype.

Constant rate infusion of deuterated phosphorylcholine to measure the effects of morphine on acetylcholine turnover rate in specific nuclei of rat brain

Abstract By using constant rate infusion of deuterated phosphorylcholine the turnover rate of acetylcholine in nucleus accumbens, n. septi, n. interpeduncularis, n. raphes dorsalis, substantia nigra, cortex, hippocampus (regio superior) and n. caudatus was measured. A dose of morphine which is the effective median dose for analgesia, failed to change the steady-state of choline and acetylcholine in the above-mentioned nuclei, but it lowered the turnover rate of acetylcholine in hippocampus, cortex and n. accumbens. The action of morphine on acetylcholine turnover rate could be antagonized by naltrexone, which per se failed to change the steady-state of acetylcholine and choline or of acetylcholine turnover rate. The possibility that opiate receptor activation in n. accumbens through inhibition of the turnover rate of acetylcholine triggers a series of events that cause analgesia and/or catatonia is discussed.

Age-dependent changes of presynaptic neuromodulation via A1-adenosine receptors in rat hippocampal slices

Thepresynaptic neuromodulation of stimulation‐evoked release of [3H]‐acetylcholine by endogenous adenosine, via A1‐adenosine receptors, was studied in superfused hippocampal slices taken from 4‐, 12‐ and 24‐month‐old rats. 8‐Cyclopentyl‐1,3‐dimethylxanthine (0.25 μM), a selective A1‐receptor antagonist, increased significantly the electrical field stimulation‐induced release of [3H]‐acetylcholine in slices prepared from 4‐ and 12‐month‐old rats, showing a tonic inhibitory action of endogenous adenosine via stimulation of presynaptic A1‐adenosine receptors. In contrast, 8‐cyclopentyl‐1,3‐dimethylxanthine had no effect in 24‐month‐old rats. 2‐Chloroadenosine (10 μM), an adenosine receptor agonist decreased the release of [3H]‐acetylcholine in slices taken from 4‐ and 12‐month‐old rats, and no significant change was observed in slices taken from 24‐month‐old rats. In order to show whether the number/or affinity of the A1‐receptors was affected in aged rats, [3H]‐8‐cyclopentyl‐1,3‐dimethylxanthine binding was studied in hippocampal membranes prepared from rats of different ages. Whereas the Bmax value was significantly lower in 2‐year‐old rats than in younger counterparts, the dissociation constant (Kd) was not affected by aging, indicating that the density rather than the affinity of adenosine receptors was altered. Endogenous adenosine levels present in the extracellular space were also measured in the superfusate by high performance liquid chromatography (HPLC) coupled with ultraviolet detection, and an age‐related increase in the adenosine level was found.

Neurochemical Changes in the Mouse Hippocampus Underlying the Antidepressant Effect of Genetic Deletion of P2X7 Receptors

Recent investigations have revealed that the genetic deletion of P2X7 receptors (P2rx7) results in an antidepressant phenotype in mice. However, the link between the deficiency of P2rx7 and changes in behavior has not yet been explored. In the present study, we studied the effect of genetic deletion of P2rx7 on neurochemical changes in the hippocampus that might underlie the antidepressant phenotype. P2X7 receptor deficient mice (P2rx7−/−) displayed decreased immobility in the tail suspension test (TST) and an attenuated anhedonia response in the sucrose preference test (SPT) following bacterial endotoxin (LPS) challenge. The attenuated anhedonia was reproduced through systemic treatments with P2rx7 antagonists. The activation of P2rx7 resulted in the concentration-dependent release of [3H]glutamate in P2rx7+/+ but not P2rx7−/− mice, and the NR2B subunit mRNA and protein was upregulated in the hippocampus of P2rx7−/− mice. The brain-derived neurotrophic factor (BDNF) expression was higher in saline but not LPS-treated P2rx7−/− mice; the P2rx7 antagonist Brilliant blue G elevated and the P2rx7 agonist benzoylbenzoyl ATP (BzATP) reduced BDNF level. This effect was dependent on the activation of NMDA and non-NMDA receptors but not on Group I metabotropic glutamate receptors (mGluR1,5). An increased 5-bromo-2-deoxyuridine (BrdU) incorporation was also observed in the dentate gyrus derived from P2rx7−/− mice. Basal level of 5-HT was increased, whereas the 5HIAA/5-HT ratio was lower in the hippocampus of P2rx7−/− mice, which accompanied the increased uptake of [3H]5-HT and an elevated number of [3H]citalopram binding sites. The LPS-induced elevation of 5-HT level was absent in P2rx7−/− mice. In conclusion there are several potential mechanisms for the antidepressant phenotype of P2rx7−/− mice, such as the absence of P2rx7-mediated glutamate release, elevated basal BDNF production, enhanced neurogenesis and increased 5-HT bioavailability in the hippocampus.

The Nootropic Drug Vinpocetine Inhibits Veratridine-Induced [Ca2+]i Increase in Rat Hippocampal CA1 Pyramidal Cells

The alkaloid derivative vinpocetine (14-ethoxycarbonyl-(3α,16α-ethyl)-14,15-eburnamine; Cavinton) has a well known beneficial effect on brain function in hypoxic and ischemic conditions. While it increases CNS blood flow and improves cellular metabolism, relatively little is known about vinpocetines underlying molecular mechanisms on the single cell level. Since apoptotic and necrotic cell damage is always preceded by an increase in [Ca2+]i, this study investigated the effect of vinpocetine on [Ca2+]i increases in acute brain slices. Sodium influx is an early event in the biochemical cascade that takes place during ischemia. The alkaloid veratridine can activate this Na+ influx, causing depolarization and increasing [Ca2+]i in the cells. Therefore, it can be used to simulate an ischemic attack in brain cells. Using a cooled CCD camera-based ratio imaging system and cell loading with fura 2/AM, the effect of vinpocetine on [Ca2+]i changes in single pyramidal neurons in the vulnerable CA1 region of rat hippocampal slices was investigated. Preperfusion and continuous administration of vinpocetine (10 μM) significantly inhibited the elevation in [Ca2+]i induced by veratridine (10 μM). When the drug was administered after veratridine, it could accelerate the recovery of cellular calcium levels. Piracetam, another nootropic used in clinical practice, could attenuate the elevation of [Ca2+]i only at a high, 1 mM, concentration. We have concluded that vinpocetine, at a pharmacologically relevant concentration, can decrease pathologically high [Ca2+]i levels in individual rat hippocampal CA1 pyramidal neurons; this effect might contribute to the neuroprotective property of the drug.

Alterations in Brain Extracellular Dopamine and Glycine Levels Following Combined Administration of the Glycine Transporter Type-1 Inhibitor Org-24461 and Risperidone

The most dominant hypotheses for the pathogenesis of schizophrenia have focused primarily upon hyperfunctional dopaminergic and hypofunctional glutamatergic neurotransmission in the central nervous system. The therapeutic efficacy of all atypical antipsychotics is explained in part by antagonism of the dopaminergic neurotransmission, mainly by blockade of D2 dopamine receptors. N-methyl-d-aspartate (NMDA) receptor hypofunction in schizophrenia can be reversed by glycine transporter type-1 (GlyT-1) inhibitors, which regulate glycine concentrations at the vicinity of NMDA receptors. Combined drug administration with D2 dopamine receptor blockade and activation of hypofunctional NMDA receptors may be needed for a more effective treatment of positive and negative symptoms and the accompanied cognitive deficit in schizophrenia. To investigate this type of combined drug administration, rats were treated with the atypical antipsychotic risperidone together with the GlyT-1 inhibitor Org-24461. Brain microdialysis was applied in the striatum of conscious rats and determinations of extracellular dopamine, DOPAC, HVA, glycine, glutamate, and serine concentrations were carried out using HPLC/electrochemistry. Risperidone increased extracellular concentrations of dopamine but failed to influence those of glycine or glutamate measured in microdialysis samples. Org-24461 injection reduced extracellular dopamine concentrations and elevated extracellular glycine levels but the concentrations of serine and glutamate were not changed. When risperidone and Org-24461 were added in combination, a decrease in extracellular dopamine concentrations was accompanied with sustained elevation of extracellular glycine levels. Interestingly, the extracellular concentrations of glutamate were also enhanced. Our data indicate that coadministration of an antipsychotic with a GlyT-1 inhibitor may normalize hypofunctional NMDA receptor-mediated glutamatergic neurotransmission with reduced dopaminergic side effects characteristic for antipsychotic medication.