Effects of serotonin depletion byp-chlorophenylalanine,p-chloroamphetamine or 5,7-dihydroxytryptamine on central dopaminergic neurons: Focus on tuberoinfundibular dopaminergic neurons and serum prolactin

Three serotonin (5-HT) neurotoxins,p-chlorophenylalanine (PCPA, 125 and 250 mg/kg, i.p.),p-chloroamphetamine (PCA, 10 mg/kg, i.p.) and 5,7-dihydroxytryptamine (5,7-DHT, 200 µg/rat, i.c.v.) were used to examine whether depletion of central 5-HT has an effect on central dopaminergic (DA) neuronal activities or on prolactin (PRL) secretion. Adult ovariectomized Sprague-Dawley rats primed with estrogen (polyestradiol phosphate, 0.1 mg/rat, s.c.) were treated with one of three neurotoxins and then decapitated in the morning after 3–7 days. Blood sample and brain tissues were collected. The acute effect of PCA (from 30 to 180 min) was also determined. The concentrations of 5-HT, DA and their metabolites, 5-hydroxyindoleacetic acid and 3,4-dihydroxyphenylacetic acid, in the median eminence, striatum and nucleus accumbens were determined by HPLC-electrochemical detection. All three toxins significantly depleted central 5-HT stores by 11–20%. Except for PCPA, neither PCA nor 5,7-DHT had any significant effect on basal DA neuronal activities or PRL secretion. PCA also exhibited an acute effect on the release and reuptake of 5-HT and DA. In summary, depletion of central 5-HT stores to a significant extent for 3–7 days did not seem to affect basal DA neuronal activity and PRL secretion.     

Effects of Single and Repeated Footshock on Dopamine Release and Metabolism in the Brains of Fischer Rats

Abstract: Changes in the tissue levels of 3-methoxytyramine (3-MT), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and dopamine in the frontal cortex, hypothalamus, nucleus accumbens, and striatum were evaluated after 0.5-4 h of footshock (2 mA, for 3 s every 30 ± 5 s) in Fischer rats. 3-MT, DOPAC, and HVA levels in the four brain areas peaked at 0.5 h and in most cases returned to baseline values within 4 h. No changes were found in dopamine levels. Repeated footshock stress was evaluated by administering 10 footshock sessions (0.5 h, two per day for 5 days). At the end of the 10th footshock session, 3-MT levels were higher than at the end of the first footshock session in three of the four brain regions, indicating sensitization of dopamine release. No differences were found between the first and 10th footshock sessions in DOPAC and HVA levels. Fourteen days after the 10th footshock session, the levels of 3-MT, DOPAC, and HVA were the same as in control rats in all four brain regions. A 0.5-h footshock challenge presented 14 days after the 10th footshock session attenuated DOPAC levels in the hypothalamus and nucleus accumbens. In contrast, DOPAC and HVA levels in the frontal cortex showed sensitization after footshock challenge, and a similar trend was apparent for 3-MT levels. These results indicate that repeated footshock stress induces generalized sensitization of dopamine release and turnover in some areas of the brain of Fischer rats. This sensitization may persist in the cortical but not subcortical dopamine neurons after discontinuation of the treatment.     

Differential Effects of Forced Locomotion, Tail-Pinch, Immobilization, and Methyl-β-Carboline Carboxylate on Extracellular 3,4-Dihydroxyphenylacetic Acid Levels in the Rat Striatum, Nucleus Accumbens, and Prefrontal Cortex: An In Vivo Voltammetric Study

In vivo voltammetry with carbon fiber electrodes was used to assess extracellular 3,4-dihydroxyphenylacetic acid (DOPAC) levels in striatum, nucleus accumbens, and anteromedial prefrontal cortex of freely moving rats subjected to altered motor activity or anxiogenic stimuli. Forced locomotion on a rotarod for 40 min caused an increase in extracellular DOPAC levels in the striatum and to a lesser extent in the nucleus accumbens but not in the prefrontal cortex. Subcutaneous injection of the anxiogenic agent methyl-beta-carboline carboxylate (10 mg/kg) increased extracellular DOPAC levels to a similar extent in prefrontal cortex and nucleus accumbens. Immobilization for 4 min augmented dopamine (DA) metabolism preferentially in the nucleus accumbens and to a lesser extent in the prefrontal cortex. Tail-pinch caused a selective activation of DA metabolism in the nucleus accumbens. None of these stimuli altered extracellular striatal DOPAC levels. These results confirm the involvement of dopaminergic systems projecting to the striatum and nucleus accumbens in motor function and suggest that mesolimbic and mesocortical dopaminergic systems can be specifically activated by certain kinds of anxiogenic stimuli; the relative activation of either of these latter systems could depend primarily on the nature (sensory modality, intensity) of the acute stressor.     

Determination of Picomole Amounts of Dopamine, Noradrenaline, 3,4-Dihydroxyphenylalanine, 3,4-Dihydroxyphenylacetic Acid, Homovanillic Acid, and 5-Hydroxyindolacetic Acid in Nervous Tissue After One-Step Purification on Sephadex G-10, Using High-Performance Liquid Chromatography with a Novel Type of Electrochemical Detection

A determination of dopamine (DA), noradrenaline (NA), 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindolacetic acid (5-HIAA) in nervous tissue is described. The method is based on a rapidly performed isolation of DA, NA, DOPA, DOPAC, HVA, and 5-HIAA from one single nervous tissue sample on small columns of Sephadex G-10, followed by reverse-phase high-performance liquid chromatography with electrochemical detection. A new type of electrochemical detector based on a rotating disk electrode (RDE) was used. The rotating disc electrode was found to be a reliable and sensitive amperometric detector with several advantages over the currently used thin-layer cells. The detector appeared very useful for routine analysis. Practical details are given for the routine use of the RDE. Brain samples containing no more than 75-150 pg (DA, DOPA, DOPAC, HVA, and 5-HIAA) or 500 pg (NA) could be reproducibly assayed with high recovery (approx. 85%) and precision (approx. 5%), without the use of internal standards. Endogenous concentrations of DA, NA, DOPA, DOPAC, HVA, and 5-HIAA were determined in eight brain structures.     

Effect of 3,4-Methylenedioxymethamphetamine on 3,4-Dihydroxyphenylalanine Accumulation in the Striatum and Nucleus Accumbens

The effect of the racemic mixture of 3,4-methylenedioxymethamphetamine (MDMA) on the synthesis of dopamine in the terminals of nigrostriatal and mesolimbic neurons was estimated by measuring the accumulation of 3,4-dihydroxyphenylalanine (DOPA) in the striatum and nucleus accumbens 30 min following the administration of the L-aromatic amino acid decarboxylase inhibitor, 3-hydroxybenzylhydrazine. MDMA produced an increase in DOPA accumulation in the striatum which was greater in magnitude and longer in duration than that in the nucleus accumbens. Although the concentrations of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in both the striatum and nucleus accumbens were reduced 3 h following an injection of MDMA (20 mg/kg), 5-HT and 5-HIAA concentrations were significantly reduced only in the striatum 7 days after the administration of MDMA. Pretreatment with a 5-HT2 antagonist, ketanserin, significantly attenuated the reduction in 5-HT concentration in the striatum 3 h following MDMA administration and completely blocked 5-HT depletion at 7 days post administration. Moreover, ketanserin completely blocked MDMA-induced DOPA accumulation in the striatum. The results obtained in these studies suggest that MDMA activates nigrostriatal dopaminergic pathways via 5-HT2 receptors. In addition, these data are supportive of the hypothesis that dopamine plays a role in MDMA-induced 5-HT depletion.     

Effect of TRH analog (DN-1417) on tyrosine hydroxylase activity in mesocortical, mesolimbic and nigrostriatal dopaminergic neurons of rat brain

Tyrosine hydroxylase (TH) was assayed in eight regions of rat brain following repeated treatment with a TRH analog, DN-1417 (gamma-butyrolactone-gamma-carbonyl-histidyl-prolinamide). Repeated DN-1417 treatment (20 mg/kg/day, IP) for 7 days increased TH activity in the ventral tegmental area and decreased in the prefrontal cortex polar, medial and lateral fields and olfactory tubercles. No significant change in TH activity was found in the nucleus accumbens, striatum and substantia nigra. Kinetic analysis showed that the increased TH activity in the ventral tegmental area was due to an increase in Vmax, but not a change in the apparent Km of TH for a cofactor, 6-methyl-tetrahydropteridine. When TH was assayed at a suboptimal pH and in the presence of a subsaturating cofactor, the striatal TH was activated significantly after DN-1417. In the prefrontal cortex medial field, nucleus accumbens and olfactory tubercles, TH activity assayed under the suboptimal condition was not modified by DN-1417 treatment. These results suggest an intimate involvement of central dopaminergic systems in the actions of DN-1417.     

Characteristics of thyrotropin releasing hormone (TRH) receptors in rat brain

Characteristics of TRH-receptors were studied in the rat central nervous system (CNS). Ion species, pH and temperature importantly influenced TRH-receptor binding. Subcellular distribution of TRH-receptor binding revealed that synaptic membranes had the greatest percentage of total sites. Scatchard analysis suggested that the rat CNS had two distinct TRH binding sites with apparent dissociation constants (Kd) of 5 X 10(09) M and 13 X 10(-8) M. Receptor activity is sensitive to trypsin and phospholipase A digestion, suggesting that protein and phospholipid moieties are essential for the binding of [3H]TRH. Thiol reagents reduced the binding activity of the receptor, suggesting that an intrachain disulfide bond may form an important constituent of the binding site for TRH. The TRH-receptor in the rat brain was successfully solubilized with non-ionic detergent Triton X-100. On gel chromatography with Sepharose 6B column, the solubilized TRH-receptor molecule eluted at the fraction corresponding to an apparent molecular weight of 300,000 daltons, with Stokes' radius of 5.8 nm. The regional distribution of TRH-receptor binding was examined to clarify the site of TRH action. The highest level of binding was in the hypothalamus, cerebral cortex and hippocampus, indicating that TRH affects the CNS function mainly through the limbic system, cerebral cortex and hypothalamus. Moreover, tricyclic anti-depressants and Li+ decreased the binding of [3H]TRH. These findings suggest that endogenous TRH and TRH receptor may play the role of a neurotransmission modulator in the brain to control emotional and mental functions.     

3-Methoxytyramine Is the Major Metabolite of Released Dopamine in the Rat Frontal Cortex: Reassessment of the Effects of Antipsychotics on the Dynamics of Dopamine Release and Metabolism in the Frontal Cortex, Nucleus Accumbens, and Striatum by a Simple Two Pool Model

Abstract: 3-Methoxytyramine (3-MT) and 3,4-dihydroxyphenylacetic acid (DOPAC) rates of formation were used, respectively, to assess the dynamics of dopamine (DA) release and turnover in the rat frontal cortex, nucleus accumbens, and striatum. Assuming total (re)uptake and metabolism of released DA are relatively uniform among the three brain regions, a simplified two pool model was used to assess the metabolic fate of released DA. Under basal conditions, 3-MT formation was found to comprise >60% of total DA turnover (sum of 3-MT plus DOPAC rates of formation) in the frontal cortex, and not more than 15% in the nucleus accumbens and striatum. Haloperidol increased the 3-MT rate of formation to a greater extent in the frontal cortex than in the two other regions. Clozapine increased the 3-MT rate of formation in the frontal cortex and decreased it in the striatum. Both drugs increased DOPAC rate of formation in the frontal cortex and nucleus accumbens. It was elevated by haloperidol but not clozapine in the striatum. It is concluded that (1) O -methylation is a prominent step in the catabolism of DA in the frontal cortex under both physiological conditions and after acute treatment with antipsychotics, (2) 3-MT is the major metabolite of released DA in the frontal cortex and possibly also in the nucleus accumbens and striatum, (3) in contrast to the frontal cortex, most of the DOPAC in the nucleus accumbens and striatum appear to originate from intraneuronal deamination of DA that has not been released, (4) because presynaptic uptake and metabolism of DA give rise to DOPAC, whereas postsynaptic uptake and metabolism produced both DOPAC and 3-MT, the ratio of 3-MT to DOPAC rates of formation can be a useful index of reuptake inhibition.     

Antagonism of ethanol-induced decrease in rat brain cGMP concentration by histidyl-proline diketopiperazine, a thyrotropin releasing hormone metabolite.

Intraperitoneal administration of thyrotropin releasing hormone (50 μmol/kg) produced an approximately 2-fold increase in rat brain cGMP concentration within 15 min. Histidyl-proline diketopiperazine, a metabolite of thyrotropin releasing hormone, produced a similar effect, but the response was faster and shorter-lasting. Intraperitoneal administration of ethanol (1.5 g/kg) decreased brain cGMP concentration approximately 50% within 10–15 min; thyrotropin releasing hormone or histidyl-proline diketopiperazine, injected 5 min after ethanol, antagonized the ethanol-induced decrease in cGMP. Antagonism of the ethanol-induced decrease in the cGMP level required 10 μmol/kg of thyrotropin releasing hormone but was observed with 5 μmol/kg of histidyl-proline diketopiperazine. These data suggest that the metabolic conversion of thyrotropin releasing hormone to histidylproline diketopiperazine might explain the previous observation that thyrotropin releasing hormone elevated the level of brain cGMP and antagonized the ethanolinduced decrease in brain cGMP concentration.     

Induction of neuronal damage in guinea pig brain by intratracheal infusion of 2-chloroethyl ethyl sulfide, a mustard gas analog

Intratracheal infusion of 2-chloroethyl ethyl sulfide (CEES), a mustard gas analog and a chemical warfare agent is known to cause massive damage to lung. The purpose of this study was to determine whether intratracheal CEES infusion causes neuronal damage. Histological, immunohistochemical, and Western blot studies indicated that CEES treatment caused dose-dependent increases in blood cell aggregation, microglial cell number, microglial activation, and brain inflammation. In addition, an increased expression of α-synuclein and a decreased expression of the dopamine transporter were observed. The results indicate that intratracheal CEES infusion is associated with changes in brain morphology mediated by an increase in α-synuclein expression, leading to neurotoxicity in a guinea pig model. These changes may be mediated by oxidative stress. Furthermore, the present study indicates for the first time that intratracheal infusion of a single dose of CEES can cause neuroinflammation, which may lead to neurological disorders in later part of life.     

Observations on N alpha-deacetylation of model amino acids and peptides: distribution and purification of a specific N-acyl amino acid releasing enzyme in rat brain

N alpha-Acyl amino acid releasing enzyme (NAARE), an enzyme cleaving acetylMet-Ala at the Met-Ala bond was purified from rat brain cytosol to apparent homogeneity by salt precipitation, gel filtration, and several steps of ion exchange. Levels of NAARE exceeded acylase measured with acetylmethionine in all brain regions and subcellular fractions examined: 60% was associated with cytosol and the remainder with debris or the crude nuclear and mitochondrial-synaptosomal subfractions. Activity was highest in pituitary and was approximately 0.5-0.6 that of liver or kidney. The purified enzyme preferentially hydrolyzed acetylmethionyl peptides: Km for acetylMet-Ala was 0.93; Vmax, 3.5 nmol-1 (kcat, 1185) with pH optimum of 8.9 as compared with 8.2 for acylases measured in cytosol. The purified enzyme was devoid of acylase and common exo- and endopeptidase contamination. Structure-activity relationships examined with synthetic formylated or acetylated peptides indicated no significant effects for di- or tripeptides if the second substituent was Ala, Ser, Asn, or Thr, but the activity was reduced 0.5-fold for Leu, a branched-chain amino acid. No hydrolysis was observed for polypeptides with five or more residues having N-terminal acetylated Tyr (enkephalin) or Ser (alpha-melanocyte-stimulating hormone, thymosin alpha 1), supporting the notion that the enzyme plays a role only in turnover of smaller peptides formed perhaps as a result of endopeptidase cleavage of proteins or polypeptides containing acetylated Met at the N terminus.     

In vitro and in vivo effects of GABA, muscimol, and bicuculline on lamprey GnRH concentration in the brain of the sea lamprey (Petromyzon marinus)

gamma-Aminobutyric acid (GABA) is a neurotransmitter with a demonstrated neuroregulatory role in reproduction in most representative species of vertebrate classes via the hypothalamus. The role of GABA on the hypothalamus-pituitary axis in lampreys has not been fully elucidated. Recent immunocytochemical and in situ hybridization studies suggest that there may be a neuroregulatory role of GABA on the gonadotropin-releasing hormone (GnRH) system in lampreys. To assess possible GABA-GnRH interactions, the effects of GABA and its analogs on lamprey GnRH in vitro and in vivo were studied in adult female sea lampreys (Petromyzon marinus). In vitro perfusion of GABA and its analogs at increasing concentrations (0.1-100 microM) was performed over a 3-h time course. There was a substantial increase of GnRH-I and GnRH-III following treatment of muscimol at 100 microM. In in vivo studies, GABA or muscimol injected at 200 microg/kg significantly increased lamprey GnRH concentration in the brain 0.5 h after treatment compared to controls in female sea lampreys. No significant change in lamprey GnRH-I or GnRH-III was observed following treatment with bicuculline. These data provide novel physiological data supporting the hypothesis that GABA may influence GnRH in the brain of sea lamprey.     

Effects of acute and sub-chronic l-dopa therapy on striatal l-dopa methylation and dopamine oxidation in an MPTP mouse model of Parkinsons disease

Aims

The molecular mechanisms for the loss of 3,4-dihydroxyphenylalanine (l-dopa) efficacy during the treatment of Parkinson's disease (PD) are unknown. Modifications related to catecholamine metabolism such as changes in l-dopa and dopamine (DA) metabolism, the modulation of catecholamine enzymes and the production of interfering metabolites are the primary concerns of this study.

Main methods

Normal (saline) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) pre-treated mice were primed with 100 mg/kg of l-dopa twice a day for 14 days, and a matching group remained l-dopa naïve. l-dopa naive and primed mice received a challenge dose of 100 mg/kg of l-dopa and were sacrificed 30 min later. Striatal catecholamine levels and the expression and activity of catechol-O-methyltransferase (COMT) were determined.

Key findings

Normal and MPTP pre-treated animals metabolize l-dopa and DA similarly during l-dopa therapy. Administration of a challenge dose of l-dopa increased l-dopa and DA metabolism in l-dopa naïve animals, and this effect was enhanced in l-dopa primed mice. The levels of 3-OMD in MPTP pre-treated animals were almost identical to those in normal mice, which we found are likely due to increased COMT activity in MPTP pre-treated mice.

Significance

The results of this comparative study provide evidence that sub-chronic administration of l-dopa decreases the ability of the striatum to accumulate l-dopa and DA, due to increased metabolism via methylation and oxidation. This data supports evidence for the metabolic adaptation of the catecholamine pathway during long-term treatment with l-dopa, which may explain the causes for the loss of l-dopa efficacy.     

Solution structures, dynamics, and lipid-binding of the sterile alpha-motif domain of the deleted in liver cancer 2

The deleted in liver cancer 2 (DLC2) is a tumor suppressor gene, frequently found to be underexpressed in hepatocellular carcinoma. DLC2 is a multidomain protein containing a sterile alpha-motif (SAM) domain, a GTPase-activating protein (GAP) domain, and a lipid-binding StAR-related lipid-transfer (START) domain. The SAM domain of DLC2, DLC2-SAM, exhibits a low level of sequence homology (15-30%) with other SAM domains, and appears to be the prototype of a new subfamily of SAM domains found in DLC2-related proteins. In the present study, we have determined the three-dimensional solution structure of DLC2-SAM using NMR methods together with molecular dynamics simulated annealing. In addition, we performed a backbone dynamics study. The DLC2-SAM packed as a unique four alpha-helical bundle stabilized by interhelix hydrophobic interactions. The arrangement of the four helices is distinct from all other known SAM domains. In contrast to some members of the SAM domain family which form either dimers or oligomers, both biochemical analyses and rotational correlation time (tau(c)) measured by backbone 15N relaxation experiments indicated that DLC2-SAM exists as a monomer in solution. The interaction of DLC2-SAM domain with sodium dodecyl sulfate (SDS) micelles and 1,2-dimyristoyl-sn-glycerol-3-phosphatidylglycerol (DMPG) phospholipids was examined by CD and NMR spectroscopic techniques. The DLC2-SAM exhibits membrane binding properties accompanied by minor loss of the secondary structure of the protein. Deletion studies showed that the self-association of DLC2 in vivo does not require SAM domain, instead, a protein domain consisting of residues 120-672 mediates the self-association of DLC2.     

Protection of cultured rat cortical neurons from excitotoxicity by asarone,a major essential oil component in the rhizomes of Acorus gramineus

Previous reports have shown that the methanol extract and the essential oil from Acori graminei Rhizoma (AGR) inhibited excitotoxic neuronal cell death in primary cultured rat cortical cells. In the present study, an active principle was isolated from the methanol extract by biological activity-guided fractionations and identified as asarone. We evaluated neuroprotective actions and action mechanisms of the isolated asarone as well as the alpha- and the beta-asarone obtained commercially. The isolated asarone inhibited the excitotoxicity induced by the exposure of cortical cultures for 15 min to 300 microM NMDA in a concentration-dependent manner, with the IC50 of 56.1 microg/ml. The commercially obtained alpha- and beta-asarone exhibited more potent inhibitions of the NMDA-induced excitotoxicity than the isolated asarone. Their respective IC50 values were 18.2 and 26.5 microg/ml. The excitotoxicity induced by glutamate (Glu) was also inhibited, but with much less potency than the toxicity induced by NMDA. The IC50 values for the alpha-, beta-, and the isolated asarone were 89.7, 121.7, and 279.5 microg/ml, respectively. Based on the receptor-ligand binding studies using a use-dependent NMDA receptor-channel blocker [3H]MK-801, asarone inhibited the specific bindings in a concentration-dependent fashion. These results indicate that asarone, the major essential oil component in AGR, exhibits neuroprotective action against the NMDA- or Glu-induced excitotoxicity through the blockade of NMDA receptor function. The alpha-asarone was found to exhibit more potent inhibition of [3H]MK-801 bindings, which is consistent with its more potent neuroprotective action than the beta- or the isolated asarone.     

Protein disulfide isomerase, but not binding protein, overexpression enhances secretion of a non-disulfide-bonded protein in yeast

In eukaryotes, secretory proteins are folded and assembled in the endoplasmic reticulum (ER). Many heterologous proteins are retained in the ER due to suboptimal folding conditions. We previously reported that heterologous secretion of Pyrococcus furiosus beta-glucosidase in Saccharomyces cerevisiae resulted in the accumulation of a large fraction of inactive beta-glucosidase in the ER. In this work, we determine the effect of introducing additional genes of ER-resident yeast proteins, Kar2p (binding protein [BiP]) and protein disulfide isomerase (PDI), on relieving this bottleneck. Single-copy expression of BiP and PDI worked synergistically to improve secretion by reverse similar 60%. In an effort to optimize BiP and PDI interactions, we created a library of beta-glucosidase expression strains that incorporated four combinations of constitutively or inducibly-expressed BiP and PDI genes integrated to random gene copynumbers in the yeast chromosome. Approximately 15% of the transformants screened had secretion level improvements higher than that seen with single BiP/PDI gene overexpression, and the highest secreting strain had threefold higher beta-glucosidase levels than the control. Nineteen of the improved strains were re-examined for beta-glucosidase secretion as well as BiP and PDI levels. Within the improved transformants BiP and PDI levels ranged sevenfold and tenfold over the control, respectively. Interestingly, increasing BiP levels decreased beta-glucosidase secretion, whereas increasing PDI levels increased beta-glucosidase secretion. The action of PDI was unexpected because beta-glucosidase is not a disulfide-bonded protein. We suggest that PDI may be acting in a chaperone-like capacity or possibly creating mixed disulfides with the beta-glucosidase's lone cysteine residue during the folding and assembly process.     

Interferon gamma promotes survival of lymphoblasts overexpressing 9-O-acetylated sialoglycoconjugates in childhood acute lymphoblastic leukaemia (ALL)

An enhanced linkage-specific 9-O-acetylated sialic acid (9-O-AcSA) on peripheral blood mononuclear cells (PBMC) of children with acute lymphoblastic leukaemia, ALL (PBMC(ALL), 9-O-AcSA+ cells) was demonstrated by using a lectin, Achatinin-H, whose lectinogenic epitope was 9-O-AcSAalpha2-6GalNAc. Our aim was to evaluate the in vitro contributory role of this glycotope (9-O-AcSAalpha2-6GalNAc) towards the survival of these 9-O-AcSA+ cells in ALL patients. For direct comparison, 9-O-AcSA- cells were generated by removing O-acetyl group of 9-O-AcSA present on PBMC(ALL) using O-acetyl esterase. An elevated level of serum interferon gamma (IFN-gamma) in affected children led us to think that PBMC(ALL) are continuously exposed specifically to this cytokine. Accordingly, 9-O-AcSA+ and 9-O-AcSA- cells were exposed in vitro to IFN-gamma. A twofold increased NO release along with inducible NO synthase (iNOS) mRNA expression by the 9-O-AcSA+ cells was observed as compared to the 9-O-AcSA- cells. The decreased viability of IFN-gamma exposed 9-O-AcSA- cells as compared to 9-O-AcSA+ cells were reflected from a 5.0-fold increased caspase-3-like activity and a 10.0-fold increased apoptosis in the 9-O-AcSA- cells when production of NO was lowered by adding competitive inhibitor of iNOS in reaction mixture. Therefore, it may be envisaged that a link exists between induction of this glycotope and their role in regulating viability of PBMC(ALL). Taken together, it is reasonable to hypothesise that O-acetylation of sialic acids on PBMC(ALL) may be an additional mechanism that promotes the survival of lymphoblasts by avoiding apoptosis via IFN-gamma-induced NO production.     

Comparative effects on blood pressure and heart rate of dynorphin A(1–13) in anterior hypothalamic area, posterior hypothalamic area, nucleus tractus solitarius, and lateral cerebral ventricle in the rat

The objective of this study was to explore the effects of the endogenous opioid peptide dynorphin A(1–13) on the CNS regulation of blood pressure and heart rate. Wistar rats, anesthetized with pentobarbital and halothane, received dynorphin A(1–13) microinjected into the anterior hypothalamus area (AHA), the posterior hypothalamic area (PHA), the nucleus tractus solitarius (NTS), or the lateral cerebral ventricle (ICV). Dynorphin A(1–13), 20 (12 nmol) or 30 μg ICV, produced significant (p < 0.05) reductions in blood pressure and heart rate. Naloxone, 50 μg/kg ICV, completely prevented the blood pressure response and significantly (p < 0.05) blunted the heart rate response to the highest dynorphin concentration, 30 μg ICV (18 nmol). Dynorphin A(1–13), 5 μg, in the NTS significantly (p < 0.05) decreased systolic and diastolic blood pressure and heart rate with the response being evident 10 min and persisting for 30 min after injection. In contrast, the same dose of dynorphin A(1–13) in the AHA produced an immediate, marked, and significant (p < 0.05) decrease in systolic and diastolic blood pressure and heart rate that attained its maximum 1–3 min and returned rapidly towards baseline levels. Dynorphin A(1–13), 5 or 10 μg in the posterior hypothalamic area, was not associated with any change in blood pressure or heart rate. Injection of the diluent at any site was not associated with any changes in blood pressure or heart rate. The maximum change in blood pressure with dynorphin was greater in the AHA than NTS, and the maximum change in heart rate was greater in the NTS than AHA. These data indicate a potential role for dynorphin as a modulator of the CNS regulation of blood pressure and cardiac rate, and this is mediated in part through different areas in the brain that maybe localized to the anterior hypothalamic area and nucleus tractus solitarius but not the posterior hypothalamic area.     

Liquid chromatographic and tandem mass spectrometric assay for evaluation of in vivo inhibition of rat brain monoamine oxidases (MAO) A and B following a single dose of MAO inhibitors: application of biomarkers in drug discovery

A simple and selective assay for the evaluation of in vivo inhibition of rat brain monoamine oxidases (MAO) A and B following a single dose of MAO inhibitors was developed through the simultaneous determination of endogenous 5-hydroxy tryptamine, 5-hydroxyindole-3-acetic acid (5-HIAA), tryptophane, and 2-phenethylamine (PEA) in rat brain using liquid chromatography-tandem mass spectrometry (LC/MS/MS). These analytes were separated on a Zorbax SB-C18 column using a gradient elution with acetonitrile and 0.2% formic acid and detected on an electrospray ionization mass spectrometer in positive-ion multiple-reaction-monitoring mode. The susceptibility and variability of these analytes as potential biomarkers in response to MAO inhibition in vivo were evaluated after application to three MAO inhibitors, tranylcypromine, clorgyline, and pargyline. A dramatic increase (about 40-fold) in PEA brain level and a decrease in 5-HIAA by more than 90% were observed after administration of 15 mg/kg of the nonselective MAO inhibitor tranylcypromine. As expected, the brain level of PEA escalated to about 6-fold, while the 5-HIAA level remained unchanged following a dose of the MAO B inhibitor pargyline at 2mg/kg. In contrast, the brain level of 5-HIAA reduced by approximately 53%, but the PEA level was unaffected following the same dose of the MAO A inhibitor clorgyline. The results indicated that 5-HIAA and PEA were susceptible and effective biomarkers in the rat brain in response to MAO A and B inhibition, respectively. The LC/MS/MS method is useful not only for the determination of inhibitory potency but also for the differentiation of the selectivity of a MAO inhibitor against rat brain MAO A and B in vivo.