Advanced intrauterine growth restriction is associated with reduced excretion of asymmetric dimethylarginine.

BACKGROUND: High blood levels of asymmetric dimethylarginine (ADMA) are associated with future development of adverse cardiovascular events. The ADMA/symmetric dimethylarginine (SDMA) ratio is a marker of ADMA catabolism, with a high ADMA/SDMA ratio being suggestive of reduced ADMA excretion. AIMS: This study aimed a) to verify the presence of a statistically significant difference between ADMA/SDMA ratio levels in a group of young adult subjects who were born preterm with an extremely low birth weight (ex-ELBW) and a group of healthy adults born at term and b) to seek correlations between ADMA/SDMA ratio levels in ex-ELBW and anthropometric and clinical parameters (gender, chronological age, gestational age, birth weight, and length of stay in the Neonatal Intensive Care Unit). SUBJECTS, STUDY DESIGN, OUTCOME MEASURES: Thirty-seven ex-ELBW subjects (11 males [M] and 26 females [F], aged 17-28 years, mean age: 22.2 ± 1.8 years) were compared with 37 controls (11 M and 26 F). ADMA/SDMA ratio levels were assessed for each patient included in the study. RESULTS: ADMA/SDMA ratio in ex-ELBW subjects was higher compared to controls (1.42 ± 0.31 vs 0.95 ± 0.14, p<0.002) and inversely correlated with birth weight (r=-0.68, p<0.0001) and gestational age (r=-0.54, p<0.0005). CONCLUSIONS: ADMA catabolism is significantly decreased in ex-ELBW subjects compared to controls, underlining a probable correlation with restriction of intrauterine growth. These results suggest the onset of early circulatory dysfunction predictive of increased cardiovascular risk in ex-ELBW.

Clinical metabolomics and hematic ADMA predict the future onset of cardiorenal syndrome in young grown-up subjects who were born preterm.

OBJECTIVES: To look for differences in the urinary metabolic profile and in the hematic asymmetric dimethylarginine (ADMA) levels between a group of young adults born preterm with an extremely low birthweight (<1000 g; ex-ELBW; n=19) and a control group of subjects born at term with a weight appropriate for their gestational age (AGA; n=13); and to look for a possible correlation between the urinary metabolic profile in ex-ELBW and their hematic levels of ADMA. DESIGN AND METHODS: Urine samples were analyzed by (1)H nuclear magnetic resonance spectroscopy, and then submitted to unsupervised and supervised multivariate analysis. Samples of blood were collected and ADMA concentration was assessed by high-performance liquid chromatography. RESULTS: Using supervised PLS-DA (partial least squares discriminant analysis) model, the authors were able to discriminate between ex-ELBW and AGA. Statistically significant differences were detected in the ADMA levels between ex-ELBW and AGA (p<0.02). Ex-ELBW metabolic profile correlated with ADMA concentrations (r=0.456, p<0.05). Conversely, ADMA levels in AGA did not correlated with their metabolic profiles. CONCLUSIONS: This study demonstrates the relevance of the metabolomic technique as a predictive tool of the metabolic status in ex-ELBW. The relationship between ex-ELBW urinary metabolic profile and their blood ADMA levels suggests the presence of a subclinical cardio-renal involvement in these subjects.

Systematic analysis of rat 12/15-lipoxygenase enzymes reveals critical role for spinal eLOX3 hepoxilin synthase activity in inflammatory hyperalgesia.

Previously, we observed significant increases in spinal 12-lipoxygenase (LOX) metabolites, in particular, hepoxilins, which contribute to peripheral inflammation-induced tactile allodynia. However, the enzymatic sources of hepoxilin synthase (HXS) activity in rats remain elusive. Therefore, we overexpressed each of the 6 rat 12/15-LOX enzymes in HEK-293T cells and measured by LC-MS/MS the formation of HXB3, 12-HETE, 8-HETE, and 15-HETE from arachidonic acid (AA) at baseline and in the presence of LOX inhibitors (NDGA, AA-861, CDC, baicalein, and PD146176) vs. vehicle-treated and mock-transfected controls. We detected the following primary intrinsic activities: 12-LOX (Alox12, Alox15), 15-LOX (Alox15b), and HXS (Alox12, Alox15). Similar to human and mouse orthologs, proteins encoded by rat Alox12b and Alox12e possessed minimal 12-LOX activity with AA as substrate, while eLOX3 (encoded by Aloxe3) exhibited HXS without 12-LOX activity when coexpressed with Alox12b or supplemented with 12-HpETE. CDC potently inhibited HXS and 12-LOX activity in vitro (relative IC50s: CDC, ~0.5 and 0.8 µM, respectively) and carrageenan-evoked tactile allodynia in vivo. Notably, peripheral inflammation significantly increased spinal eLOX3; intrathecal pretreatment with either siRNA targeting Aloxe3 or an eLOX3-selective antibody attenuated the associated allodynia. These findings implicate spinal eLOX3-mediated hepoxilin synthesis in inflammatory hyperesthesia and underscore the importance of developing more selective 12-LOX/HXS inhibitors.

Could ADMA levels in young adults born preterm predict an early endothelial dysfunction?

BACKGROUND: Sporadic data present in literature report how preterm birth and low birth weight are risk factors for the development of cardiovascular diseases in later life. High levels of asymmetric dimethylarginine (ADMA), a strong inhibitor of nitric oxide synthesis, are associated with the future development of adverse cardiovascular events and cardiac death. AIMS: 1) to verify the presence of a statistically significant difference between ADMA levels in young adults born preterm at extremely low birth weight (<1000 g; ex-ELBW) and those of a control group of healthy adults born at term (C) and 2) to seek correlations between ADMA levels in ex-ELBW and anthropometric and clinical parameters (gender, chronological age, gestational age, birth weight, and duration of stay in Neonatal Intensive Care Unit). METHODS: Thirty-two ex-ELBW subjects (11 males [M] and 21 females [F], aged 17-29years, mean age 22.2 ± 2.3 years) were compared with 25 C (7 M and 18F). ADMA levels were assessed by high-performance liquid chromatography with highly sensitive laser fluorescent detection. RESULTS: ADMA levels were reduced in ex-ELBW subjects compared to C (0.606+0.095 vs 0.562+0.101 µmol/L, p<0.05), and significantly correlated inversely with gestational age (r=-0.61, p<0.00001) and birth weight (r=-0.57, p<0.0002). CONCLUSIONS: Our findings reveal a significant decrease in ADMA levels of ex-ELBW subjects compared to C, underlining a probable correlation with preterm birth and low birth weight. Taken together, these results may underlie the onset of early circulatory dysfunction predictive of increased cardiovascular risk.

Evidence for co-release of noradrenaline and dopamine from noradrenergic neurons in the cerebral cortex.

The aim of this study was to determine whether extracellular dopamine (DA) in the prefrontal cortex (PFC) might originate other than from DA neurons, also from noradrenergic (NA) ones. To this aim, we compared the levels of DA and NA in the dialysates from the PFC, a cortical area innervated by NA and DA neurons, and cortices that receive NA but minor or no DA projections such as the primary motor, the occipital-retrosplenial, and the cerebellar cortex. Moreover, the effect of alpha(2)-ligands and D(2)-ligands that distinctly modify NA and DA neuronal activity on extracellular NA and DA in these areas was studied. Extracellular NA concentrations were found to be similar in the different cortices, as expected from the homogeneous NA innervation, however, unexpectedly, also DA concentrations in the PFC were not significantly different from those in the other cortices. The alpha(2)-adrenoceptor agonist clonidine, intraperitoneally (i.p.) injected or locally perfused into the PFC, reduced not only extracellular NA levels, as expected from its ability to inhibit NA neuron activity, but also markedly reduced extracellular DA levels. Conversely, the alpha(2)-adrenoceptor antagonist idazoxan, i.p. injected or locally perfused into the PFC, not only increased extracellular NA levels, in line with its ability to activate NA neuron activity, but also increased those of DA. Conversely, in contrast to its ability to inhibit DA neuronal activity, the D(2) receptor agonist quinpirole only modestly and transiently reduced extracellular DA levels, while gamma-butyrolactone failed to modify DA levels in the PFC; conversely, haloperidol, at variance from its ability to activate DA neurons, failed to significantly modify extracellular DA levels in the PFC. Both haloperidol and quinpirole were totally ineffective after local perfusion into the PFC. Systemically injected or locally perfused, clonidine and idazoxan also modified both DA and NA concentrations in dialysates from primary motor, occipital-retrosplenial and cerebellar cortices as observed in the PFC. Finally, i.p. injected or locally perfused, clonidine reduced and idazoxan increased extracellular NA levels in the caudate nucleus, but neither alpha(2)-ligand significantly modified extracellular DA levels. Our results suggest that extracellular DA in the PFC, as well as in the other cortices, may depend on NA rather than DA innervation and activity. They suggest that dialysate DA reflects the amine released from NA neurons as well, where DA acts not only as NA precursor but also as co-transmitter. The co-release of NA and DA seems to be controlled by alpha(2)-receptors located on NA nerve terminals.

Lead intoxication during intrauterine life and lactation but not during adulthood reduces nucleus accumbens dopamine release as studied by brain microdialysis.

Environmentally relevant levels of lead (Pb) have been demonstrated to have a neurotoxic action, especially on children. In this study, Long-Evans rats were continuously exposed to Pb acetate in drinking water from early gestational days (2-6) or from 28 days of age. At the 13th week of age, the functional activity of the nucleus accumbens (NAC) dopaminergic system was studied by means of transversal microdialysis. Neither Pb treatment regimen modified dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) extracellular concentrations, with respect to control rats. However, neuronal depolarisation, induced by perfusion with 60 mM KCl, increased extracellular DA levels to a significantly minor degree in rats exposed to Pb during the intrauterine life, with respect to both control and adult Pb treated rats. The in utero treated rats also responded with a lower DA release to amphetamine (1 mg/kg ip) administration. On the other hand, no difference in NAC DA level was found amongst treatment groups in response to different concentrations of the D(2)-D(3) dopaminergic agonist quinpirole, locally administered by means of inverse dialysis. These data indicate a preferential impairment of NAC DA synthesis and/or release in rats exposed to Pb acetate during their intrauterine life.

Dissociation of haloperidol, clozapine, and olanzapine effects on electrical activity of mesocortical dopamine neurons and dopamine release in the prefrontal cortex.

The aim of the present study was to compare the effects of the typical antipsychotic haloperidol and the atypical antipsychotics clozapine and olanzapine on both extracellular dopamine (DA) levels in the medial prefrontal cortex (mPFC) as well as electrical activity of mesoprefrontal DA (mPFC-DA) neurons. Extracellular single unit recordings and microdialysis experiments were carried out in different groups of chloral hydrate anesthetised rats under identical experimental conditions. Intravenous administration of haloperidol, clozapine, and olanzapine increased the firing rate and burst activity of antidromically-identified mPFC-DA neurons; maximal increase in firing rate of approximately 140, 155, and 70 %, was produced by haloperidol, clozapine, and olanzapine at doses of 0.2, 2.5, and 1 mg/kg, i.v., respectively. Intravenous administration of the same doses increased extracellular DA levels in mPFC by 20%, 190%, and 70%, respectively. Moreover, while haloperidol and olanzapine increased extracellular levels of the deaminated DA metabolite DOPAC, by 60% and 40%, respectively, clozapine was totally ineffective. The D1 receptor antagonist SCH 23390 modified neither DA output nor neuronal firing. To determine whether the effect of the three antipsychotics on DA release might depend on a direct action on the mPFC, rats were perfused locally via inverse dialysis in the mPFC at concentrations ranging from 10(-6) to 10(-4)M. While clozapine and olanzapine increased extracellular DA concentrations by up to 400% of basal level, haloperidol was totally ineffective. The results obtained from this study indicate that the rank potency of the three antipsychotics in stimulating the firing rate of DA neurons projecting to mPFC, correlates with their affinity for D2 receptors and doses used clinically. On the other hand, their stimulating effect on DA release does not correlate with their effect on neuronal firing but depends on a direct action on the mPFC.

Antagonism by abecarnil of enhanced acetylcholine release in the rat brain during anticipation but not consumption of food.

Changes in the extracellular concentration of acetylcholine (ACh) were evaluated in the prefrontal cortex and hippocampus of freely moving rats habituated for 35 days to consume their daily meal during a fixed 2-h period. During the 40 min immediately before presentation, ACh output increased by 49 and 55% in the prefrontal cortex and hippocampus, respectively. ACh release increased further during the first 40 min of consumption phase in the prefrontal cortex (+220%) and hippocampus (175%). Administration of abecarnil (0.1 mg/kg, IP) 40 min before food presentation prevented the increase in ACh output in both brain regions during the anticipatory phase. In contrast, although abecarnil reduced the ACh content achieved during the consummatory phase, it did not prevent the increase in ACh release in the prefrontal cortex or hippocampus induced by food intake. Finally, the binding of [35S]TPBS to cerebral cortex, hippocampus, or septum of rats killed 20 min before food presentation was significantly higher than the values for animals killed 2 h after food presentation. These results suggest that during ingestive behavior ACh release is regulated by at least two independent mechanisms: one, associated with the anticipatory phase, that is sensitive to the activation of GABA(A) receptors. and a second, associated with the consummatory phase, that is insensitive to abecarnil.

Inhibition of basal and stress-induced dopamine release in the cerebral cortex and nucleus accumbens of freely moving rats by the neurosteroid allopregnanolone.

The neurosteroid allopregnanolone is a potent and efficacious modulator of γ-aminobutyric acid (GABA) type A receptors. The effects of intracerebroventricular injection of allopregnanolone (5 to 15 µg in 5 µl) on basal and stress-induced changes in the extracellular concentrations of dopamine were investigated by microdialysis in various brain areas of freely moving rats and compared with those of the benzodiazepine midazolam (1 to 10 µg in 5 µl). Allopregnanolone reduced (by a maximum of 65 to 75%) basal dopamine content in the prefrontal cortex and nucleus accumbens in a dose-dependent manner, but had no effect on dopamine output in the striatum. Allopregnanolone (10 to 15 µg) also completely prevented the increase in extracellular dopamine concentrations in the nucleus accumbens and cerebral cortex induced by foot-shock stress. Midazolam reduced basal dopamine content in all three brain regions studied as well as the stress- induced increase in dopamine content in the nucleus accumbens and cerebral cortex with a greater potency than allopregnanolone. These results suggest that endogenous neurosteroids may participate in the GABAergic modulation of dopaminergic transmission in the rat cerebral cortex and nucleus accumbens, two brain areas which are important in the regulation of emotional processes. These agents do not appear to affect striatal dopaminergic transmission which modulates motor function.