Relationship between hypothalamic noradrenergic activity and the sympathetic activity in interscapular brown adipose tissue after cold-swim stress in rats.

Noradrenaline (NA) activities in both hypothalamus and interscapular brown adipose tissue (IBAT) were simultaneously assessed before and after cold-swim stress in rats. The technique of gas chromatography-mass spectrometry was employed for the analysis of NA and its primary neuronal metabolite, 3,4-dehydroxy-phenylethylenglycol (DHPG), and the ratio of DHPG to NA was used as an index of NA activity. The ratios of DHPG/NA in both hypothalamus and IBAT were significantly elevated 5 and 20 min after cold-swim stress. Moreover, we found that there is a highly significant positive relationship between the hypothalamic DHPG/NA ratio and the ratio of DHPG/NA in IBAT (r = 0.872, p less than 0.0001). This observation strongly supports the concept in which hypothalamic NA neurons play an important role in modulating the sympathetic outflow.     

Dopamine-beta-hydroxylase inhibition acutely stimulates rats hypothalamic noradrenaline and dopamine neuronal activity as assessed from metabolic ratios and circulating glucose and ACTH responses

Because central noradrenaline neuronal activity is tonically inhibited by noradrenaline (NA) itself via an action at prejunctional alpha 2-adrenoceptors, it was hypothesised that the blockade of central NA synthesis following acute dopamine-beta -hydroxylase (DBH) inhibition might primarily deplete prejunctional NA levels and result in an increase in central NA neuronal activity through reduced NA autoinhibition. This hypothesis was tested in the rat following the acute administration of the DBH inhibitors diethyldithiocarbamate (DDC) and cysteamine (CSH). Computerised gas chromatography/mass spectrometry was used to precisely measure the hypothalamic levels of NA and dopamine (DA) together with those of their primary neuronal metabolites dihydroxyphenylethyleneglycol (DHPG) and dihydroxyphenylacetic acid (DOPAC), respectively. Both DDC (at 4 h) and CSH (at 30 min.) caused approximately a 50% reduction of hypothalamic NA concentrations. However this was associated with marked and highly significant increases in hypothalamic DHPG levels (by 50-100%) and in the hypothalamic ratio DHPG/NA. Also, when measured after CSH, the hypothalamic levels of the DHPG metabolite 3-methoxy-4-hydroxyphenylethyleneglycol were highly significantly increased. Consistent with increased DA neuronal activity, both DBH inhibitors raised DA and DOPAC levels and also the ratio DOPAC/DA in the hypothalami of treated rats and markedly suppressed serum prolactin levels (all p less than 0.01). The rise in hypothalamic concentrations of DHPG indicates that an increase in hypothalamic NA neuronal activity occurs following DBH inhibition. Significant elevations of blood glucose, corticosterone and ACTH were also observed after DBH inhibition. As we have previously demonstrated that increased central NA activity is associated with elevations of blood glucose, corticosterone and ACTH, these data provide further evidence for a functional increase in central NA activity caused by acute DBH inhibition. It is proposed that the increase in hypothalamic NA activity after DBH inhibition results from a primary depletion of the prejunctional alpha 2-active autoregulatory pool of NA.     

Brain noradrenergic neuronal activity affects 3,4-dihydroxyphenylethyleneglycol (DHPG) levels

Brain regional DHPG levels were determined following pharmacological manipulations that are known to alter brain noradrenergic neuronal activity. In rats given the α-adrenergic antagonist yohimbine (1, 5 and 10 mg/kg, i.p.) 2 h prior to sacrifice, there was a dose-dependent increase in cortical, midbrain, pons + medulla, hypothalamic and spinal total DHPG and MHPG concentrations. In contrast, cortical and spinal total DHPG and MHPG concentrations were markedly decreased 2 h following the α-adrenergic agonist, clonidine (10 and 250 μg/kg, i.p.). These findings indicate that rat brain DHPG formation is also sensitive to changes in brain noradrenergic neuronal impulse flow.     

Different acute effects of the tyrosine hydroxylase inhibitors alpha-methyl-p-tyrosine and 3-iodo-L-tyrosine on hypothalamic noradrenaline activity and adrenocorticotrophin release in the rat

Computerized gas chromatography-mass spectrometry techniques using selected ion monitoring and deuterated internal standards were used to assay simultaneously the medial basal hypothalamic concentrations of dopamine (DA) and noradrenaline (NA) and their major metabolites in individual rats 30 min after the administration of two different inhibitors of tyrosine hydroxylase, alpha-methyl-p-tyrosine (alpha-MT) and 3-iodo-L-tyrosine (MIT). Consistent with inhibition of DA synthesis, administration of both alpha-MT and MIT resulted in marked reductions (P less than 0.005) in the hypothalamic concentrations of DA and its metabolite homovanillic acid as well as in highly significant increases in prolactin secretion. alpha-MT administration, but not MIT, resulted in a highly significant decrease in NA concentration and a highly significant increase in the concentration of the NA metabolite 3,4-dihydroxyphenylethyleneglycol (DHPG). The hypothalamic ratio DHPG/NA was thus markedly increased (P less than 0.005) by alpha-MT indicating increased NA neuronal activity. alpha-MT administration also resulted in increased ACTH secretion (P less than 0.0005), an effect not observed following MIT. It is proposed that the effects on hypothalamic NA activity and ACTH secretion caused by alpha-MT are stress-mediated and unrelated to tyrosine hydroxylase inhibition. MIT is devoid of these effects but exhibits blockade activity, thus indicating it to be a preferable drug for the acute inhibition of tyrosine hydroxylase in neuroendocrine investigations.     

Relative Importance of 3-Methoxy-4-Hydroxyphenylglycol and 3,4-Dihydroxyphenylglycol as Norepinephrine Metabolites in Rat, Monkey, and Humans

Abstract: A gas chromatographic-mass spectrometric assay, which allowed simultaneous measurement of 3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxyphenylglycol (DHPG), was used to show that the concentration of MHPG in primate CNS far exceeded that of DHPG and that both metabolites were mainly in the unconjugated form. In rat brain, DHPG concentration was generally higher than that of MHPG, and both existed predominantly as conjugates. Rat and primate plasma contained more MHPG than DHPG. In plasma of primates but not of rats, higher proportions of the metabolites were conjugated, compared to those in brain. Significant correlations existed between MHPG and DHPG in rat brain, monkey brain, human plasma, and both monkey CSF and plasma. In monkeys, a significant CSF-plasma correlation was found for MHPG, but not for DHPG. Acute administration of piperoxane raised rat brain MHPG and DHPG concentration; desipramine prevented this rise in DHPG, but not in MHPG. Desipramine alone decreased DHPG, but not MHPG, concentration. Piperoxane increased monkey brain MHPG, but not DHPG, concentration. These data suggest that DHPG is a valuable metabolite to measure when assessing norepinephrine metabolism in the rat. Under certain conditions, measurement of rat brain MHPG and DHPG may provide information concerning the site of norepinephrine metabolism. However, in primates the importance of monitoring DHPG, in addition to MHPG, is uncertain.     

Effects of U-50,488H and U-50,488H withdrawal on catecholaminergic neurons of the rat hypothalamus

Previous report from our laboratory showed that morphine produces a stimulatory effect of hypothalamic noradrenaline (NA) turnover concurrently with enhanced pituitary-adrenal response after its acute injection and during withdrawal. In the present work we have studied the effects of acute and chronic administration of the kappa agonist U-50,488H as well as the influence of U-50,488H withdrawal on the activity of hypothalamic NA and dopamine (DA) neurons and on the activity of hypothalamic-pituitary-adrenal (HPA) axis. A single dose of U-50,488H (15 mg/kg i.p.) significantly increased hypothalamic NA and decreased DA turnover at the time of an enhanced corticosterone release. Rats rendered tolerant to the kappa agonist by administration of U-50,488H twice a day for 4 days showed no changes in corticosterone secretion. Additionally, a decrease in both hypothalamic MHPG (the cerebral NA metabolite) production and NA turnover was observed, whereas DOPAC concentration and DA turnover were enhanced, which indicate the development of tolerance towards the neuronal and endocrine actions of U-50,488H. After naloxone (3 mg/kg s.c.) administration to U-50,488H-tolerant rats, we found neither behavioural signs of physical dependence nor changes in hypothalamic catecholaminergic neurotransmission. In addition, corticosterone secretion was not altered in U-50,488H withdrawn rats. Present data clearly indicate that tolerance develops towards the NA turnover accelerating and DA turnover decreasing effect of U-50,488H. Importantly and by contrast to mu agonists, present results demonstrate that U-50,488H withdrawal produce no changes in hypothalamic catecholamines turnover or in corticosterone release (an index of the hypothalamus-pituitary-adrenal activity), which indicate the absence of neuroendocrine dependence on the kappa agonist. As has been proposed, this would suggest that the mu and the kappa receptor be regulated through different cellular mechanisms, as kappa agonists have a lower proclivity to induce dependence.     

Stimulation of the hypothalamic-pituitary-adrenal axis and inhibition of growth hormone release via increased central noradrenaline neuronal activity by urethane anaesthesia in the rat: blockade by clonidine

Computerized gas chromatography-mass spectrometry was used to measure precisely the hypothalamic levels of noradrenaline (NA), dopamine and serotonin together with those of their major neuronal metabolites 3,4-dihydroxyphenylethyleneglycol (DHPG), 3,4-dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid in normal male rats 45 min after stimulation of hypothalamic-pituitary-adrenal function by urethane (1.3 g/kg) administration. Urethane treatment resulted in a significant elevation of central noradrenergic neuronal activity (NNA) as assessed from marked rises in hypothalamic DHPG concentrations and the ratio (DHPG/NA). At the same time there was significant stimulation of ACTH and corticosterone release and inhibition of growth hormone release. These hormonal and central effects of urethane (but not anesthesia) were inhibited when the alpha 2-agonist clonidine (150 micrograms/kg) was co-administered. Urethane had no major effect on hypothalamic dopamine or serotonin status. We propose that the release of ACTH and the suppression of growth hormone release following urethane anaesthesia is a result of activation of central NNA and suggest that the hormonal responses are mediated via hypothalamic noradrenergic facilitation of corticotrophin releasing factor and somatostatin release to the anterior pituitary.     

The stimulatory effect of chronic lithium treatment on basal thyrotropin secretion in rats: In vivo antagonism by methylparaben

Chronic treatment of rats with lithium chloride was examined in order to determine its effect on hypothalamic monoamine and metabolite content, basal thyrotropin (TSH) secretion and thyroid function. The hypothalamic concentrations of noradrenaline (NA), dopamine (DA) and its metabolites, dihydroxyphenylacetic acid. (DOPAC) and homovanillic acid (HVA) in the lithium treated rats remained unaltered when compared to control levels. NA turnover and the NA metabolite, 3-methoxy-4-hydroxyphenylglycol (total MHPG), were significantly lower (p<0.01), whereas both serotonin (5-HT) and its metabolite, 5-hydroxyindole-3-acetic acid (5-HIAA), were significantly higher (p<0.01 and p<0.02, respectively) in the lithium treated rat hypothalami than in controls. Chronic lithium treatment significantly elevated basal TSH levels (p<0.05). This effect was antagonized by methylp-hydroxybenzoate (methylparaben, p<0.01), which did not itself affect basal TSH levels. Free serum T₃ and T₄ levels were not significantly affected by chronic lithium treatment, although T₄ tended to be slightly lower than control levels. The monoamine changes observed in the hypothalamus of lithium treated rats did not appear to account for the elevated TSH levels observed in these rats since NA activity which is generally regarded as stimulatory was decreased and 5-HT which has an inhibitory effect on TSH secretion, was increased. The elevated TSH levels may have been due to a reduced negative feedback inhibition of TSH release by the mildly reduced circulating T₄ levels caused by chronic lithium treatment. A further possibility is that the pituitary cGMP (and hence TSH) response to TRH may have been enhanced by chronic lithium treatment and methylparaben may have antagonized this effect.     

Neurotransmitters and neuropeptides in blood pressure regulation in the spontaneously hypertensive rat

Studies of the roles played by neurotransmitters in the development of hypertension in the spontaneously hypertensive (SHR) rat are complicated by the presence of genetic differences between SHR and normotensive control rats, which are not related to differences in blood pressure. One approach that may be used in an attempt to overcome this difficulty is to study the manner in which neurotransmitter and metabolite levels change with age, and to relate these changes to alterations in blood pressure with ageing. Noradrenaline (NA) levels in the brainstem and spinal cord of SHR and Wistar Kyoto rats fell with age, while 3,4-dihydroxyphenylethyleneglycol (DHPG) levels (a neuronal metabolite of noradrenaline) remained constant. Similar changes were seen when NA and DHPG levels were measured in the discrete brainstem A1, A2, and C2 region, and when adrenaline, NA, and DHPG levels were examined in the C1 region. Differences in age-related changes of neuropeptide Y (NPY) levels were also found in the ventromedial nucleus of the hypothalamus and the locus coeruleus, and of beta-endorphin in the anterior hypothalamic nucleus, the paragigantocellular nucleus of the brainstem, and the locus coeruleus. These changes may indicate either a progressive increase in the activity of neurons in the sympathoexcitatory C1 region or a progressive reduction in the activity of vasodepressor A1, A2, and C2 regions with ageing, or both. However, changes in catecholamines and metabolites with age were similar in both strains and therefore cannot readily explain the more rapid rise in blood pressure with ageing in SHR rats.     

Asymmetric metabolism of hypothalamic catecholamines alternates with side of ovulation in a lizard (Anolis carolinensis).

We determined levels of monoamines and their metabolites in 2 hypothalami dissected from the right and left hemibrains of 15 females during the right-left alternating ovulatory cycle of Anolis carolinensis. Tissue contents of the following were measured using HPLC and electrochemical (coulometric) detection: dopamine (DA) and its metabolite 2,4-dihydroxyphenylacetic acid (DOPAC), norepinephrine (NE) and its metabolites 3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxyphenylglycol (DHPG), and serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA). An asymmetry ratio (AR) was determined by subtracting hypothalamic content (pM/mg) on the larger ovary (LO) side from that on the smaller ovary (SO) side, divided by the sum of the 2 sides (AR = SO - LO/SO+LO). The Ar of MHPG and DHPG both decreased as the largest follicle in the LO grew during the cycle, from greater than 0 (content higher on the SO side) at the beginning of the cycle to less than 0 (content higher on the LO side). The average content of MHPG in the 2 sides significantly increased during the cycle. There were no significant asymmetric changes in hypothalamic DA or DOPAC. The average content of DA increased during the cycle, whereas the content of DOPAC, as well as DOPAC/DA, did not change. The average content of 5-HT increased, and the average metabolite ratio of 5-HIAA/5-HT decreased during the cycle without significant asymmetries. The metabolite ratios of NE and DA, but not 5-HT, were asymmetric on the same side in a given female.(ABSTRACT TRUNCATED AT 250 WORDS)     

Postmortem Stability of Brain 3-Methoxy-4-Hydroxyphenylethyleneglycol and 3,4-Dihydroxyphenylethyleneglycol in the Rat and Mouse

Abstract: To assess the postmortem stability of brain 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) and 3,4-dihydroxyphenylethyleneglycol (DHPG) levels, groups of rats and mice were killed by cervical dislocation and left at either 21° or 4°C for intervals of up to 24 h until removal and freezing of whole brain. Whole brain free and total MHPG and DHPG levels were determined simultaneously by gas chromatography-mass fragmentography (GC-MF). By 2 h after death, statistically significant decrements occurred in rat brain free DHPG (20%), total MHPG (21%), and total DHPG (11%) at 4°C, but free MHPG increased significantly (50%) compared with controls. At 21°C, rat brain total MHPG increased compared with controls at 2 h (15%) but decreased at 4 h (15%) and 8 h (15%), whereas free MHPG levels were increased at these times. Although brain total and conjugated DHPG levels showed little change, free DHPG levels were reduced at all times. In mouse brain no significant changes occurred in free MHPG and DHPG by 24 h at 4°C. At 21°C, mouse brain DHPG levels decreased whereas MHPG concentrations increased over the 8-h period of study. These findings demonstrate the occurrence of significant postmortem time- and temperature-dependent changes in brain MHPG and DHPG concentrations and indicate caution in the interpretation of changes in these metabolites in studies employing human postmortem brain tissue.     

Interspecies differences in the metabolism of brain norepinephrine to glycol metabolites

Using a highly sensitive and specific gas chromatography-mass spectrometric assay, the glycol metabolites of norepinephrine (NE), 3,4-dihydroxyphenylethyleneglycol (DHPG) and 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) were determined simultaneously in brain and body fluids of several mammalian species, including humans. Highest molar ratios of DHPG to MHPG were found in rat brain (1.20), a species in which these glycol metabolites were primarily conjugated. In mouse, guinea pig, hamster, monkey, and human brain, DHPG and MHPG were mostly unconjugated, and DHPG concentrations were about 30–60% of the respective MHPG levels. In dog cortex, MHPG occurred predominantly as conjugates, whereas DHPG could only be detected in its unconjugated form. In all species studies, highest DHPG and MHPG concentrations occurred in hypothalamus followed, in general, by midbrain and brainstem whereas cerebral cortex, caudate and cerebellum had the lowest values. These results demonstrate substantial differences in the degree of conjugation and relative abundance of brain DHPG compared to MHPG between the rat and other animal species studied.     

Plasma 3,4-dihydroxyphenylglycol (DHPG) and 3-methoxy-4-hydroxyphenylglycol (MHPG) are insensitive indicators of alpha 2-adrenoceptor mediated regulation of norepinephrine release in healthy human volunteers.

The usefulness of the plasma concentrations of two major metabolites of norepinephrine (NE), 3,4-dihydroxyphenylglycol (DHPG) and 3-methoxy-4-hydroxyphenylglycol (MHPG), as indicators of neuronal NE release was investigated. The potent alpha 2-adrenoceptor agonist, dexmedetomidine, induced only about 15% maximal reductions in the metabolite concentrations, in spite of almost total inhibition of neuronal NE release, as evidenced by 90% reductions in plasma NE concentrations. Similarly, administration of the alpha 2-adrenoceptor antagonist atipamezole was followed by only small increases in plasma DHPG and no change in MHPG levels, in spite of almost six-fold, albeit short-lasting, increases in plasma NE. In contrast, a single dose of the reversible monoamine oxidase type A (MAO-A) inhibitor moclobemide reduced plasma DHPG levels by 78% and MHPG levels by 51%. It is concluded that the plasma concentrations of DHPG and MHPG are largely determined by intraneuronal, MAO-A-dependent metabolism of NE, and do not accurately reflect acute alterations in neuronal NE release. The concentration of NE in venous plasma is clearly a more sensitive indicator of alpha 2-adrenoceptor-mediated regulation of NE release.     

Rat Brain and Plasma Norepinephrine Glycol Metabolites Determined by Gas Chromatography-Mass Fragmentography

Abstract: A gas chromatographic-mass fragmentographic (GC-MF) procedure is described for the simultaneous quantitation of 3,4-dihydroxyphenyl-ethyleneglycol (DHPG) and 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) in brain tissue and plasma. DHPG and MHPG were assayed as their respective acetyl-trifluoroacyl esters, using [²H₂]DHPG and [²H₃]MHPG as internal standards. Assay sensitivities of at least 1 ng per sample were attainable for the quantitation of free glycols, whereas for determination of total DHPG, assay sensitivity was 2.5 ng. Whole rat brain total (99.2 ±4.11 ng/g) and free (13.0 ± 1.14 ng/g) DHPG concentrations were similar to respective total (86.0 ± 3.70 ng/g) and free (12.3 ± 0.41 ng/g) MHPG levels. Total DHPG concentrations exceeded total MHPG levels in hypothalamus (3.0:1), midbrain (1.4:1), pons plus medulla (1.3:1), and hippocampus (1.5:1), whereas in other brain regions the levels of these metabolites were similar. In plasma, however, total DHPG levels were only 20% as high as MHPG concentrations. In mouse brain, DHPG and MHPG occurred almost entirely in free form (>90%), but total DHPG levels were only 50% as high as respective MHPG concentrations. These results emphasize the substantial formation of DHPG compared with MHPG in rat and mouse brain and suggest that DHPG formation and eMux may be of equal or greater importance than MHPG in the metabolic clearance of CNS norepinephrine in some species.     

Ventricular Cerebrospinal Fluid Monoamine Transmitter and Metabolite Concentrations Reflect Human Brain Neurochemistry in Autopsy Cases

Concentrations of dopamine (DA), its metabolites 3-methoxytyramine and homovanillic acid (HVA), noradrenaline (NA), its metabolites normetanephrine (NM) and 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-hydroxytryptamine (5-HT, serotonin), and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were measured in 14 brain regions and in CSF from the third ventricle of 27 human autopsy cases. In addition, in six cases, lumbar CSF was obtained. Monoamine concentrations were determined by reversed-phase liquid chromatography with electrochemical detection. Ventricular/lumbar CSF ratios indicated persistence of rostrocaudal gradients for HVA and 5-HIAA post mortem. Ventricular CSF concentrations of DA and HVA correlated positively with striatal DA and HVA. CSF NA correlated positively with NA in hypothalamus, and CSF MHPG with levels of MHPG in hypothalamus, temporal cortex, and pons, whereas CSF NM concentration showed positive correlations with NM in striatum, pons, cingulate cortex, and olfactory tubercle. CSF 5-HT concentrations correlated positively with 5-HT in caudate nucleus, whereas the concentration of CSF 5-HIAA correlated to 5-HIAA levels in thalamus, hypothalamus, and the cortical areas. These data suggest a specific topographic origin for monoamine neurotransmitters and their metabolites in human ventricular CSF and support the contention that CSF measurements are useful indices of central monoaminergic activity in man.     

Effects of chlorpromazine on hypothalamic aminergic neurons and stress responses in moderate cold

Guinea-pigs were treated with chlorpromazine or 0.9% NaCl and exposed to +4 degrees C or +23 degrees C for 2 h. Hypothalamic noradrenaline (NA), dopamine (DA), 5-hydroxytryptamine (5-HT), 3-methoxy-4-hydroxyphenylethylene-glycol (MHPG), homovanillinic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) were determined by high-performance liquid chromatography. Serum and urinary catecholamines, muscle and liver glycogen and blood glucose were also measured. Chlorpromazine caused deep hypothermia at this moderately cold temperature and slight hypothermia at room temperature. Cold increased the activity of noradrenergic and serotonergic neurons, as indicated by the increase in hypothalamic MHPG and 5-HIAA and also the MHPG:NA and 5-HIAA:5-HT ratios. A tendency towards drug-induced inhibition of hypothalamic serotonergic neurons was seen, although this was not significant. A drug-induced inhibition of noradrenergic neurons could not be ruled out. Increased drug-induced turnover of DA was observed in the cold, and a tendency in the same direction was seen at room temperature. Excretion of DA into the urine was induced by chlorpromazine. The hypothermic guinea-pigs had low serum catecholamines, indicating diminished sympathetic activity, but high urinary catechols, a sign of cold stress.     

Rat Brain Norepinephrine Metabolism: Substantial Clearance Through 3,4-Dihydroxyphenylethyleneglycol Formation

To assess whether the metabolic clearance of rat brain norepinephrine (NE) through 3,4-dihydroxyphenylethyleneglycol (DHPG) formation is quantitatively comparable or greater than through 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) production, we studied the accumulation rates of conjugated DHPG and MHPG following probenecid administration in whole brain as well as in several brain regions. Administration of increasing doses of probenecid (100-500 mg/kg, i.p.) 1.5 h before sacrifice produced a dose-dependent increase of conjugated DHPG and MHPG levels. The maximum increment of these conjugated metabolites occurred at a dose of 300 mg/kg or higher. During the first hour following probenecid administration (300 mg/kg, i.p.), rat brain conjugated DHPG and MHPG levels accumulated linearly at a rate of 646 and 319 pmol/g/h, respectively. With the probenecid technique, the estimated appearance rates of conjugated DHPG significantly exceeded those of conjugated MHPG in hypothalamus, midbrain, brainstem, hippocampus, and cerebral cortex. These results clearly indicate that under resting conditions, formation and efflux of conjugated DHPG is the major route of metabolic clearance of rat brain NE.     

Involvement of noradrenergic transmission in the PVN on CREB activation, TORC1 levels, and pituitary-adrenal axis activity during morphine withdrawal

Experimental and clinical findings have shown that administration of adrenoceptor antagonists alleviated different aspects of drug withdrawal and dependence. The present study tested the hypothesis that changes in CREB activation and phosphorylated TORC1 levels in the hypothalamic paraventricular nucleus (PVN) after naloxone-precipitated morphine withdrawal as well as the HPA axis activity arises from α(1)- and/or β-adrenoceptor activation. The effects of morphine dependence and withdrawal on CREB phosphorylation (pCREB), phosphorylated TORC1 (pTORC1), and HPA axis response were measured by Western-blot, immunohistochemistry and radioimmunoassay in rats pretreated with prazosin (α(1)-adrenoceptor antagonist) or propranolol (β-adrenoceptor antagonist). In addition, the effects of morphine withdrawal on MHPG (the main NA metabolite at the central nervous system) and NA content and turnover were evaluated by HPLC. We found an increase in MHPG and NA turnover in morphine-withdrawn rats, which were accompanied by increased pCREB immunoreactivity and plasma corticosterone concentrations. Levels of the inactive form of TORC1 (pTORC1) were decreased during withdrawal. Prazosin but not propranolol blocked the rise in pCREB level and the decrease in pTORC1 immunoreactivity. In addition, the HPA axis response to morphine withdrawal was attenuated in prazosin-pretreated rats. Present results suggest that, during acute morphine withdrawal, NA may control the HPA axis activity through CREB activation at the PVN level. We concluded that the combined increase in CREB phosphorylation and decrease in pTORC1 levels might represent, in part, two of the mechanisms of CREB activation at the PVN during morphine withdrawal.     

Simultaneous determination of noradrenaline and 3-methoxy-4-hydroxyphenylglycol in plasma with high-performance liquid chromatography using electrochemical detection

We herein report the simultaneous determination of the levels of noradrenaline (NA), and 3-methoxy-4-hydroxyphenylglycol (MHPG), a major metabolite of NA. The sample was subjected to a Sep Pak C18 cartridge prior to the NA and MHPG assay by high-performance liquid chromatography with an electrochemical detector. The results correlated well with the established methods. The average percentage of recovery was 91.2 and 98.7% for NA and MHPG, respectively. The intraassay coefficients of variation were 3.7 and 4.6% for NA and MHPG. The interassay coefficients of variation were 3.5 and 7.5% for NA and MHPG, respectively.     

Effects of naloxone-precipitated withdrawal after a single dose of morphine on catecholamine concentrations in guinea-pig brain

The effect of naloxone-precipitated withdrawal after acute morphine was studied on the concentrations of noradrenaline (NA), 4-hydroxy-3-methoxyphenylethyleneglycol (MHPG), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and on the metabolite/parent amine ratios MHPG/NA, DOPAC/DA and HVA/DA, in eight regions of the guineapig brain. Guinea-pigs were treated with a single dose of morphine sulphate (15 mg/kg s.c.) or saline (control) and 2h later with naloxone hydrochloride (15 mg/kg s.c.) to precipitate withdrawal. The animals were decapitated at 0.5 h or 1 h after naloxone injections and their brains analysed for monoamine concentrations by HPLC-ECD. At 0.5 h after naloxone-precipitated withdrawal NA and MHPG levels, and the MHPG/NA ratio, were increased in the hypothalamus, and the NA levels were increased in the hypothalamus, medulla/pons and cortex 1 h after naloxone. Naloxoneprecipitated withdrawal also produced increased DA metabolism in the cortex, midbrain and medulla 0.5 h later, and in the cortex, hypothalamus and striatum 1 h later. Hence naloxone-precipitated withdrawal from acute morphine treatment produced a complex pattern of increased synthesis and metabolism of NA and DA which varied over time and with the brain region examined.