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We investigated lithium-induced changes in norepinephrine (NE) catabolism. NE and its major metabolites 3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxyphenyl glycol (DHPG), ions such as lithium (Li(+)), magnesium (Mg(2+)), and potassium (K(+)) were measured in rat plasma and cerebral cortex using an HPLC method with electrochemical detection for amines. The results obtained with a group of rats treated by lithium chloride (2 mmol/kg/IP) were compared with a control group receiving sodium chloride (2 mmol/kg/IP). Animals were killed at different times over a period of six hours in the morning following salt administration to minimize possible chronobiological effects. There are two pathways leading to MHPG formation: way A, without DHPG, and way B, with DHPG. In plasma and cerebral cortex of lithium treated rats, way A catabolism seems to be preferential. Lithium increases Mg(2+) and K(+) plasma levels. These results suggest that lithium may increase inactivation of NE and decrease NE available for adrenergic receptors.

In healthy men, a decrease in plasma testosterone levels was observed in the context of metabolic stress. While physiological mechanisms underlying this response are unclear, there are several lines of evidence suggesting circulating epinephrine's influence on plasma testosterone levels. The purpose of this study was to directly relate stress-induced changes in plasma testosterone and epinephrine. The stressor used was blockade of glucose metabolism with pharmacological doses (40 mg/kg) of 2 deoxyglucose (2DG). Arterial plasma samples from 10 healthy males were assayed at 20 minutes intervals for 60 minutes for the concentrations of testosterone, epinephrine and related biochemicals. Bolus administration of 2DG resulted in progressive decline in testosterone and increases in epinephrine and norepinephrine plasma levels (mean change from baseline: 29, 2530 and 186%, respectively). Inverse correlation was detected between both absolute (r(s)=-0.72; df=8; p=0.017) and baseline-corrected testosterone concentrations at the 60 minute time point and epinephrine area under the curve values. Our results suggest that adrenomedullary activation may be involved in stress-induced testosterone effects. The implications of these data for the understanding of the role of catecholamines in glucoprivic stress response are discussed.

Mean arterial blood pressure (MAP), heart rate (HR) and arterial plasma levels of corticotropin (ACTH), renin activity (PRA) and catechols [norepinephrine (NE), epinephrine (EPI), and the intraneuronal NE metabolite dihydroxyphenylglycol (DHPG)] at baseline and in response to the serotonin-1C/2 (5-HT1C/5-HT2) agonist 1-(2,5-dimethoxy-4-iodophenyl)2-aminopropane (DOI, 1.0 mg/kg i.a.) in conscious, freely-moving, juvenile (4 week old) spontaneously hypertensive rats (SHR's) and age-matched Wistar-Kyoto (WKY) normotensive control rats were measured simultaneously. Baseline levels of MAP, NE, DHPG, and EPI all were significantly higher in the SHR's. There was a similar trend for PRA, but ACTH did not differ between the two strains. DOI produced marked increases in levels of MAP, ACTH, EPI, and also PRA but did not affect NE or DHPG concentrations. HR decreased only in the WKY group after administration of DOI. The magnitudes of the EPI and ACTH responses did not differ between the rat strains. Responses of MAP and PRA were significantly larger in SHR's. These results suggest that there is a selective hyperresponsiveness of PRA and blood pressure to 5-HT2 receptor stimulation parallel to a deficient baroreceptor reflex in juvenile SHR's.

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.

The plasma concentration of the dopamine (DA) metabolite, homovanillic acid (HVA), is used as an indicator of central nervous system dopaminergic activity. Using percutaneously inserted catheters we were able to obtain blood samples simultaneously from the right and left internal jugular veins. Veno-arterial HVA plasma concentration differences combined with adjusted organ plasma flows were used, according to the Fick Principle, to determine the HVA overflow from the brain. The HVA overflow from the liver was also measured. HVA overflow from the brain represented 12% of the total body HVA production. A similar amount was released from the liver, illustrating the limited validity of peripheral plasma HVA measurements as an indicator of central dopaminergic activity. HVA release from the human brain displayed a degree of asymmetry, the overflow into the left internal jugular vein being 36% greater than that into the right. Cerebral venous blood flow scans indicated that cortical cerebral regions drained preferentially into the right internal jugular; by inference the higher HVA overflow on the left originated from dopamine-rich subcortical brain areas. Since HVA in plasma may arise from the metabolism of DA existing either as a neurotransmitter or a norepinephrine (NE) precursor we measured the internal jugular vein plasma concentrations of NE, and its metabolite dihydroxyphenylglycol (DHPG), to determine whether they displayed a similar pattern of release to HVA. The overflow of both NE and DHPG into the right internal jugular vein was approximately double that on the left. Since the overflow of HVA did not parallel that of NE and DHPG it may be inferred that the origin of much of the subcortically produced HVA is from dopaminergic neurons and not from the metabolism of precursor DA in noradrenergic neurones or cerebrovascular sympathetic nerves.

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.

The serotonin receptor antagonist methysergide was used to investigate the mechanism mediating stimulation of prolactin release after single doses of the reversible MAO-A inhibitor moclobemide. Eight healthy male volunteers participated in a placebo-controlled cross-over study, where pretreatment with methysergide almost totally prevented the moclobemide-induced increase in plasma prolactin levels. MAO-A inhibition, as evidenced by up to 80% decreases in the plasma concentration of 3,4-dihydroxyphenylglycol, a deaminated metabolite of norepinephrine, was similar after both pretreatments. This result suggests that moclobemide stimulates prolactin release through activation of serotonergic receptors, and provides evidence that the drug is capable of augmenting central serotonergic neurotransmission in humans.

The magnitude and duration of effects of a single intravenous injection of 4'-amino MPTP, an analogue of the dopamine neurotoxin, MPTP, on plasma levels of catechols and normetanephrine were examined in conscious dogs. Plasma samples were collected prior to treatment with intravenous saline or 4'-amino MPTP.2HCl (22.5 mg/kg) and at weekly intervals for six weeks following treatment. Saline treatment had no effect on plasma levels of any of the measured compounds. Following 4'-amino MPTP, plasma DHPG fell to 14% of the pre-injection value and remained decreased for the full 6-week test period, with partial recovery by week 6 to 42% of the pre-injection value. Plasma DOPAC levels fell to 28% of pre-injection values 1 week after treatment with 4'-amino MPTP and showed no evidence of recovery during the 6-week test period. Plasma DOPA fell to 58% of the pre-injection level, while concentrations of the catecholamines NE, EPI, and DA were generally unaffected. The plasma concentration of the O-methylated NE metabolite, normetanephrine, was also unchanged by 4'-amino MPTP treatment. There were no differences in the concentrations of DA, NE or EPI within the adrenal medulla between saline and 4'-amino MPTP treated groups. This pattern of changes in plasma levels of catechols, which is consistent with presynaptic inhibition of MAO within sympathetic terminals, may be a useful indicator of exposure to MPTP-like neurotoxins.

The effect of manipulations aimed to alter brain noradrenergic neuron activity on the levels of free and conjugated DOPEG in discrete brain areas was studied in the rat. Electrical stimulation of the medial forebrain bundle for 10-20 min produced a frequency dependent elevation of free and conjugated DOPEG concentrations in the anterior cerebral cortex and in the hippocampus. In contrast, acute interruption of noradrenergic nerve impulse flow by application of tetrodotoxin (50-100 ng) into the medial forebrain bundle markedly diminished cortical and hippocampal DOPEG levels at 0.5-2 h post-injection. Cortical conjugated and free DOPEG levels were also reduced (by 80-96%) 2-3 weeks after bilateral electrolytic lesion of the locus coeruleus, 6-hydroxydopamine-induced lesion of the ascending noradrenergic pathways or noradrenergic denervation by the neurotoxic agent DSP4. Finally, the alpha-adrenoceptor blocking agents yohimbine (1-10 mg/kg, ip) and RX 781094 (3-10 mg/kg, ip) increased whereas the alpha-adrenergic agonist clonidine (0.01-1 mg/kg, sc) decreased DOPEG levels in the cerebral cortex, hypothalamus and septal areas. These data indicate that free and conjugated DOPEG formation is dependent on, and may serve as an index of, central noradrenergic neuron activity.

One-hour immobilization stress increased levels of the major metabolite of brain noradrenaline (NA), 3-methoxy-4-hydroxyphenyl-ethyleneglycol sulfate (MHPG-SO4), in nine brain regions of rats. Diazepam at 5 mg/kg attenuated the stress-induced increases in MHPG-SO4 levels in the hypothalamus, amygdala, hippocampus, cerebral cortex and locus coeruleus (LC) region, but not in the thalamus, pons plus medulla oblongata excluding the LC region and basal ganglia. The attenuating effects of the drug on stress-induced increases in metabolite levels in the above regions were completely antagonized by pretreatment with Ro 15-1788 at 5 or 10 mg/kg, a potent and specific benzodiazepine (BDZ) receptor antagonist. When given alone, Ro 15-1788 did not affect the increases in MHPG-SO4 levels. Behavioral changes observed during immobilization stress such as vocalization and defecation, were also attenuated by diazepam at 5 mg/kg and this action of diazepam was antagonized by Ro 15-1788 at 10 mg/kg, which by itself had no effects on these behavioral measurements. These findings suggest: (1) that diazepam acts via BDZ receptors to attenuate stress-induced increases in NA turnover selectively in the hypothalamus, amygdala, hippocampus, cerebral cortex and LC region and (2) that this decreased noradrenergic activity might be closely related to relief of distress-evoked hyperemotionality, i.e., fear and/or anxiety in animals.

Eleven normal volunteers were given an acute and two chronic doses of desipramine (DMI). The plasma norepinephrine (NE), 3-methoxy-4-hydroxyphenylglycol (MHPG), and dihydroxyphenylglycol (DHPG) concentrations were measured before and during drug administration. DMI reduced plasma concentrations of MHPG by 13% and DHPG by 17%. After two weeks of drug administration, the MHPG/NE ratio was reduced, and there was a significant negative correlation with the concurrent drug concentration. These results suggest that DMI: (1) reduces the turnover of NE; and (2) diminishes the oxidative deamination of NE. In addition, the drug concentration response relationship indicates that the effects of uptake inhibition may not be maximal until concentrations in the apparent therapeutic range are achieved.

Exposure of rats to an acute, uncontrollable stressor can increase brain norepinephrine (NE) turnover and decrease locomotor and exploratory behavior. We examined the ability of exogenous tyrosine, NE's amino acid precursor, to protect rats from developing these neurochemical and behavioral changes when stressed. Animals pretreated with saline or tyrosine (200 mg/kg, i.p.) were subjected to tail shock (15 v, 2 mA, 5 sec/30 sec) or to no shock during a 60-min period. Exposure to shock depleted NE and increased its turnover [as indicated by altered NE and 3-methoxy-4-hydroxy-phenylene-glycol sulfate levels (MHPG-SO4)] within the locus coeruleus, the hippocampus and the hypothalamus. Behavioral deficits were observed using measures of locomotion, standing on hind legs, and hole-poking in an open-field apparatus. Animals given tyrosine before shock displayed neither shock-induced NE depletion nor the deficits in locomotion and hole-poking; brain MHPG-SO4 levels tended to be greater than after shock alone. These observations suggest that the stress caused NE to be released from some neurons more rapidly than it could be restored by synthesis or reuptake, thereby impairing noradrenergic transmission and NE-dependent exploratory behaviors. Tyrosine administration presumably enhanced the transmitter's synthesis in stressed animals, thereby preventing both the neurochemical and the behavioral deficits.

We designed a rapid, simple and sensitive method for the determination of norepinephrine (NE) and its metabolites by reversed-phase high-performance liquid chromatography (HPLC) with electrochemical detection. NE, 3,4-dihydroxymandelic acid (DOMA), and 3,4-dihydroxyphenylglycol (DOPEG) were adsorbed on alumina and eluted with 0.2 N HCl. From the remaining solution, normetanephrine and 3-methoxy-4-hydroxyphenylglycol (MOPEG) were extracted with ethyl acetate in the presence of both borate buffer and K2HPO4. Vanillylmandelic acid was extracted with ethyl acetate after acidification of the solution with concentrated HCl. The combined ethyl acetate phase was evaporated and the residue was dissolved in 0.1 N HCl. A 50 mu1 aliquot of each eluate or solution was injected onto the HPLC. Detection limits ranged from 300 pg to 1 ng per initial sample. We used this method to determine substances in the medium following incubation of the rat vas deferens. Approximately 110 and 80 ng/g/10 min of DOPEG and MOPEG, respectively, were present under normal conditions. The electrical stimulation of tissues from the rat vas deferens led to increases in the levels of NE, DOPEG, DOMA and MOPEG. Normetanephrine and vanillylmandelic acid were not detected in the medium. This is probably the first documentation of the endogenous levels of NE and all its metabolites in medium containing tissue of the sympathetic nervous system.

An adaptation of the single-isotope radioenzymatic catecholamine assay technique allows simultaneous quantitation of free dihydroxyphenylglycol (DHPG), dihydroxymandelic acid (DOMA), and dihydroxyphenylacetic acid (DOPAC) in small volumes of plasma. Incubation of sample with catechol-O-methyltransferase and S-adenosylmethionine is followed by acidic extractions of metabolites and thin-layer chromatography. O-methylated products of the beta-hydroxylated metabolites DHPG and DOMA are further subjected to periodate cleavage to improve sensitivity. Quantitation by liquid scintillation counting with internal standardization results in a sensitivity of approximately 8, 30 and 70 pg for DHPG, DOMA and DOPAC. Normal values for these three metabolites in resting humans are (mean +/- S.D., pg/ml): 1010 +/- 280 for DHPG, 2090 +/- 720 for DOMA and 3270 +/- 1470 for DOPAC.

The immobility-reducing action of desipramine (DMI) in forced swimming rats was attenuated by intracerebroventricular (i.c.v.) injection of isoproterenol (ISO) and potentiated by i.c.v. atenolol (ATE), a beta 1-adrenoceptor antagonist. The effect of ISO was blocked by ATE. When administered i.c.v. in normal rats, ISO reduced the contents of 3-methoxy-4-hydroxyphenylethyleneglycol sulfate (MHPG-SO4), a major metabolite of noradrenaline, in the septal area, thalamus and hypothalamus while ATE had no effect in most of the brain regions. However, in forced swimming rats treated with DMI, ISO reduced MHPG-SO4 in 6 out of 8 brain regions tested and conversely, ATE increased the levels in the amygdala, septal area and hypothalamus. Similar to the behavioral effect, the effect of ISO was antagonized by ATE. These results support the hypothesis that central beta 1-adrenergic mechanisms inhibit the immobility-reducing action of DMI by reducing the activity of noradrenergic neurons in the brain.

Rats were given FLA-63, followed by R04-1284 0.5 h later to relatively selectively deplete brain NA. After 8 h, some animals were examined for regional brain NA and MHPG-SO4, while some were subjected to 3 h of cold-restraint stress and then examined. All brain regions examined showed significant NA and MHPG reduction. Specific NA depletion markedly exacerbated restraint ulcer formation and plasma corticosterone levels. NA depletion without restraint stress did not induce ulcers or elevate corticosterone. Intact brain NA activity appears to be essential for coping with stress.

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.

It has been suggested that the circulating levels of 3-methoxy-4-hydroxyphenylethylene glycol (MHPG), a major end metabolite of noradrenaline (NA), may provide an index of central NA neuronal activity. The aim of this study was to examine in the rat the relationship between serum MHPG and hypothalamic NA neuronal activity during basal conditions, and when hypothalamic NA neuronal activity was stimulated or suppressed. Hypothalamic NA neuronal activity was assessed from the concentrations of the primary neuronal NA metabolite 3,4-dihydroxyphenylethyleneglycol (DHPG), MHPG, and the DHPG/NA and MHPG/NA ratios. Following 2-deoxyglucose (2DG) and cysteamine administration, hypothalamic NA neuronal activity and serum MHPG rose significantly. In contrast, hypothalamic NA neuronal activity and serum MHPG fell significantly in gentled rats. Serum MHPG correlated significantly with hypothalamic DHPG and the DHPG/NA ratio in control rats, and with hypothalamic DHPG, MHPG, and the DHPG/NA and MHPG/NA ratios in gentled, 2DG- and cysteamine-treated rats. In the latter two groups, serum MHPG also correlated significantly with serum glucose, which is itself closely related to hypothalamic NA neuronal activity. These studies demonstrate a significant relationship between serum MHPG and hypothalamic NA neuronal activity in the rat, so that serum MHPG provides an index of hypothalamic NA neuronal activity in the rat.

We investigated the utility of deaminated norepinephrine (NE) metabolites in the study of human sympathetic nervous pathophysiology. Plasma levels of the NE metabolite dihydroxyphenylglycol (DHPG) appear to be related to intraneuronal NE stores. Plasma DHPG increases when sympathetic nervous activity or circulating NE increase and decreases when neuronal NE is depleted or neuronal NE reuptake is blocked. Changes in plasma dihydroxymandelic acid (DOMA) related less closely to changes in plasma NE. The coupling of measurements of plasma NE with its deaminated metabolites and DHPG may improve understanding of human NE metabolism and neuronal NE reuptake.

The effects of DSP-4 on brain NE levels and turnover in rats were investigated in six brain regions: cortex, hippocampus, cerebellum, brainstem, hypothalamus and locus coeruleus. Administration of 50 mg/kg of DSP-4 significantly decreased NE levels in all brain regions; greatest reductions occurred in the cortex (86% decrease) and in the hippocampus (91% decrease). Doses of DSP-4 less than 50 mg/kg did not significantly lower NE levels in other brain regions, except within the cerebellum. Levels of the NE metabolite 3-methoxy, 4-hydroxyphenylethylene glycol sulfate (MHPG-S04) declined in parallel with those of NE, except within the brainstem and the locus coeruleus. NE turnover, expressed as the ratio of the MHPG-S04 concentration to that of NE, was higher in the cortex and hippocampus than other regions in control animals, and NE turnover significantly increased only in these two areas after the administration of 50 mg/kg of DSP-4 (p less than 0.01). There were no significant changes in the levels of dopamine and a significant decrease of serotonin only in the striatum. These results indicate that DSP-4 is a neurotoxin with a strong predilection for noradrenergic neurons, that its effects vary according to brain region and that its administration increases NE turnover in those brain regions showing the greatest depletion of NE.

We describe a sensitive specific and simple high pressure liquid chromatographic procedure for determining 3,4-dihydroxyphenyl-ethylene glycol (DHPG) in both plasma and tissue. DHPG is extracted from plasma or tissue extracts by adsorption onto alumina. DHPG in the alumina eluate is detected electrochemically following chromatography on a C18 reverse phase column. The method is sensitive enough to detect approximately 20 pg/ml of plasma DHPG. Both clonidine (100 micrograms/kg) and desmethylimipramine (2.5 mg/kg) when administered to rabbits for 3 days induced significant falls in both cardiac and plasma DHPG concentrations. These experiments indicate that both tissue and plasma DHPG concentrations may be of value in assessing both the release and re-uptake of norepinephrine at peripheral sympathetic nerve endings in vivo.

Performance of microbore reverse phase HPLC coupled with amperometric detection is detailed for the analysis of catecholamines in small tissue samples and human blood plasma and cerebrospinal fluid. Extraction procedures for pre-concentration and clean-up of these samples are described. Marked signal enhancement is observed due to the smaller column volume as well as the increased coulometric yield which results from the lower flow rates used with this technique. Detection limits of 0.2 to 0.5 picograms are obtained allowing analysis of catecholamines in extremely small tissue samples or small volumes of cerebrospinal fluid or plasma.

A high performance liquid chromatographic method involving electrochemical detection is described which permits the assay of noradrenaline (NA), adrenaline (A), dopamine (DA), and dihydroxyphenylethyleneglycol (DOPEG) in human plasma and brings about analytical recoveries of 70% and more. This method was used to assess the effects of graded orthostasis and mental stress on the plasma levels of these catechols. Orthostasis elicited increases in plasma NA and DOPEG, but did not cause any change in plasma A and DA. The increases in NA and DOPEG were dependent on the degree of orthostasis and correlated closely (rs = 0.724; n = 30, P less than 0.001). Pretreatment with desipramine abolished the DOPEG response to standing, indicating that orthostasis - induced increases in plasma DOPEG are presynaptic in origin. Mental stress evoked pronounced increases in plasma A, less pronounced increases in plasma NA and no changes in plasma DA and DOPEG. Hence, the simultaneous measurement of plasma NA and DOPEG may help to distinguish between various types of activation of the sympathetic nervous system.