Effects of oral clonidine on plasma 3-methoxy-4-hydroxyphenethyleneglycol (MHPG) in man: Preliminary report

Repeated (N=15) administration of clonidine (0,1,5 μg/kg,p.o.) to three normotensive male subjects resulted in significant decreases in plasma free 3-methoxy-4-hydroxyphenethyleneglycol (MHPG) at three hours for both the 1 μg/kg dose (p < .05) and the 5 μg/kg dose (p < .01) when compared to concentrations following placebo. The mean decrement in plasma free MHPG following a 5 μg/kg dose was 36%. Systolic blood pressure fell a mean of 17 mmHg after 1 μg/kg and 37 mmHg after 5 μg/kg of clonidine. The application of a clonidine challenge test to assess noradrenergic receptor sensitivity $\text{in}$$\text{vivo}$ is discussed.     

Yohimbine induced anxiety and increased noradrenergic function in humans: effects of diazepam and clonidine

Yohimbine (30 mg) produced significant increases in subjective anxiety, autonomic symptoms, blood pressure, and plasma 3-methoxy-4-hydroxy-phenylethyleneglycol (MHPG) in ten healthy subjects. The effects of pretreatment with diazepam (10 mg) or clonidine (5 micrograms/kg) on these yohimbine induced changes was examined. Both diazepam and clonidine significantly antagonized yohimbine-induced anxiety, but only clonidine significantly attenuated the yohimbine induced increases in plasma MHPG, blood pressure, and autonomic symptoms. When given alone, clonidine significantly decreased plasma MHPG and blood pressure, whereas diazepam did not. These findings indicate that: (1) noradrenergic hyperactivity may be a factor in the production of some anxiety states; (2) the anti-anxiety effects of clonidine appear to result from its actions on receptors which decrease noradrenergic activity; (3) diazepam reverses yohimbine-induced anxiety without effects on several physiological or biochemical indicators of noradrenergic activity in humans.     

Agmatine attenuates neuropathic pain in rats: possible mediation of nitric oxide and noradrenergic activity in the brainstem and cerebellum

Effect of agmatine (10-400 mg/kg) on neuropathic pain in a rat model produced by loose ligatures around the common sciatic nerve was studied. The involvement of possible alterations in nitric oxide (NO) levels [measured as its stable metabolites nitrate + nitrite] and in noradrenergic activity [measured as norepinephrine and 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) levels] in this effect was also investigated biochemically in the brainstem and cerebellum. Agmatine increased the neuropathic pain threshold at 300 and 400 mg/kg. There was almost a twofold increase in nitrate + nitrite levels in the brainstem and cerebellum of the rats with neuropathic pain and agmatine decreased the high nitrate + nitrite levels only in the brainstem at 300 mg/kg and both in the brainstem and cerebellum at 400 mg/kg. Ligation of sciatic nerve resulted in almost twofold increase in norepinephrine and MHPG levels only in the brainstem of the rats. Agmatine decreased MHPG levels at 300 and 400 mg/kg, however it decreased norepinephrine levels only at the higher dose. These findings indicate that agmatine decreases neuropathic pain, an effect which may involve the reduction of NO levels and noradrenergic activity in the brain.     

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.     

Effects of acutely and chronically administered antidepressants on the clonidine-induced decrease in rat brain 3-methoxy-4-hydroxyphenylethylene-glycol sulphate content.

A study was undertaken of the effects of acutely and chronically administered agents on rat brain MHPGSO₄ content and on the ability of a low dose of clonidine (25 μg/kg) to lower brain MHPGSO₄ levels. This effect of clonidine is attributed to an activation of presynaptic α-adrenoceptors. The agents studied were desipramine and nisoxetine, both inhibitors of NA uptake, Org 6582, a specific inhibitor of 5-HT uptake, and iprindole and mianserin, two atypical antidepressants. In all studies, a dose of 10 mg/kg was used. Acutely administered mianserin increased rat brain MHPGSO₄ levels and prevented the clonidine-induced reduction. The clonidine-induced fall in MHPGSO₄ levels was also absent following the acute administration of desipramine or nisoxetine. However, the observed effect cannot be unequivocally attributed to a blockade of presynaptic α-adrenoceptors since both drugs decreased basal levels of MHPGSO₄. For chronic studies drugs, other than Org 6582, were injected every 12 h for 14 days and experiments were undertaken 12 h after the last injection. Org 6582 was injected once daily for 14 days and experiments undertaken 24 h after cessation of administration. Chronic mianserin and nisoxetine increased MHPGSO₄ levels. Only chronic desipramine blocked the clonidine-induced fall, the phenomenon developing between 5 and 9 days of chronic desipramine administration. This study indicates that, under the experimental conditions employed, the ability of chronic desipramine to elicit subsensitivity of presynaptic α-adrenoceptors does not extend to the other four agents studied.     

Increase in norepinephrine turnover after tyrosine or DL-threo-3,4-dihydroxyphenylserine (DL-threo-DOPS)

The amino acids tyrosine and DL-threo-3,4-dihydroxyphenylserine (DL-threo-DOPS) were compared for their effectiveness in increasing central nervous system norepinephrine (NE) turnover in both saline and DSP-4 pretreated mice. NE was decreased significantly in cortex, hippocampus and cerebellum, and only slightly in hypothalamus and brainstem two weeks after a single intraperitoneal injection of the neurotoxin DSP-4. Levels of the major NE metabolite, 3-methoxyl-4-hydroxyphenylethylene glycol (MHPG), decreased in parallel in these five brain regions. Neither administration of tyrosine (250 mg/kg, as the ethyl ester, i.p.) nor DL-threo-DOPS (200 mg/kg, i.p.) affected regional NE concentration. However, after tyrosine administration, MHPG levels increased significantly in cortex in control mice and in cortex and hippocampus of DSP-4 pretreated mice. In all five brain noradrenergic regions MHPG level increased after DL-threo-DOPS administration and this increase was enhanced (approximately doubled) in DSP-4 pretreated mice. Thus, both amino acids may be useful as precursors of central NE when its level is depleted (e.g. following administration of DSP-4); DL-threo-DOPS producing a generalized increase in brain NE turnover, while increases following tyrosine are specific to those areas in which neuronal activity is increased i.e. cortex and hippocampus.     

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.     

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.     

Clonidine Prevents Transient Loss of Noradrenaline in Response to Cholinergic Hypofunction Induced by Ethylcholine Aziridinium (AF64A)

Intracerebroventricular injection of ethylcholine aziridinium (AF64A) (2 nmol/ventricle) induced a considerable decrease in the level of acetylcholine (ACh) in hippocampus (from 21.14 +/- 0.84 to 10.04 +/- 0.59 pmol/mg of tissue; p less than 0.001) 4 days after application. The reduction of cholinergic function was accompanied by a decrease in the level of noradrenaline (NA) (from 1.96 +/- 0.08 to 1.41 +/- 0.06 pmol/mg of tissue; p less than 0.001). Two days after administration of AF64A (1 or 2 nmol/ventricle), the dose-dependent decrease in NA level was associated with an increase in the level of its major metabolite, 3-methoxy-4-hydroxyphenylglycol (MHPG), resulting in a considerable increase in the MHPG/NA molar ratio (from 0.84 +/- 0.06 to 1.62 +/- 0.17; p less than 0.002). Chronic treatment of AF64A-injected rats with clonidine (0.02-0.2 mg/kg, i.p., every 8-12 h) had no significant effect on the loss of ACh content, whereas the decrease in NA content in hippocampus was completely prevented. Clonidine induced aggressive behavior in the AF64A-treated rats, in contrast to sedation in vehicle-injected rats. The response to clonidine under these experimental conditions and the increased MHPG/NA molar ratio in response to AF64A suggest that the transient loss of NA content following AF64A administration results from increased NA release. The increased noradrenergic activity in hippocampus may be linked to the reduction of tonic inhibitory cholinergic input. These results are discussed in relation to possible implications for senile dementia of the Alzheimer type.     

A new approach to biochemical evaluation of brain dopamine metabolism

1. Dopaminergic neurotransmission in brain is receiving increased attention because of its known involvement in Parkinson's disease and new methods for the treatment of this disorder and because of hypotheses relating several psychiatric disorders to abnormalities in brain dopaminergic systems. 2. Chemical assessment of brain dopamine metabolism has been attempted by measuring levels of its major metabolite, homovanillic acid (HVA), in cerebrospinal fluid, plasma, or urine. Because HVA is derived in part from dopamine formed in noradrenergic neurons, plasma levels and urinary excretion rates of HVA do not adequately reflect solely metabolism of brain dopamine. 3. Using debrisoquin, the peripheral contributions of HVA to plasma or urinary HVA can be diminished, but the extent of residual HVA formation in noradrenergic neurons is unknown. By measuring the levels of methoxy-hydroxyphenylglycol (MHPG) in plasma or of urinary norepinephrine metabolites (total MHPG in monkeys; the sum of total MHPG and vanillyl mandelic acid (VMA) in humans) along with HVA, it is possible to estimate the degree of impairment by debrisoquin of HVA formation from noradrenergic neuronal dopamine and thereby better assess brain dopamine metabolism. 4. This method was applied to a monkey before and after destruction of the nigrostriatal pathway by the administration of MPTP.     

Influence of clonidine on the acute dependence response elicited in naive rats by naloxone

Clonidine has been used successfully in the treatment of opiate dependence. The discomforting effects of withdrawal are attenuated by the drug. The question of whether the more central process of dependence is affected by clonidine was tested in the present study. Change in plasma corticosterone was used as the indication of the stress of acute withdrawal from morphine. Conscious, unrestrained male rats showed a dose-related, though somewhat delayed, increase in plasma corticosterone after clonidine (0.01-0.1 mg/kg). The suggested mechanism for this effect involves presynaptic inhibition of noradrenergic neurons inhibiting CRF (corticotropin-releasing factor) release. Similar animals showed an elevation of plasma corticosterone after naloxone (0.4 mg/kg) was administered 3 hrs following a single morphine-priming (10 mg/kg). The naloxone-precipitated response was unaffected by clonidine (0.04 mg/kg). This dose of clonidine did not substitute for morphine-priming to produce the naloxone-precipitated response. The data suggests that clonidine elevated plasma corticosterone by an indirect mechanism. Further, the stress associated with acute withdrawal is unaffected by clonidine suggesting that the drug does not alter dependence development.     

Naltrexone precipitated opiate withdrawal in methadone addicted human subjects: Evidence for noradrenergic hyperactivity

The noradrenergic alpha-2 receptor agonist, clonidine, suppresses many of the effects of opiate withdrawal in both humans and other animals and this is consistent with the data indicating important interactions of opiate and noradrenergic systems in brain. This evidence supports the hypothesis that central noradrenergic hyperactivity is involved in the expression of major signs and symptoms of the opiate withdrawal syndrome, but to date clinical studies have not provided biochemical data consistent with this idea. In order to assess whether naltrexone precipitated opiate withdrawal in methadone addicted human subjects is associated with changes in noradrenergic function, a double-blind study was completed in which 15 methadone-dependent subjects received naltrexone and 8 subjects received placebo. Signs and symptoms of the opiate abstinence syndrome increased significantly in the 15 subjects who received naltrexone compared with the 8 who received placebo. Plasma concentrations of free MHPG also increased significantly in those subjects who took naltrexone compared to the placebo treated subjects. In addition, withdrawal signs and symptoms were significantly correlated with plasma MHPG concentration. Since several compounds which suppress central noradrenergic activity also reduce the severity of the withdrawal syndrome, the noradrenergic hyperactivity suggested by the present clinical study may be a functional mechanism for at least part of the opiate withdrawal syndrome.     

Dorsal noradrenergic bundle lesions fail to alter opiate withdrawal or suppression of opiate withdrawal by clonidine

Previous work has shown that clonidine effectively supresses many of the signs of opiate withdrawal. The present study was designed to test the hypothesis that the supression of opiate withdrawal by clonidine is mediated by forebrain noradrenergic projections of the locus coeruleus. Two groups of 24 rats each were subjected to either a 6-hydroxydopamine lesion of the dorsal noradrenergic bundle (Lesion group) or a sham, vehicle injection (Sham group). All rats were made dependent on morphine by subcutaneous implantation of one 75 mg silastic morphine pellet for three days followed by 3 more days with two additional 75 mg pellets. Following removal of the morphine pellet, withdrawal was precipitated in all rats by subcutaneous injection of 4 mg/kg of naloxone. Pretreatment 10 min. before withdrawal with clonidine (0.1 or 0.2 mg/kg) produced a significant attenuation of withdrawal signs as compared to saline injected rats; this effect was equally significant in both sham and lesion groups. Lesions of the locus coeruleus had no effect on withdrawal, nor did they affect the ameliorating action of clonidine. These results substantiate the observation that clonidine can effectively attenuate signs of opiate withdrawal in the rat, but fail to support the hypothesis that these effects are mediated by the forebrain projections of the locus coeruleus.     

Acute estrogen treatment facilitates recognition memory consolidation and alters monoamine levels in memory-related brain areas

Acute effects of estrogens on mnemonic processes were examined at the behavioral and neurochemical levels. 17β-estradiol and 17α-estradiol influences on memory consolidation were assessed using object placement (OP) and object recognition (OR) tasks. Subjects received treatment immediately after a sample trial (exploring two novel objects), and memory of objects (OR memory) or location of objects (OP memory) was tested 4 h later. Both isomers of estradiol enhanced memory. For spatial memory, 15 and 20 µg/kg of 17β-estradiol facilitated OP, while lower and higher doses were ineffective. 17α-estradiol had a similar pattern, but a lower dose was effective. When treatment was delayed until 45 min after a sample trial, memory was not enhanced. For non-spatial memory, OR was facilitated at 5 µg/kg of 17β-estradiol and at 1 and 2 µg/kg of 17α-estradiol and, similar to OP, lower and higher doses were ineffective. These data demonstrate that beneficial effects of estrogens are dose, time and task dependent, and the dose-response pattern is an inverted U. Because monoamines are known to have contributions to memory, brains were removed 30 min after treatment for measurements of dopamine (DA), norepinephrine (NE), serotonin (5-HT), and metabolites. Estrogen elevated 5HT, NE metabolite MHPG, turnover ratio of NE to MHPG, and DA metabolite DOPAC levels in the prefrontal cortex, while NE and MHPG were decreased in the hippocampus. Thus, acute estrogens exert rapid effects on memory consolidation and neural function, which suggests that its mnemonic effects may involve activation of membrane associated estrogen receptors and subsequent signaling cascades, and that monoamines may contribute to this process.     

Catecholamine metabolites in human plasma as indices of brain function: Effects of debrisoquin

Metabolites of catecholamine neurotransmitters in plasma are, potentially, an easily available indicator of brain function in man. The peripheral contribution to these metabolites was lowered by debrisoquin sulfate, a monoamine oxidase inhibitor that does not enter the brain. In the monkey, it had been shown that debrisoquin decreased peripheral production of the dopamine metabolite, homovanillic acid (HVA), without changing production by brain; production of the norepinephrine metabolite, 3-methoxy-4-hydroxyphenethyleneglycol (MHPG) was decreased peripherally and in brain. Low-dose debrisoquin administration in man eliminated about 80% of the peripheral contribution to HVA and MHPG in plasma, resulting in a situation in which at least 75% of these metabolites in plasma were from the brain. Under these conditions, HVA and MHPG in plasma had a significant correlation. It could also be estimated that production of MHPG by brain was reduced 55%. Debrisoquin potentially provides a method for studying brain catecholamines through their metabolites in plasma and for treating conditions of brain noradrenergic excess.     

The effects of caffeine on plasma MHPG,subjective anxiety,autonomic symptoms and blood pressure in healthy humans

The effects of oral administration of caffeine (10 mg/kg) on plasma free 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) levels, behavioral ratings, blood pressure, and autonomic symptoms was determined in eleven healthy subjects. Caffeine produced robust increases in subject rated anxiety and nervousness and small elevations in blood pressure and a decrease in heart rate. Caffeine did not alter plasma MHPG in a consistent fashion and there was no correlation between changes in plasma MHPG and changes in anxiety or other ratings. Caffeine may produce symptoms of anxiety-nervousness without increasing central norepinephrine turnover.     

Clonidine increases food and protein consumption in rats

Rats were given clonidine or its diluent, and allowed to eat freely from two isocaloric diets that differred in protein or carbohydrate content. Low clonidine doses (25–50 μg/kg) significantly increased total food and protein intake by rats given access to a high and a low protein diet. Several pairs of diets, differing in protein contents, were tested; clonidine's effect was greatest when a diet containing 30–45% protein was paired with one that was very low (5%) in protein. Higher clonidine doses (200 μg/kg) failed to modify either total food or protein intake. Clonidine had no effects on food or nutrient intake among animals given access to diets that differed in carbohydrate content (25 or 70% carbohydrate, plus 25% protein). In rats given access to only one diet, clonidine administration decreased food consumption when the diet was low in protein (5%), but increased consumption when the diet contained 25 or 50% protein. These data suggest that central noradrenergic synapses participate in the mechanisms controlling appetites for proteins. Clonidine may enhance protein intake by stimulating presynaptic alpha receptors, thus diminishing central noradrenergic tone. This effect on noradrenergic transmission is probably partly overcome by protein consumption, which increases brain tyrosine levels and thus can accelerate norepinephrine synthesis. Clonidine or related drugs may be useful clinically in treating diseases characterized by impaired appetite or increased need for protein.     

Formation and Clearance of Norepinephrine Glycol Metabolites in Mouse Brain

To determine the degree of conversion of 3,4-dihydroxyphenylethyleneglycol (DHPG) to 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) and the amount of DHPG eliminated unchanged from the brain, we have examined the kinetics of formation and disappearance of mouse brain MHPG and DHPG following clorgyline (10 mg/kg, i.p.) and/or tropolone (75 mg/kg, i.p.) treatment. During the first 10 min after tropolone, brain DHPG levels accumulated linearly at a rate of 1,300 pmol/g/h, whereas MHPG disappeared exponentially at a rate of 411 pmol/g/h. Following clorgyline administration, brain DHPG declined exponentially at a rate of 1,240 pmol/g/h. In contrast, the elimination of MHPG became a first-order process only when catechol-O-methyltransferase (COMT) was also inhibited in addition to monoamine oxidase. Thus, combined clorgyline and tropolone treatment resulted in an exponential decline of MHPG levels at a rate of 524 pmol/g/h, whereas DHPG levels were slightly but significantly elevated compared to control values. When the animals were treated with pargyline (75 mg/kg, i.p.) in combination with clorgyline and tropolone, brain DHPG and MHPG disappeared at rates of 40 and 660 pmol/g/h, respectively. The above observations suggest that mouse brain DHPG is cleared primarily through O-methylation with minimal direct elimination from brain. Assuming the disposition and clearance of norepinephrine metabolites are similar in mouse and human brain, peripherally measured DHPG in humans is likely derived principally from extracerebral sources and reflects peripheral sympathetic function.     

Norepinephrine-dependent and independent mechanisms of persistent effects of amphetamine in rat cerebellum

Previous studies of the effects of chronic low-dose amphetamine (2 mg/kg per day X 21 days) on the spontaneous discharge rate of cerebellar Purkinje neurons have shown persistent depressant effects for up to 50 days after cessation of drug administration. The depression of spontaneous discharge observed was only partially reversible by various pharmacological agents which disrupt noradrenergic neurotransmission in cerebellum. In the present study, several additional approaches were used to investigate further this persistent effect. Rats were treated, either before or after chronic treatment with amphetamine, with intracisternal 6-hydroxydopamine at doses which destroy most noradrenergic fibers in cerebellum. In either case Purkinje neurons were still significantly slowed after cessation of amphetamine treatment, although the depression was not as great as previously observed. In another experiment, cerebellar cortical levels of 3-methoxy, 4-hydroxy phenyl glycol (MHPG) were measured after cessation of amphetamine administration, to determine if there was biochemical evidence for increased noradrenergic neurotransmission. At ten days, MHPG levels were elevated by 36%, and they returned to control values by 30 days. The evidence obtained in these studies suggests that chronic amphetamine treatment causes a persistent increase in noradrenergic neurotransmission, but non-noradrenergic mechanisms may also be important mechanisms in the long-lasting depression of activity of cerebellar Purkinje neurons.     

Significance of ED50 and therapeutic indexes of various radiation-protective agent groups]

Radioprotective agents are divided in 3 groups: (1) cystamine, AET, cystaphos, gammaphos, and thiogammaphos with ED50 (the dose that gives a half of the maximal protective effect) of 10(3)-10(1.6) mumol/kg and therapeutic index K = LD50/ED50 = 10(0)-10(1.6); (2) 5-methoxytryptamine, phenylephrine, serotonin, and norepinephrine with ED50 = 10(1)-10(0) mumol/kg and K = 10(1.8)-10(2,6); (3) clonidine and isoprenaline with ED50 = 10(-0.5)-10(-0.8) mumol/kg and K = 10(3)-10(4). Possible causes of these differences and advantages of low ED50 and high K are discussed.