Assay of norepinephrine and dopamine in the rat brain after microwave irradiation

Rapid inactivation of enzymes prior to the assay of rat brain catecholamines was evaluated. Regional levels of norepinephrine and dopamine were measured by high performance liquid chromatography with electrochemical detection after enzyme inactivation by microwave irradiation at levels of 1.3 kw and 5 kw, and compared with decapitation. The differences found in regional levels of catecholamines between the two methods of euthanasia indicate that rapid inactivation of brain enzymes is necessary for accurate analysis of catecholamines in rat brain.     

Species differences in epinephrine concentration and norepinephrine N-methyltransferase activity in hypothalamus and brain stem

1. The concentration of epinephrine, norepinephrine and dopamine, and the activity of norepinephrine N-methyltransferase, the epinephrine-forming enzyme, were determined in hypothalamus and brain stem in several species. 2. Epinephrine concentration in hypothalamus, a nerve terminal region, varied in the order frog greater than turtle greater than chicken greater than cat greater than dog greater than pigeon greater than rat greater than ferret greater than hamster greater than mouse, with concentrations being undetectable in rabbits, horses and guinea pigs. 3. Epinephrine concentration was lower than norepinephrine concentration in all species except the frog. 4. NMT activity was detected in all species except guinea pigs. 5. Epinephrine concentration was lower in brain stem, a cell body region, than in hypothalamus in all species. Only in the frog brain stem was there more epinephrine than norepinephrine. 6. No epinephrine or NMT activity was detected in either brain region in guinea-pigs.     

Assay of free and conjugated catecholamines by high-performance liquid chromatography with electrochemical detection

A rapid and simple method for the analysis of free and conjugated catecholamines in body tissues and fluids is described. The free catecholamines were isolated by standard alumina procedures before and after hydrolysis of the conjugated compounds to free compounds by heating the samples in perchloric acid. Free catecholamines were then separated by high-performance liquid chromatography and detected by electrochemical detection. Conjugated compound was the difference between the total and free amount in each sample. This method was utilized to measure free and conjugated norepinephrine, epinephrine, and dopamine in human urine and rat adrenal gland, and to measure free and conjugated dopamine in rat whole brain and kidney.     

Epinephrine, norepinephrine and dopamine concentrations in amphibian brain

The whole brain concentrations of epinephrine, norepinephrine and dopamine in North American amphibians, orders Caudata and Salientia, are reported. Epinephrine is the major catecholamine in the Salientia while norepinephrine and epinephrine concentrations are roughly equivalent in suborders of Caudata. Relative regional concentrations are similar to other classes (mammals, reptiles and birds) although the absolute concentration of epinephrine is considerably higher in amphibians than in most other species.     

Effect of dietary proteins and carbohydrates on urinary and sympathoadrenal catecholamines

We previously observed that administration of tyrosine to rats or humans elevated urinary dopamine, norepinephrine and epinephrine levels. The present studies examine the effects on these urinary catecholamines of varying the ratio of protein to carbohydrate in the diets.Rats consumed diets containing 0, 18 or 40% protein (76, 58 and 36% carbohydrate respectively) for 8 days. The stress of consuming the protein-free food was associated with a 16% weight reduction, and with significantly lower serum, heart and brain tyrosine levels than those noted in rats eating the 18 or 40% protein diets. Absence of protein from the diet also decreased urinary levels of dopamine and DOPA but increased urinary norepinephrine and epinephrine, probably by increasing sympathoadrenal discharge; it also increased the excretion of DOPA in animals pretreated with carbidopa, a DOPA decarboxylase inhibitor. Carbidopa administration decreased urinary dopamine, norepinephrine and epinephrine as expected; however, among carbidopa-treated rats urinary norepinephrine and epinephrine concentrations were highest for animals consuming the protein-free diet, again suggesting enhanced release of stored catecholamines from sympathoadrenal cells. The changes in urinary catecholamines observed in animals eating the protein-free diet were similar to those seen in rats fasted for 5 days: dopamine levels fell sharply while norepinephrine and epinephrine increased.These data indicate that the effects of varying dietary protein and carbohydrate contents on dopamine secretion from peripheral structures differ from its effects on structures secreting the other two catecholamines. Protein consumption increases dopamine synthesis and release probably by making more of its precursor, tyrosine, available to peripheral dopamine-producing cells; it decreases urinary norepinephrine and epinephrine compared with that seen in protein-deprived animals, probably by diminishing the firing of sympathetic neurons and adrenal chromaffin cells.     

CATECHOLAMINES IN FETAL AND NEWBORN RAT BRAIN

The levels of dopamine and norepinephrine were determined in the brains of fetal and newborn rats by means of a sensitive, radiometric-enzymatic assay. Catecholamines were converted to their 3-O-methylated derivatives in the presence of catechol-O-methyl transferase (EC 2.1.1.1) and [³ H-methyl]S-adenosylmethionine; and the [³H]-derivatives were isolated by selective extraction. The assay had a sensitivity for dopamine and norepinephrine of 100 picograms and was linear to at least 30 nanograms of catecholamines. Both amines were present at 15 days of gestation and increased 15-fold in content during the last week of gestation. The regional distribution of these neurotransmitters in the brain of the newborn rat correlated with the distribution of their biosynthetic enzymes. An investigation of the effects of reserpine, pheniprazine, α-methyl-para-tyrosine, diethyldithiocarbamate and l -DOPA on the levels of dopamine and norepinephrine in the brains of the 18-day gestational fetus indicated that the levels of these neurotransmitters are under controls similar to those known to occur in the brain of the adult rat.     

Catecholamine Release and Excretion in Rats with Immunologically Induced Preganglionic Sympathectomy

Abstract: Plasma and urinary catecholamines were quantified to assess global sympathoadrenal function in rats with preganglionic lesions caused by antibodies to acetyl-cholinesterase (AChE). Rats were given intravenous injections of normal mouse IgG or murine monoclonal anti-acetylcholinesterase IgG (1.5 mg). Five or 16 days afterward, basal blood samples were taken through indwelling arterial cannulae. A few hours later, the rats were immobilized for 10 min in padded restrainers, and another blood sample was drawn. HPLC determinations showed low basal levels of norepinephrine and epinephrine (<0.2 ng/ml in all rat plasma samples). In control rats, immobilization stress increased levels of plasma catecholamines up to 35-fold. In rats tested 5 days after injection of antibody, the norepinephrine response was much smaller (15% of control), and (he epinephrine response was nearly abolished (5% of control). There was some recovery at 16 days after antibody treatment, but stress-induced catecholamine release was still markedly impaired. Reduced stress-induced release: was not accompanied by major changes in tissue epinephrine or norepinephrine (heart, spleen, adrenal glands, and brain), although adrenal dopamine content dropped by 60%. Urinary excretion was studied in parallel experiments to gain insight into the effects of AChE anti-bodies on basal sympathoadrenal activity. Epinephrine, norepinephrine, dopamine, and selected metabolites were quantified in 24-h urine samples collected at frequent intervals for 30 days after antibody injection. No statistically gnificant changes were detected in the urinary output of dopamine, 3-methoxytyramine, normetanephrine, or 3-methoixy-4-hydroxyphenylglycol. On the other hand, epinephrine and norepinephrine output increased sharply at the time of antibody injection and then fell significantly below control levels. Norepinephrine output returned to normal after 2 weeks, but epinephrine output remained depressed. These results are consistent with previous evidence of widespread and persistent antibody-mediated βmade to the preganglionic sympathetic system.     

Adrenomedullary chromaffin cells of the rat. An ultrastructural study

Adrenomedullary chromaffin cells of the rat were studied at the ultrastructural level. Chromaffin cells contained a large population of electron-dense-core vesicles of two types, one very electron-dense (norepinephrine) and the other moderately electron-dense (epinephrine). The vesicles showed an even distribution pattern in the cytoplasmic matrix. No physical contacts were observed between cytoplasmic and vesicle membranes to indicate exocytosis, a mechanism frequently observed in the hamster. For chromaffin cells to be used as a transplantable source of dopamine, both the cells and vesicles must survive the trauma of denervation and vascular elimination or alternatively acquire the two factors from the transplantation site.     

Regional distribution of monoamines and monoamine metabolites in the brain of the crucian carp (Carassius carassius L.)

1. Monoamines (which all demand oxygen for their synthesis) and monoamine metabolites were quantified in 6 brain regions of the extremely anoxia tolerant crucian carp (Carassius carassius L.).2. The norepinephrine levels were generally twice as high as the dopamine levels. No epinephrine was found.3. The major dopamine metabolite seemed to be homovanillic acid. No 3,4-dihydroxyphenylacetic acid was found.4. Serotonin occurred at about the same levels as dopamine. The levels of the main serotonin metabolite, 5-hydroxyindole-3-acetic acid, were about 10% of that of serotonin.5. All three monoamines had a similar distribution, with the highest concentrations in hypothalamus and the lowest in cerebellum and vagal lobes.6. The distribution and levels of monoamines agreed with that of anamniote vertebrates in general, suggesting that the crucian carp has not adapted to anoxia by abandoning or minimizing the use of monoaminergic systems.     

The convulsive effects of dopamine sulfate conjugates in rat brain

The administration of 40 μg or more of dopamine sulfate conjugate, the predominant form of dopamine in the peripheral nervous system, into the left lateral ventricle of Sprague Dawley rat led to seizures. The severe and generalized convulsions with clonic extension of forelimbs and hindlimbs occurred within 2–6 minutes of the injection, and lasted for 10–20 minutes. No convulsion was observed when dopamine, norepinephrine, nitrocatechol sulfate or a hydrolyzed solution of dopamine sulfate conjugate were similarly injected. The dopamine sulfate- induced seizures were not blocked by pretreatment of rats with phenoxybenzamine, metoclopramide or haloperidol but were reduced by propranolol and suppressed by diazepam. Dopamine sulfate conjugates which were recently detected in discrete areas of rat brain and in human cerebrospinal fluid may thus have certain function in the brain different from that of free dopamine.     

Presence of Conjugated Catecholamines in Rat Brain: A New Method of Analysis of Catecholamine Sulfates

Abstract: A new method of measuring catecholamine (CA) sulfate permitted us to detect its presence in rat brain for the first time. The procedure consisted of separating the CA sulfate from the free CA by alumina adsorption followed by passage through Dowex, and measuring the CA sulfate by a radioenzymatic assay in the presence of a sulfatase. This method permitted demonstration of the presence of dopamine sulfate, and occasionally, of norepinephrine and epinephrine sulfate in the hypothalamus, striatum, and hippocampus of rat brain.     

Regional development of catecholamine biosynthesis in rat brain

Abstract— The ontogenetic development of norepinephrine and dopamine and their associated biosynthetic and degradative enzymes was investigated in five anatomical regions of the rat brain. Clear regional differences were found in the development of both norepinephrine and tyrosine hydroxylase (EC 1.14.3.1). In the case of both norepinephrine and tyrosine hydroxylase, brainstem structures achieved adult levels well before forebrain structures. The development of DOPA decarboxylase (EC 4.1.1.26), monoamine oxidase (EC 1.4.3.4) and catechol-0-methyl transferase (EC 2.1.1.6) did not appear to differmarkedly from area to area. Further analysis of the data revealed that in forebrain structures both the amines and the biosynthetic enzymes developed concurrently. By contrast, in the brainstem structures, there was a dissociation of amine and enzyme development with development of tyrosine hydroxylase, in particular, markedly preceding that of norepinephrine and of DOPA decarboxylase. The bases for both the lower amine levels in the infant brain and the regional developmental differences are discussed in relation to the anatomical organization of the central catecholamine-containing neurons.     

Influence of age on orthostatic changes in plasma renin activity and urinary catecholamines in man.

The authors studied plasma renin activity (PRA), urinary epinephrine, norepinephrine and dopamine excretion and their mutual relationships in 54 healthy subjects under basal (recumbent) conditions and age-related orthostatic changes in these parameters. The test subjects were divided into six 10-years groups, according to their year of birth (1901-1910 to 1951-1960). In the oldest groups (1901-1910 and 1911-1920), both basal PRA values and norephrine and epinephrine excretion and their postural increase were smaller than in younger subjects. Conversely, urinary dopamine excretion and the dopamine/norepinephrine and epinephrine ratio rose with advancing age. There were no significant differences between the plasma sodium and potassium concentrations in the various groups. Urinary aldosterone excretion was slightly higher in the oldest group than in the others, but was still within the control value limits. The intravenous administration of Inderal reduced both resting PRA values and the orthostatic increase in the youngest age groups, so that their PRA approached the values in older subjects. Higher norepinephrine and epinephrine excretion and the lower dopamine/norepinephrine and epinephrine in young subjects may play a role in their higher PRA, especially in the orthostatic reaction. Diminution of sympathetic activity, with lower norepinephrine and epinephrine excretion and relatively high dopamine excretion, may have a direct bearing on the lower PRA values in older subjects. The diminished capacity of older subjects for catecholamine mobilization and raised renin secretion during an orthostatis stress may be related to the higher incidence of orthostatic forms of hypotension in old age.     

The simultaneous quantification of dopamine,norepinephrine and epinephrine in micropunched rat brain nuclei by on-line trace enrichment HPLC with electrochemical detection: Distribution of catecholamines in the limbic system

A method is described for the simultaneous quantification of dopamine, norepinephrine and epinephrine in microdissected rat brain nuclei using on-line trace enrichment high performance liquid chromatography (HPLC) and electrochemical detection (EC). The method is specific, sensitive and rapid with lower limits of detection comparable to those of radioenzymatic and mass fragmentographic techniques. The ideal application of the method to determinations in discrete microdissected brain nuclei is illustrated by mapping the topographic distribution of these three catecholamines in the limbic system of the rat. Dopamine is well represented and remarkably heterogeneously distributed in the amygdaloid nuclei. Norepinephrine concentrations are high in the interstitial (bed) nucleus of the stria terminalis and the paraventricular, periventricular, dorsomedial and medial preoptic hypothalamic nuclei, though generally more evenly distributed among the limbic nuclei than dopamine. Epinephrine was reliably quantified in a minority of the limbic nuclei examined with the highest concentration found in the paraventricular hypothalamic nuclei. The method described is ideally suited for examination of the pharmacological and functional heterogeneity of dopamine-, norepinephrine- and epinephrine-containing neuronal systems at the level of discrete brain nuclei.     

Development of α-Noradrenergic and Dopaminergic Receptor Systems Depends on Maturation of Their Presynaptic Nerve Terminals in the Rat Brain

To study the relationship between ontogeny of rat brain catecholamine nerve terminals and the receptor systems for the catecholamine transmitters, the developmental patterns of synaptosomal uptake mechanisms were compared with those of alpha-noradrenergic and dopaminergic receptor-mediated effects. Uptakes of [(3)H]dopamine or [(3)H]norepinephrine into dopaminergic and noradrenergic nerve terminals were low during the 1st week postpartum and increased rapidly during the 2nd week. A similar pattern was obtained for ontogeny of dopaminergic receptor binding sites, as evaluated by [(3)H]domperidone binding. Stimulation of incorporation of (33)P(i) into brain phospholipids (elicited by intracisternal injection of dopamine), which is mediated by dopaminergic receptors, was shown to be highly correlated with the maturation of both receptor binding sites and presynaptic nerve terminal uptake. A similar result was seen with norepinephrine, in that the synaptosomal uptake mechanism and norepinephrine-induced stimulation (33)P(i) incorporation into phospholipids, an alpha-noradrenergic effect, developed in a parallel fashion. To test the hypothesis that development of the receptor systems is linked to nerve terminal ontogeny, presynaptic nerve terminals were destroyed in neonates by intracisternal administration of 6-hydroxydopamine. The lesions prevented the maturational increase in the number of dopamine receptor binding sites and produced a defect in development of the dopamine- and norepinephrine-induced stimulation of (33)P(i) incorporation. The results suggest that ontogeny of both dopaminergic and alpha-noradrenergic receptor systems depend upon development of the presynaptic nerve terminals containing the transmitters.     

Excretion of vanillic acid and homovanillic acid and tissue distribution of catecholamines and their metabolites in mice with various levels of pigmentation]

Studies concerning metabolism of catecholamines in mice differing with respect to the degree of pigmentation were based on determination of daily excretion of vanillylmandelic and homovanillic acid and tissue content of epinephrine, norepinephrine, dopamine and their methoxy derivatives. It was found that pigmented mice excrete more homovanillic acid, the metabolite of dopamine, than do albinotic mice. Tissue studies have shown that the brain of albinotic mice contains more dopamine and kidneys more epinephrine, norepinephrine and their methoxy derivatives than the respective organs of the pigmented mice. The probable reasons of differences in the rate of inactivation of catecholamines in albinotic and pigmented mice have been discussed.     

Rat serum stimulates serotonin N-acetyltransferase activity in pineal monolayer cultures

The effects of rat serum on serotonin N-acetyltransferase (NAT) activity and indole synthesis in monolayer cultures of neonatal rat pineal glands was examined. The addition of 5% rat serum to these cultures resulted in stimulation of NAT activity equal to that obtained with optimal concentrations of the adrenergic agonist norepinephrine (NE). Rat serum also increased the synthesis of both N-acetylserotonin and melatonin from tryptophan. Stimulation of NAT activity by rat serum was partially blocked by metoprolol and propranolol, but not by phentolamine or butoxamine. The serum factor responsible for the stimulation was stable to both freezing and boiling. No significant amounts of epinephrine, norepinephrine, or dopamine were detected in the serum.     

Effect of chronic dietary treatment with L-tryptophan on the maintenance of hypertension in spontaneously hypertensive rats

Chronic dietary administration of L-tryptophan at 2.5 and 5.0% by weight reduced the elevated systolic blood pressure of spontaneously hypertensive (SH) rats. Blood pressure was reduced significantly by 3 weeks after initiation of treatment and continued to fall during the course of the 15 weeks of treatment. Body weights of the treated rats were not affected significantly by treatment, nor were daily food and fluid intakes and urine outputs. SH rats, treated with the higher dose of tryptophan, also significantly reduced their urinary outputs of epinephrine and norepinephrine compared with SH controls, while both doses of tryptophan increased urinary outputs of dopamine significantly above that of SH controls. Treatment with tryptophan increased significantly the specific binding of [125I]angiotensin II (Ang II) to membranes from the diencephalon in a dose-dependent manner. Measurement of catecholamine concentration of the supernatant from homogenates used for the Ang II binding assay revealed a significant correlation between the specific binding of Ang II to brain membranes of the two tryptophan-treated groups and the concentration of norepinephrine in the supernatant. There was also a significant correlation between the specific binding of Ang II and the concentration of dopamine in the supernatant of the control group and the group treated with the higher dose of tryptophan. These results show that chronic dietary administration of tryptophan can reduce the elevated blood pressure of SH rats and support the possibility that this neutral amino acid may act via its effect on the concentration of the neurohormones, norepinephrine and dopamine, in the diencephalon to regulate the binding of Ang II to its receptors.     

Effect of catecholamines on ornithine decarboxylase activity in the testis of immature rat

Intratesticular injection of epinephrine and norepinephrine caused stimulation of ornithine decarboxylase (ODC) activity in the testis of immature rat. The effect of epinephrine was time and dose dependant. The minimal effective dose for epinephrine was found to be 100 pg and optimal stimulation was observed with 500 ng of the drug. Maximal stimulation of ODC occurred at 2 h after the treatment and reduced significantly at 4 h reaching to control levels at 6 h. Simultaneous injection of epinephrine with dibutyryl cAMP, luteinizing hormone, follicle stimulating hormone or prostaglandin E₂ caused additional stimulation of the enzyme activity. Injection of epinephrine to norepinephrine treated animals caused additional effect. Both epinephrine and norepinephrine were found to stimulate the enzyme activity in leydig cell and seminiferous tubule fractions. These results suggest that catecholamines are also involved in the regulation of ODC activity in the testis of rat.