Rate Of Norepinephrine Synthesis Research Articles

Stress promotes development of ovarian cysts in rats: the possible role of sympathetic nerve activation.

Activation of the sympathetic innervation precedes the induction of polycystic ovaries in rats given estradiol valerate (EV). The mechanism of induction by EV may thus involve both direct and neurogenic components. We tested this hypothesis using a combined cold and restraint stress to induce an increase in sympathetic tone, including that of the ovarian sympathetic nerves. Three weeks after the start of stress we found: 1. An increase in the content of norepinephrine (NE) in the celiac ganglion. 2. An increase in the release of NE from the ovary. 3. An unchanged NE uptake by the ovary. 4. An unchanged content of NE in the ovary. The ovarian content of neuropeptide Y (NPY) (colocalized with NE) was significantly decreased. These results suggest that NE synthesis and its secretion are increased during this period and correlate with the increase in secretion of androgens and estradiol, the development of precystic follicles, and a decrease in the ovulatory rate. After 11 wk, NE release had returned to control values, whereas the ovarian NE content had risen significantly, suggesting a maintained high rate of NE synthesis. In the ovary, NPY contents, steroid secretion, morphology, and ovulation had returned to the control state. These results suggest the participation of an extraovarian factor that might act locally to control the release of NE from the ovary, and further support the hypothesis that increased sympathetic activity plays a role in the development and maintenance of ovarian cysts.

Functional and high level expression of human dopamine beta-hydroxylase in transgenic mice.

Dopamine beta-hydroxylase (DBH; EC 1.14.17.1) catalyzes the production of the neurotransmitter and hormone norepinephrine in the third step of the catecholamine biosynthesis pathway. Transgenic mice were generated with multiple copies of a human DBH minigene construct containing the full-length cDNA connected downstream of the 4-kilobase upstream promoter region to achieve overexpression of DBH. Human DBH mRNA and immunoreactivity were detected tissue-specifically in the brain and adrenal gland of these transgenic mice. The transgene products were correctly processed to a glycosylated mature polypeptide with a molecular mass of 72 kDa and existed in the secretory vesicles as both soluble and membrane-bound forms. We detected a marked increase in DBH activity in various catecholamine-containing tissues of the mice that occurred as a consequence of expression of the catalytically active human DBH enzyme. However, in these transgenics the steady-state levels of norepinephrine and epinephrine were normally maintained without the acceleration of the catecholamine turnover rate, suggesting that there are some regulatory mechanisms to preserve a constant rate of norepinephrine synthesis in spite of the increased amount of DBH protein. These transgenic mice with the minigene construct provide one approach to study the mechanisms underlying biogenesis of the DBH polypeptide and regulation of norepinephrine synthesis.

Reduced Brain Norepinephrine Metabolism in Obese (ob/ob) Mice is Not Normalized by Tyrosine Supplementation

Five-week-old female lean and obese (ob/ob) mice were fed a 20% protein diet (1.1% tyrosine) or a 20% protein diet supplemented with tyrosine (4% tyrosine). On d 4 of supplementation, brain norepinephrine (NE) synthesis rate and brain efflux of 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG), a major metabolite of NE in mouse brain, were measured after administration of the monoamine oxidase inhibitor pargyline. Control obese mice had a lower rate of NE synthesis and a lower MHPG efflux than lean controls. Tyrosine supplementation elevated brain tyrosine concentration twofold, but had no effect on NE synthesis rate or MHPG efflux in either group. Likewise, tyrosine supplementation for up to 1 mo had no effect on food intake, oxygen consumption or body weight in obese mice and only a transient effect in lean mice, in which oxygen consumption was higher on d 2 and 3 and food intake was higher on d 3 and 4 compared with nonsupplemented lean mice. We conclude that tyrosine availability is not a limiting factor in the reduced brain noradrenergic activity of obese mice.

Norepinephrine in the rat ovary: ontogeny and de novo synthesis.

The aims of this study were to characterize the ontogeny of catecholamines (CA) in the rat ovary, to determine the ability of the immature ovary to synthetize norepinephrine (NE) in vitro, and to correlate between ovarian CA and plasma pituitary hormones. Ovaries, spleen (as a control tissue not subjected to endocrine regulation), and trunk blood were collected at 5-day intervals between days 5 and 40. Ovarian NE concentration increased markedly between days 20 and 35 of life, whereas the major rise in splenic NE concentration occurred between days 10 and 15. Ovarian and splenic tissues from neonatal females were capable of de novo synthesis of NE from tritiated tyrosine without an appreciable accumulation of L-Dopa and dopamine. The rate of NE synthesis by ovarian tissue taken from 20-day-old rats was significantly lower than that from 30- and 40-day-old rats, whereas NE production by splenic tissue from 20-, 30-, and 40-day-old rats were similar. Plasma FSH concentration was significantly elevated between days 10 and 20, whereas the major rise in plasma LH and PRL occurred between days 25 and 40. The following conclusions were reached. The delayed elevation of ovarian NE, compared to splenic NE, is attributable to a decreased production of NE by the ovary on day 20 and may involve a suppression or a delay in development of the activity of a key CA biosynthetic enzyme such as tyrosine hydroxylase. Given the temporal relationship between plasma gonadotropins, in particular FSH, and changes in ovarian NE, it is postulated that ovarian CA during neonatal development are subjected to regulation by circulating pituitary hormones.

Norepinephrine turnover in the heart and blood vessels of rats subjected to bilateral renal infarction.

The total norepinephrine (NE) content, the uptake of [3H]NE, the turnover rate and the synthesis rate of the neurotransmitter at the heart and blood vessels have been studied during the development of hypertension in rats subjected to bilateral renal infarction. Normal and sham-operated rats were used as controls. Fifty percent of the rats with renal infarction became hypertensive. The weight of the hearts and blood vessels of the experimental animals was significantly increased 15 days after renal infarction. Changes were greater in hypertensive animals. NE concentration in the heart was slightly decreased without achieving statistical significance, while total NE content was unchanged. In the artery wall NE concentration was significantly decreased in normotensive and hypertensive operated rats. [3H]NE uptake in the heart and blood vessels was similar in experimental and control animals. In relation to NE turnover, in both the heart and blood vessels, normal and sham-operated animals behaved as one population while normotensive and hypertensive rats behaved as another population. The rate constant of NE turnover was increased in both tissues of operated experimental animals without achieving statistical significance in the case of the heart. NE synthesis rate was unchanged in the cardiac muscle but was significantly increased in the blood vessels of operated animals. Present data indicate that results describing NE dynamics in the heart cannot be extrapolated for the blood vessels level; on the other hand changes in the neurotransmitter do not seem to be related to the development of high blood pressure after renal infarction in the rat.

Increased dopamine in the failing hamster heart: Transvesicular transport of dopamine limits the rate of norepinephrine synthesis

An earlier study demonstrated an increase in dopamine and a decrease in norepinephrine in the failing myopathic hamster heart. There was evidence to suggest that this abnormal distribution of catecholamines was secondary to a marked increase in cardiac sympathetic nerve traffic rather than a peculiarity of hamster cardiomyopathy. In this study the hamster model was used to dissect further the step limiting the conversion of dopamine into norepinephrine. In heart failure, cardiac norepinephrine was reduced from a mean value (± standard error of the mean) of 1,192 ± 176 to 441 ± 61 ng/g (probability [p] < 0.005) and cardiac dopamine was increased from 51 ± 5 to 458 ± 70 ng/g (p < 0.001). Cardiac decompensation also induced increases in both cardiac tyrosine hydroxylase (84 percent, p < 0.001) and cardiac dopamine-beta-hydroxylase (46 percent, p < 0.001) relative to values In the control hearts. In preparations of noradrenergic granules 44 percent of norepinephrine stores and 33 percent of dopamine stores were associated with the vesicular pellet of normal hearts. Congestive heart failure led to a significant decrease in norepinephrine, particularly in the intravesicular compartment, whereas dopamine exhibited an approximately 20-fold rise, reflected exclusively by the supernatant (extravesicular) fraction. Ganglionic blockade restored catecholamine content and distribution to normal. Inhibition of monoamine oxidase doubled the norepinephrine content of failing hearts (p < 0.01) in the absence of a change in dopamine. Catecholamine histofluorescence observations revealed a marked reduction in the number of nerves in apposition to the muscle of myopathic hearts In addition to a sprouting of new adrenergic fibers into connective tissue and in the lipofuscin pigment debris. It is concluded that an increase in sympathetic activity, at least in the myopathie hearts, can lead to a shift in the rate-limiting step for norepinephrine synthesis from the hydroxylation of tyrosine to the transport of dopamine across the membrane of the noradrenergic granule. Furthermore, it appears that one or more deaminated metabolites of dopamine in the extravesicular space contribute to this shift by inhibiting the dopamine uptake system.

Purification and characterization of avian dopamine .beta.-hydroxylase

Dopamine beta-hydroxylase (EC 1.14.17.1) has been purified from the chromaffin granules of avian adrenals. The enzyme has a molecular mass of approximately 320K daltons and consists of four apparently identical subunits joined in pairs by disulfide bonds. Analysis of the products formed from dopamine tritiated in the beta position indicated that 1.72 times as much tritium was retained in norepinephrine as was released as water. Ferrocyanide could serve as a reductant, but ascorbate at equal concentrations afforded higher rates. The enzyme had a pH optimum of 5-6 and was activated by either fumarate or acetate, with fumarate being far more effective. Kinetic experiments varying the concentrations of the substrates ascorbate and dopamine and those of the products dehydroascorbate and norepinephrine suggested that the mechanism was un-uni bi-uni ping pong. By this mechanism, the enzyme released dehydroascorbate after being irreversibly reduced by ascorbate and then sequentially bound oxygen and dopamine and released the product norepinephrine. The enzyme was inhibited by high but probably physiological concentrations of the substrate ascorbate and was activated by low concentrations of the product dehydroascorbate. Ascorbate inhibition was noncompetitive with dopamine, and dehydroascorbate activation was due to an increase in the enzyme's affinity for ascorbate with little or no change in its Vmax. Substrate inhibition by ascorbate and product activation by dehydroascorbate might together ensure that the rate of norepinephrine synthesis in vivo remains relatively unaffected by changes in the ratio of ascorbate to dehydroascorbate within chromaffin granules.

Analysis of fluctuations in the activity of rat pineal tyrosine hydroxylase

Tyrosine hydroxylase was measured in pineals removed from light-adapted and dark-adapted rats. The animals were adapted to the appropriate light situation for approximately 5 h before removal of the pineals. Bilateral ganglionectomy confirmed that all detectable enzyme activity in the pineal appears present in sympathetic fibers originating in the superior cervical ganglia. An analysis of tyrosine hydroxylase at subsaturating cofactor concentrations in dark-adapted rats revealed activity which was 60% higher than in appropriate light control animals. Further kinetic analysis showed that the K m for the synthetic cofactor, 6-methyltetrahydropterin, for dark-adapted animals was 0.8 ± 0.1 mM which was significantly lower than a K m value of 1.4 ± 0.2 mM for light adapted animals. Thus the rate of norepinephrine synthesis in vivo could increase due to an alteration in the enzyme molecule, which affects its interaction with substrate. These findings support previously documented results which show both an increased turnover and an increase in the concentration of norepinephrine in the pineals of dark-adapted rats.

Compensatory increase in tyrosine hydroxylase activity in rat brain after intraventricular injections of 6-hydroxydopamine.

The neurotoxin 6-hydroxydopamine produced a permanent loss of endogenous norepinephrine and of 3H-labeled norepinephrine uptake sites in the hippocampus within 5 days. These losses were initially accompanied by parallel decreases in tyrosine hydroxylase activity and synaptosomal norepinephrine synthesis. Within 21 days, however, hippocampal tyrosine hydroxylase activity and norepinephrine synthesis rate increased three- to fivefold. These data suggest a novel form of plasticity in brain-damaged animals characterized by an increase in the capacity for transmitter biosynthesis in residual neurons.

EFFECT OF CONCUSSIVE HEAD INJURY ON CENTRAL CATECHOLAMINE LEVELS AND SYNTHESIS RATES IN RAT BRAIN REGIONS 1

Abstract—Unanesthetized male Sprague‐Dawley rats were subjected to head injury using a unique acceleration‐deceleration model designed to mimic the most prevalent form of clinical head injury. Endogenous tyrosine, dopamine and norepinephrine were determined fluorometrically and catecholamine synthesis rates were determined by a radioisotopic method. These values were determined in four brain regions: cortex‐striatum, midbrain‐hypothalamus, medulla‐pons, and cerebellum, and were performed at 5 min, 15 min, 1 h, and 2h post‐trauma. Dopamine levels were elevated in the medulla pons region at 5 min after trauma and in the midbrain‐hypothalamus at the 15 min and 1 h time periods. This increase in dopamine levels may reflect disruption of dopamine beta hydroxylase activity. Norepinephrine synthesis rate in the cerebellum was elevated at 2 h after trauma. Changes in the other parameters were observed but appeared to be related to stress, and the effect of stress in this model is discussed.

Plasma prolactin levels and hypothalamic catecholamine synthesis during suckling.

The effects of suckling on the release of prolactin and the synthesis rates of hypothalamic catecholamines were determined in lactating rats. 3H-tyrosine was administered to rats at the onset of suckling and the rate of its incorporation into 3H-dopamine and 3H-norepinephrine was measured at various intervals. Suckling caused an increase in the plasma prolactin concentration within 4 min. Although an increase in the rate of dopamine synthesis did occur during suckling, it was not significant until after 15–25 min of suckling. There were no significant changes in the rate of norepinephrine synthesis. The results suggest that the increase in prolactin release was probably not caused by the increase in dopaminergic neuron activity.

Effects of 2-mercaptoethylguanidine and other compounds on norepinephrine synthesis by adrenal medullary granules

The effects of 2-mercaptoethylguanidine and other compounds on dopamine uptake and norepinephrine synthesis by adrenal medullary granules were studied. The effects of these materials on crude dopamine-β-hydroxylase were also tested. A microanalytical technique was developed which allows the use of the natural precursor, dopamine, in studies of uptake and synthesis by adrenal medullary granules. The conversion of dopamine to norepinephrine by dopamine-β-hydroxylase was also studied with this technique. Catecholamines were assayed as their dansyl (5-dimethylaminonaphthalene-1-sulfonyl) derivatives, thereby increasing greatly the resolution and sensitivity of detection. The effects of 2-mercaptoethylguanidine and reserpine on dopamine uptake and norepinephrine synthesis by medullary granules were compared. Reserpine inhibited norepinephrine synthesis indirectly through inhibition of dopamine uptake. 2-Mercaptoethylguanidine, on the other hand, depressed norepinephrine synthesis in intact granules by a direct inhibitory effect on dopamine-β-hydroxylase rather than on the uptake mechanism. 2-Mercaptoethylguanidine increased the rate of norepinephrine synthesis by crude dopamine-β-hydroxylase in the presence of added Cu 2+ and inhibited norepinephrine synthesis in the absence of added Cu 2+. The nature of the latter effect was resolved by demonstrating the formation of a Cu-2-mercaptoethyl-guanidine complex in pure solutions. Since dopamine-β-hydroxylase is a Cu-containing enzyme, the mechanism of the 2-mercaptoethylguanidine inhibition of dopamine-β-hydroxylase appears to be through the binding of enzymic Cu.

Rate of norepinephrine synthesis after tyramine-induced depletion of norepinephrine in rat heart.

Repeated injections of sufficient doses of tyramine (20 mg/kg i.p.) to rats at 15-min intervals for a total of 12 doses, deplete the heart norepinephrine (NE) stores by about 90 %. Following the last dose of tyramine, the concentration of heart NE increased progressively as the net result of the processes of synthesis and loss. The net rate of NE repletion is given by the equation: log[(([NE]∞ – [NE]) [NE]∞)/[NE]∞] –kt/2.303 The calculated ‘k’ constant times the NE steady-state level corresponds to the NE synthesis rate ‘K’. This rate is identical with that determined from the decline in levels after α-methyltyrosine (α-MT) and from the loss of ³H-NE after labeling with tracer doses.

Adrenergic activity in hypothalamus and ovulation.

The turnover of norepinephrine (NE) and its synthesis in the hypothalamus in rats just prior to and subsequent to ovulation was studied. Hypothalamic NE concentration in proestrus (preovulatory) was higher than in estrus rats (3.48 plus or minus) .09 mcg/gm vs. 2.13 plus or minus .04 mcg/gm). When methylester hydrochloride (MPT), a blocker of catecholamine biosynthesis, was injected, proestrus rats NE dropped 62% vs. estrus rats' drop of 28.6%. Tritiated NE injected to show synthesis rates showed a higher rate of NE synthesis produced in the hypothalamus during proestrus vs. estrus. In addition there was an increase in NE levels between diestrus Day 2 and proestrus localized to the anterior and middle hypothalamus.

Catecholamine Metabolism in Hypertensive Rats

The possible role of catecholamines in two forms of experimental hypertension in rats was investigated further. Inbred, spontaneously hypertensive (SH) Wistar rats had unchanged endogenous levels of norepinephrine (NE) in the three tissues studied, and a significantly ( P &lt;0.05) decreased rate of NE synthesis ( 14 C-tyrosine technique) in heart and brainstem but not in gut, in comparison to normotensive Wistar rats. Also, the levels of free fatty acids (FFA) and major urinary catecholamine metabolites in plasma revealed no evidence of increased catecholamine turnover or release. Sprague-Dawley rats rendered hypertensive by treatment with desoxycorticosterone acetate and 1% salt had decreased cardiac NE concentration and increased cardiac NE turnover ( 3 H-NE technique) and cardiomegaly, confirming the work of others. However, urinary normetanephrine and plasma FFA did not differ from those in normal rats. These and other results fail to support but do not completely exclude a primary role for catecholamines in either type of hypertension.

Influence of ganglionic blockade on catecholamine and metabolite excretion.

Abstract : Rats were placed at 28C for a control period of 4 hr prior to treatment with mecamylamine or saline. After injection the rats were divided into two groups with one group remaining at 28C and the other being placed at 15C. Ganglionic blockade with mecamylamine decreased the urinary excretion of norepinephrine and normetanephrine in both groups of rats. 3-methoxy-4-hydroxyphenyl glycol (MHPG) excretion fell in the mecamylamine-treated group kept at 28C but rose slightly in the group transferred to 15C. The fall in norepinephrine and normetanephrine excretion, seen after mecamylamine treatment, reflects a decrease in norepinephrine secretion. The increase in MHPG in the cold indicates a rise in the synthesis and intraneuronal degradation of norepinephrine. These results support our earlier postulate that the rate of norepinephrine synthesis is dependent, at least partially, on the ambient temperature and can dissociate itself from the rate of secretion. (Author)

On the use of synthesis inhibitors to estimate brain norepinephrine synthesis in gonadectomized rats

Brain norepinephrine synthesis rate in gonadectomized and control rats treated with alpha-methyl paratyrosine

Influence of ambient temperature on excretion of catecholamines and metabolites.

Abstract : The study demonstrates the influence of ambient temperature on the excretion of epinephrine, norepinephrine, and their major metabolites. As a result of this work it is concluded that norepinephrine secretion increases in a cold environment and decreases during moderate warm exposure. Heat stress, 36 C, however, increases norepinephrine excretion. The rate of norepinephrine synthesis is inversely proportional to the ambient temperature and in three instances in our experiments the quantity of amine formed was not related to the amount secreted. It is thus concluded that the rate of norepinephrine synthesis is dependent, at least partially, on the ambient temperature and can dissociate itself from the rate of norepinephrine secretion. (Author)

The prolongation of reserpine-induced cardiac norepinephrine depletion by metabolic inhibitors

Levels of cardiac norepinephrine (NE) in the rat were determined at various times after the intraperitoneal administration of 2 mg/kg reserpine phosphate. The rate of repletion of NE was decreased by the administration of certain metabolic inhibitors [thioacetamide; 2,2-dichloro- N-(β-hydroxy-α-(hydroxymethyl) - p-methylsulfonyl-phenethyl) acetamide; β-diethylaminoethyl-diphenylpropyl acetate (SKF 525-A); actinomycin d] in proper dosage schedules. None of these agents significantly affected the endogenous NE concentration in the heart of the nonreserpinized rat. In animals pretreated with phenobarbital prior to reserpine, the levels of cardiac NE returned to normal more rapidly than did those of animals receiving no prior barbiturate. SKF 525-A decreased the levels of 3H-reserpine found in the heart and liver, and did not affect the concentration of 3H-reserpine in the carcass of animals 4 days after 2 mg/kg 3H-reserpine. Thioacetamide did not alter the capacity of the heart to retain tritiated NE after i.v. administration. Actinomycin d did not interfere with the rate of NE synthesis in the heart, nor did it change the characteristics of the NE uptake process of isolated perfused hearts from reserpinized animals. Repletion of adrenal NE in reserpinized animals was not affected by administration of actinomycin d. It was concluded that the slow repletion of the cardiac NE after reserpine administration may be due to the necessity for the synthesis of new NE storage sites in the heart, and that the metabolic inhibitors retard the return of normal NE levels by inhibiting the synthesis of these storage sites.

Acceleration of norepinephrine synthesis in the rat submaxillary gland in vivo during sympathetic nerve stimulation.

Abstract In nembutal-anesthetized rats, the sympathetic nerves to the submaxillary gland were stimulated electrically during simultaneous intravenous infusion of tyrosine-C14 and dopa-H3. Stimulation resulted in a four- to five-fold increase in norepinephrine synthesis from tyrosine-C14 but not from dopa-H3. Endogenous norepinephrine content and tyrosine specific activity were not significantly altered. The results suggest an impulse-induced acceleration of norepinephrine synthesis in the adrenergic neurone. Synthesis is apparently stimulated at or before the tyrosine hydroxylation step, although there are no changes in the content of tyrosine hydroxylase. The rate of norepinephrine synthesis might be controlled by tyrosine transport, product inhibition, endogenous inhibitors or cofactors of tyrosine hydroxylase.