Changes of norepinephrine levels, tyrosine hydroxylase and dopamine-beta-hydroxylase activities after castration and testosterone treatment in vas deferens of adult rats

Norepinephrine levels and tyrosine hydroxylase and dopamine-beta-hydroxylase activities have been used to evaluate the effect of castration and testosterone treatment on the sympathetic innervation of the adult vas deferens. Castration was followed by a decrease in both norepinephrine content and tyrosine hydroxylase activity, even though the changes were not concomitant. Treatment of castrated animals with testosterone reversed the effect of castration on organ weight and norepinephrine content, but only a short-lasting increase in tyrosine hydroxylase activity occurred at the beginning of testosterone treatment. In contrast, the testosterone-induced recovery of norepinephrine content observed at this time was accompanied by a marked increase in dopamine-beta-hydroxylase activity. The results suggest that in rat vas deferens, norepinephrine levels are under androgenic control and that this regulation mainly involves changes in dopamine-beta-hydroxylase activity rather than a modulation of tyrosine hydroxylase.     

Mechanism of release of norepinephrine from peripheral adrenergic neurones by the calcium ionophores X 537A and A 23187

The effects of the calcium ionophores, X 537A and A 23187, in causing release of norepinephrine (NE) were examined in isolated guinea-pig vas deferens and rat atrial segments. Examination of calcium dependence, dopamine-β-hydroxylase (DBH) release and release of deaminated metabolites of NE suggest that while A 23187 releases by both an exocytotic and non-exocytotic mechanism, X 537A releases NE predominantly through a non-exocytotic mechanism. The relative impotence of A 23187 in effecting release of NE may explain its inactivity in alteration of cardiac responses.     

CHANGES IN VESICULAR DOPAMINE-β-HYDROXYLASE RESULTING FROM TRANSMITTER RELEASE

—Exposure of rats to 3°C for up to 30 min leads to a decrease of 30 per cent in the dopamine-β-hydroxylase activity of the vesicular pellet of the heart; this is greater than can be accounted for by loss of soluble DBH from the two populations of noradrenaline storage vesicles known to be present in the heart. Cold exposure in the presence of α-methyltyrosine causes a much smaller reduction in dopamine-β-hydroxylase activity; this suggests that there is a decrease in transmitter release when synthesis is inhibited. The noradrenaline concentration of the vesicular pellet rises briefly during cold exposure and is then maintained at control levels; the early rise is absent in the presence of α-methyltyrosine. The use of the noradrenaline : dopamine-β-hydroxylase ratio as an index of saturation of vesicular storage capacity suggests that during cold exposure an increased synthesis rate leads to increased filling of vesicles.     

Formation in vitro of matrix-like aggregates of chromogranins and phospholipids derived from chromaffin granules of the bovine adrenal medulla

Matrix-like particles were formed in sonicated suspensions of soluble chromogranins and phospholipids extracted from the isolated membrane phase of chromaffin granules. The artificial particles which sedimented into 0.4–0.6 M sucrose layers during isopycnic centrifugation, consisted of liposomal vesicles and granular aggregates indicative of lamellar and possibly hexagonal organization patterns of phospholipids and the aqueous phase containing the soluble chromogranins. The ultrastructure of the granular aggregates was strikingly similar to the matrix phase of the intact chromaffin granule.The dopamine-β-hydroxylase activity (EC 1.14.2.1) of the soluble chromogranin preparation was potentiated by the addition of the phospholipids and further enhanced by the sonication procedure. The enzymic activity was highest in the 0.4–0.6 M sucrose layers, rich in the artificial matrix-like particles.The artificially formed particles incorporated ATP by further sonication and the presence of ATP in the particle fractions did not inhibit the specific dopamine-β-hydroxylase activity, even at ratios of 4 and 2 μmoles ATP/mg protein in the 0.4 and 0.6 M sucrose layers, respectively. Noradrenaline was incorporated into the particle fractions in presence of ATP. However, the presence of noradrenaline above 3·10^?6 M resulted in a 50–75% inhibition of the dopamine-β-hydroxylase activity of the artificial matrices.     

SUBCELLULAR DISTRIBUTION OF DOPAMINE-β-HYDROXYLASE AND INHIBITORS IN THE HIPPOCAMPUS AND CAUDATE NUCLEUS IN SHEEP BRAIN

—The enzyme dopamine-β-hydroxylase (EC 1.14.17.1) which converts dopamine to noradrenaline was found to be present in substantial amounts in sheep brain hypothalamus and caudate nucleus and was located to the synaptic vesicle fractions in these two brain regions by subcellular fractionation. This dopamine-β-hydroxylase was associated with paniculate matter in these two brain regions since it was resistant to solubilization with butan-1-ol and 0.1% Triton X-100. As highly significant levels of dopamine-β-hydroxylase were present in the caudate nucleus, factors other than a simple lack of this enzyme must operate to maintain the low levels of noradrenaline and high levels of dopamine in the caudate nucleus. Purified adrenal dopamine-β-hydroxylase was substantially inhibited by two factors prepared from sheep brain hypothalamus and caudate nucleus. These were found to be cupric ions and a sulphydryl inhibitor. High levels of the sulphydryl inhibitor of dopamine-β-hydroxylase were found in synaptosomal fractions from sheep brain hypothalamus and caudate nucleus and the levels were comparable in both regions. Upon subfractionation of a synaptosome-containing fraction from the hypothalamus, the inhibitor was located predominantly in the soluble fraction, although there were significant levels in the synaptic vesicle fraction. Therefore, the sulphydryl inhibitor must be considered as a possible regulator of dopamine-β-hydroxylase activity. Free cupric ion concentrations as low as 2·5 μM were found to inhibit purified adrenal dopamine-β-hydroxylase in vitro and the concentration of copper in the soluble tissue component of hypothalamus and caudate nucleus was well above this minimal copper concentration. The percentage content of soluble copper in the caudate nucleus was significantly higher than in the hypothalamus. The importance of the soluble to particulate-bound ratio of copper in brain was shown in studies of the developing rat brain. A rapid increase in the level of copper in brain was found in the first 4 weeks but the level was constant by 2 months of age. The percentage of soluble copper, however, was maximal soon after birth and had declined to a constant figure by 2 months of age. A scheme for the regulation of dopamine-β-hydroxylase activity involving these factors is proposed.     

Changes in noradrenergic vesicle markers of rabbit oviducts during progesterone treatment

The effect of progesterone (P) on norepinephrine (NE), [3H] norepinephrine ([3H]NE) and dopamine beta-hydroxylase (DBH) in noradrenergic vesicles from rabbit oviducts was studied after daily injections of the hormone during different periods (4, 7 and 15 days). Progesterone induced a concomitant increase in NE and DBH activity and [3H]NE uptake. To study the mechanism involved in such effects, 4 tissue fractions were obtained by differential centrifugation of the oviducts of which the vesicular fraction was applied over continuous sucrose gradients (0.3-2 M). The changes induced by P in markers of tissue and gradient fractions showed an increase of the NE storage capacity which could be ascribed to an increase in the number of storage vesicles, and/or to a higher extravesicular storage capacity. The occurrence of these mechanisms during pregnancy or after P treatment could account for the (long-lasting) high levels of NE observed in such instances.     

Preliminary characterization, androgen-dependence and ontogeny of an abundant protein from mouse vas deferens

Polyacrylamide gel electrophoresis analysis revealed that the vas deferens of adult mouse contains a major protein. Mouse vas deferens protein is a basic glycoprotein with a molecular weight of 34,800 +/- 300. The protein represents 17 +/- 0.7% and 42 +/- 2.4% of soluble proteins from homogenate and luminal fluid respectively, an estimate based on densitometric scanning of polyacrylamide gels. The protein originated from the vas deferens since it was not detected in blood plasma or in sexual organs and it was still present after ligation of the epididymis. Changes in androgen status of the animal markedly affected the vas deferens protein. After castration a progressive decrease in the protein was observed and its relative percentage dropped to 2 +/- 0.4% after 45 days. The concentration of the protein returned to precastration levels after 2 weeks of testosterone treatment but oestradiol, progesterone and corticosterone were ineffective in this respect. The vas deferens protein was not synthesized in significant amounts until animals were 20 days old and its concentration increased rapidly from 20 to 30 days in concert with the pubertal increase of androgens in the vas deferens.     

Direct comparison of the rapid axonal transport of norepinephrine and dopamine-β-hydroxylase activity

Stop-flow techniques were used to examine the rapid axonal transport of norepinephrine in rabbit sciatic nerves. When the midpoint of a nerve incubated in vitro was cooled to 2°C while the remainder was kept at 37°C, norepinephrine accumulated proximal to the cooled region at a rate corresponding to an average transport velocity between 5 and 6 mm/hr in a distal direction. Since only about half of the norepinephrine appeared to be free to move, the mean velocity of the moving fraction was probably twice as great. No norepinephrine accumulated distal to a broad cooled region under conditions in which there would have been a significant accumulation of dopamine-β-hydroxylase activity. Therefore, unlike dopamine-β-hydroxylase, norepinephrine may not be subject to rapid retrograde transport. When nerves that had been locally cooled for 1.5 hr were rewarmed uniformly to 37°C, a wave of norepinephrine moved exclusively in a distal direction. The peak of this wave moved at a velocity of 12.2 ± 0.5 mm/hr or 293 ± 12 mm/day; the front of the wave moved at about 18 mm/hr. or 430 mm/day; and the tail probably moved faster than 6 mm/hr. This spectrum of velocities was virtually identical to the one displayed by the wave of dopamine-β-hydroxylase activity that was generated under the same conditions. Our results are consistent with the conclusion that all axonal structures containing norepinephrine also contain dopamine-β-hydroxylase, but they are not consistent with the converse.     

Effect of cold pressor stimulation on plasma norepinephrine, dopamine-beta-hydroxylase, and renin activity

Changes in plasma levels of norepinephrine, dopamine-β-hydroxylase (DβH), and renin activity were observed in nine healthy volunteers during cold pressor stimulation. Increases in mean arterial blood pressure and heart rate during cold stimulation were accompanied by a sharp rise in plasma norepinephrine, while plasma DβH and renin activity showed little or no change. The results indicate that plasma norepinephrine accurately reflects acute activation of the sympathetic nervous system in contrast to plasma DβH and renin activity.     

Axonal transport of muscarinic receptors in vesicles containing noradrenaline and dopamine-β-hydroxylase

Presynaptic muscarinic receptors labeled with [³H]dexetimide and noradrenaline in dog splenic nerves accumulated proximally to a ligature at the same rate of axonal transport. After fractionation by differential centrifugation, specific [³H]quinuclidinyl benzilate or [³H]dexetimide binding revealed a distribution profile similar to that of dopamine-β-hydroxylase and noradrenaline. Subfractionation by density gradient centrifugation showed two peaks of muscarinic receptors; the peak of density 1.17 contained noradrenaline and dopamine-β-hydroxylase whereas that of density 1.14 was devoid of noradrenaline. Therefore the foregoing experiments provide evidence that presynaptic muscarinic receptors are transported in sympathetic nerves in synaptic vesicles which are similar to those containing noradrenaline and dopamine-β-hydroxylase. This suggests a possible coexistence of receptor and neurotransmitter in the same vesicle.     

3H-noradrenaline metabolism in the isolated epididymal and prostatic portions of the rat vas deferens

The spontaneous ³H-noradrenaline efflux from the isolated epididymal and prostatic portions of the rat vas deferens, was investigated. The spontaneous tritium efflux was higher in the epididymal portion than in the prostatic one from normal animals. Such differences were abolished after castration. On the other hand, acetylsalicylic acid enhanced the total tritium efflux only in the epididymal portion, whereas phentolamine and phenoxybenzamine increased the total tritium efflux from the two portions of the rat vas deferens in both experimental groups. The prostatic portion released a similar amount of ³H normetanephrine than the apididymal end, whereas other metabolic fractions (3,4-dihydroxyphenylglycol; 3,4-dihydroxymandelic acid and O-methylated deaminated metabolites) were smaller in the prostatic portion in comparison with the epididymal portion from control vas deferens. The results presented in the isolated rat vas deferens suggest the existence of a prostaglandin as well as an alpha adrenoceptive modulation of the spontaneous total tritium efflux.     

EVIDENCE FOR EXTRANORADRENERGIC DOPAMINE-β-HYDROXYLASE ACTIVITY IN RAT SALIVARY GLAND

—Three days after superior cervical ganglionectomy of adult Sprague-Dawley rats, the levels of endogenous norepinephrine, the uptake process for [³H]norepinephrine and the activity of tyrosine hydroxylase decreased 99 per cent in the ipsilateral salivary gland. In contrast, the activity of dopamine-β-hydroxylase and DOPA decarboxylase fell to 30 per cent of the activity of the contralateral innervated gland. Examination of the cofactor requirements, the characteristics of activation by cupric ion and the immunologic identity of this residual hydroxylase activity indicated that it was authentic dopamine-β-hydroxylase. The residual dopamine-β-hydroxylase in the denervated gland had the same subcellular distribution as the enzyme in the innervated salivary gland. Procedures that caused atrophy or hypertrophy of the acinar cells did not affect the total content of dopamine-β-hydroxylase in the denervated salivary gland. Chemical sympathectomy with 6-hydroxy-dopamine caused a 40 per cent decrement in the serum levels of dopamine-β-hydroxylase but a 30 per cent increase in its activity in the denervated salivary gland. Although denervation caused a complete loss of endogenous norepinephrine in the salivary gland, it resulted in only a 15 per cent decrement in the levels of endogenous octopamine and β-phenylethanolamine, two other products of dopamine-β-hydroxylase.     

Hormonal influence on rat vas deferens responses induced by field stimulation

The effect of castration and treatment with testosterone propionate (PT), estradiol benzoate (E2) and ciproterone acetate (CPA) on the responses of the vas deferens in rat has been studied. Castration produces a time-dependent decrease of the response amplitude. PT augments the response amplitude in normal rats and reverses the effect of castration. E2 augments the response amplitude in normal rats without modifying the castration effect. CPA does not change the response of the vas deferens. The results suggest that PT and E2 through possible different mechanisms, facilitate the transmission in rate vas deferens, whereas castration obstructs it.     

Androgen and estrogen metabolism in the reproductive tract and accessory sex glands of the domestic boar (Sus scrofa).

Steroid metabolism in target tissues has relevance in assessing biological response. We have investigated the metabolism of testosterone and estrogens in the reproductive tract and accessory sex glands in the boar. Seminal vesicles were taken from four 6-mo-old animals; and seminal vesicles, prostate, vas deferens, and regions of the epididymis were taken from two mature boars (10 and 24 mo old). Tissues were incubated in 5 ml medium (TC-199) at 34 degrees C under 5% CO(2) and 95% air for 2 h with (3)H-labeled testosterone, estrone, and estradiol-17beta. Aliquots of spent media were taken to measure radioactivity before separation of unconjugated and conjugated steroids on Waters C(18) Sep-Pak cartridges. Sulfoconjugated steroids and glucuronidates were recovered in series from C(18) cartridges after solvolysis and enzyme hydrolysis, respectively. Profiles of metabolites for free and hydrolyzed fractions were obtained from gradient HPLC with acetonitrile:water on a reversed-phase C(18) column. No clear evidence of conjugation was seen for testosterone metabolites. 5alpha-Dihydrotestosterone was the principal metabolite, but the amounts formed depended on the source, with little from the epididymal tissues and seminal vesicles, but greater quantities from the vas deferens (>25%) and prostate (>30%). The most noteworthy feature of estrogen metabolism was the extent of conjugation by all tissues. Almost all radioactivity in the conjugate fractions for the epididymis and vas was present as sulfates. Glucuronidates were seen for the prostate and were the dominant form of conjugation (about 60%) for the seminal vesicles. A striking parallel existed for the profiles of estrogen metabolites from all tissues for unconjugated and hydrolyzed fractions. Only in quantitative terms were some distinctions noted. These overall findings underscore a need to consider local metabolism of steroid hormones in target tissues of the male reproductive system.     

Pre- and postsynaptic adrenergic activation by norepinephrine reuptake inhibitors in the field-stimulated rat vas deferens

Desipramine (DMI), protriptyline, chlorpromazine, amitriptyline and cocaine, alone or in the presence of prazosin, produced a dose-related inhibition of contractions induced by field stimulation of the rat vas deferens. The inhibition of contractions was readily reversed by yohimbine. In contrast, when yohimbine was first added to the bath, all agents, except chlorpromazine, produced a dose-related enhancement of contractions which were readily reversed by prazosin. The potencies of these agents for induction of contractile inhibition, after prazosin, and contractile enhancement, after yohimbine, were similar. Both of the latter contractile responses of DMI were markedly attenuated or absent in tissues taken from rats pretreated with reserpine and alpha-methyl-para-tyrosine. The data indicate that, in the rat vas deferens, inhibition of norepinephrine reuptake results primarily in presynaptic (α₂) receptor activation. Postsynaptic (α₁) adrenergic activation by inhibition of norepinephrine reuptake can be demonstrated in this tissue only after presynaptic (α₂) receptor blockade. The possible implications of the present studies to the delayed clinical onset of action of tricyclic antidepressants is discussed.     

Solubilization of histamine H-1, GABA and benzodiazepine receptors.

Dopamine 3-0-sulfate is present in considerable amounts in mammalian plasma and peripheral tissues. Incubation of dopamine 3-0-sulfate (0.1 μmole) with purified bovine dopamine-β-hydroxylase resulted in the formation of free norepinephrine (7.3 × 10^?3 μmole). The conversion to norepinephrine was inhibited by 0.6 mM of fusaric acid, an inhibitor of dopamine-β-hydroxylase. The reaction of dopamine 3-0-sulfate with dopamine-β-hydroxylase followed Michaelis-Menten kinetics. The calculated K_m was 17 mM, different from the K_m for free dopamine (0.1 mM). The incubation medium does not contain any sulfatase activity.     

Biochemical evidence for the presence of small and large noradrenergic storage vesicles isolated from cat ovary in isoosmotic conditions. Distribution of dopamine-β-hydroxylase activity

The cat ovary presents unusually high levels of noradrenaline that change according to the endocrine status of the animal. Their functional meaning remains unknown. The cat ovary innervation, unlike that of other organs receiving noradrenergic innervation, has been poorly characterized on biochemical grounds. We present here a biochemical characterization of the neurotransmitter storage. By using hyperosmotic and isoosmotic gradients evidence is presented that noradrenaline is associated to two different populations of vesicles. In hyperosmomolarity conditions (sucrose gradients) “light” vesicles (density 1.12 g/ml) and “heavy” vesicles (density 1.17 g/ml) appeared. In both vesicles, noradrenaline and dopamine-β-hydroxylase were found. In isoosmotic Percoll gradients distribution of the markers also suggested the presence of two vesicle populations. Light vesicles (density 1.033 g/ml) with high dopamine-β-hydroxylase activity but very low levels of noradrenaline and adenosine triphosphate; [³H]noradrenaline, used as a specific exogenous vesicle marker, was feebly incorporated in this fraction. Heavy vesicles (density 1.041 g/ml) containing high levels of noradrenaline, adenosine triphosphate, low levels of dopamine-β-hydroxylase activity are able to incorporate high amounts of [³H]noradrenaline. In these gradients, Mg²⁺ activated ATPase activity was present in both vesicle fractions.

Sedimentation analysis by analytical differential centrifugation also disclosed two types of vesicles: large vesicles with a sedimentation coefficient between 348 and 308 and small vesicles with a sedimentation coefficient of 96 . Large vesicles were associated with noradrenaline-β-hydroxylase activity, while small vesicles were associated only with noradrenaline.

In isoosmotic conditions the use of other microsomal markers allowed us to define the degree of contamination of the vesicle fractions. It was found that the noradrenergic heavy vesicles fraction presented under 11% of 5′-nucleotidase activity of the total activity present in the gradient and less than 5% of acid phosphatase, NADH-cytochrome c reductase and monoaminooxidase of the total activities in the gradients.

In isoosmotic conditions the physical properties of presumed vesicles were apparently undisturbed supporting the current morphometric observations. Our results then suggest prevailing roles for each type of vesicle: synthesis for light vesicles, and storage and/or release for heavy ones.     

Different behavior of the rat vas deferens of castrated animals and those treated with cyproterone acetate

The effects of castration and treatment with the antiandrogen cyproterone acetate (CPA) on the responses of the vas deferens of the rat induced by phenylephrine, KCl and BaCl2 has been studied. Both castration and CPA induced a spontaneous motility in the rat vas deferens. Castration produces a decrease of the response amplitude induced by phenylephrine and KCl and an increase of those induced by BaCl2 in animals killed 30 days after castration. CPA increases the response amplitude induced by phenylephrine and KCl without modifying those induced by BaCl2. These results suggest that the antiandrogen CPA produces modifications qualitatively different from castration.     

Vesicular and Ganglionic Norepinephrine in the Rat: Progressive Increase with Age

This study analyzed dopamine (DA) and norepinephrine (NE) in the synaptic vesicles and cytoplasm of brains of rats of 2 months and 14 months. The data revealed a clear NE increase in the synaptic vesicles of the 14-month-old rats, contrasting with NE in the cytoplasmic fraction of the rat brain, which remained unchanged with age. Synaptic vesicles from different regions of rat brain, including those from the striatum, consistently exhibited higher NE than DA concentrations, suggesting that they are predominantly noradrenergic. In the brain, DA concentrations in vesicular and cytoplasmic fractions did not vary with age, whereas in the superior cervical ganglia DA and NE concentrations increased in the older rats. L-3,4-Dihydroxyphenylalanine administration significantly increased DA without affecting NE in the ganglia of rats of all ages. In the brain, such a treatment significantly raised DA only in the synaptic vesicles of the older rats, suggesting an increased facilitation of DA transport into the synaptic vesicles with age, which may account for the higher vesicular NE in the older rats.