CYCLOHEXIMIDE ALTERS AXONAL TRANSPORT AND SUBCELLULAR DISTRIBUTION OF DOPAMINE-β-HYDROXYLASE ACTIVITY

—Administration of cycloheximide, 10 mg/kg s.c. led within 4 h to an approx 30% reduction of dopamine-β-hydroxylase (DBH) activity in the abdominal portion of rat sciatic nerves. At least two more hours elapsed before DBH activity in the distal part of these nerves began to fall. This pattern suggests reduced synthesis or delivery of DBH into axons but continued transport of previously delivered enzyme. Coinciding with the time at which DBH activity began to fall in distal segments of sciatic nerve, there was a marked reduction in the accumulation of DBH activity above a ligature in this region. Between 4 and 8 h after administration of cylcoheximide, 10 mg/kg, accumulation above a ligature was 70% less than in untreated nerves (P < 0.001), a reduction significantly greater (P < 0.05) than the accompanying 28% loss of baseline DBH activity. At the same time, the clearance of DBH activity from nerve regions distal to a ligature was greatly reduced. This pattern is consistent with the depletion of a minor but rapidly transported compartment of DBH. Six hours after administration of cylcoheximide, 10 mg/kg, the apparent subcellular distribution of DBH in distal regions of sciatic nerve was altered by a significant 36% loss in sedimentable DBH activity, with non-significant changes in othcr fractions. This suggests that rapidly transported DBH, depleted from the nerve by cycloheximide-induced inhibition of protein synthesis, is more highly associated with intraneuronal particles than is slowly transported or stationary DBH.     

TRANSPORT AND TURNOVER OF DOPAMINE-β-HYDROXYLASE (EC 1.14.2.1) IN SYMPATHETIC NERVES OF THE RAT

Axoplasmic transport of dopamine-β-hydroxylase (DBH), a marker enzyme for catecholamine storage vesicles, was studied in sympathetic nerves of the rat. At 24 h after ligation of the sciatic nerve, there was a marked accumulation of DBH activity in the first 3 mm proximal to the ligature. Immediately distal to the ligature, a slight accumulation took place. Accumulation proximal to the ligature was a linear function of time for at least 6 h; the velocity of transport was calculated as 4.6 mm/h. Local application of 1 ·l of 0.1 M colchicine, caused a rapid increase in DBH activity in superior cervical ganglia. This increase remained linear for 22 h and its rate indicated a turnover time of 12 h for DBH in these ganglia. After application of colchicine to the ganglia, there was a decrease in DBH activity in the submaxillary salivary glands. The initial rate of this decrease was less than the rate of increase in the ganglia and probably reflected the normal turnover of the enzyme. Our results indicated that the turnover time for DBH in salivary glands ranged between 3.6 and 6.3 days.     

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 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.     

SERUM DOPAMINE-β-HYDROXYLASE IN SCHIZOPHRENIC PATIENTS

Abstract— Dopamine-β-hydroxylase (DBH) activity in serum was decreased significantly in schizophrenic patients (16.17 ± 12.60 μmol/min/1 of serum, mean ± S.D., n = 149) when compared with that of normal controls (42.53 ± 30.94 μmol/min/1 of serum, mean ± S.D., n= 153) and neurotic patients. Long duration of disease did not cause any significant changes in serum DBH activity except a tendency for increase in patients of lodger than 18 years duration. We also examined the possibility that the serum DBH deficiency in the schizophrenic group was an artifact of treatment with antipsychotic drugs, especially phenothiazines. No significant difference was observed between the patients treated with the drugs and the patients not receiving the drugs.     

DOPAMINE-β-HYDROXYLASE IN THE RAT BRAIN: DEVELOPMENTAL CHARACTERISTICS

Abstract— A sensitive and specific assay for dopamine-8-hydroxylase (DBH) in the rat brain has been developed. The enzyme in the brain has requirements for cofactors and affinity for substrate similar to DBH in the adrenal medulla. DBH activity was demonstrable in the brain of the fetal rat at 15 days of gestation; there was an increase in DBH activity with maturation that preceded and paralleled the rise in levels of endogenous norepinephrine until 3 weeks after birth. There was a shift in the distribution of total DBH activity from the caudal to the rostral regions of the brain during development. In the adult brain, DBH was highly localized in the nerve terminals. Between 17 days of gestation and adult-hood, there was 2300-fold increase in the DBH activity that sedimented with sheared-off nerve terminals.     

LOCALIZATION OF EXTRANEURONAL DOPAMINE-β-HYDROXYLASE IN RAT SALIVARY GLAND BY IMMUNOFLUORESCENCE HISTOCHEMISTRY

Abstract— Superior cervical ganglionectomy results in a complete noradrenergic neuronal denervation of the rat sublingual-submaxillary salivary gland. Dopamine-β-hydroxylase activity in the serous submaxillary gland falls approximately 90% after noradrenergic denervation; but in the mucinproducing sublingual gland dopamine-β-hydroxylase activity is reduced by only 33%. Dopamine-β-hydroxylase immunofluorescence in the submaxillary gland is distributed with noradrenergic neurons and is eliminated by superior cervical ganglionectomy. In the sublingual gland dopamine-β-hydroxylase immunofluorescence is localized within mucinous acini and small ducts, and the disposition and intensity of staining materials is not affected by noradrenergic denervation for up to 30 days. DBH protein in the sublingual gland had little physiologic activity in vivo. Low levels of authentic dopamine-β-hydroxylase activity were detected in saliva. Thus, dopamine-β-hydroxylase protein is present in the sublingual gland in an extraneuronal location and appears to be a secretory product of the gland.     

AXOPLASMIC TRANSPORT OF AROMATIC l-AMINO ACID DECARBOXYLASE (EC 4.1.1.26) AND DOPAMTNE β-HYDROXYLASE (EC 1.14.2.I) IN RAT SCIATIC NERVE

The axoplasmic transport of aromatic l -amino acid decarboxylase and dopamine β-hydroxylase, two enzymes involved in the biosynthesis of catecholamines, was studied in rat sciatic nerve. The two enzymes exhibited markedly different axoplasmic flow characteristics, since dopamine β-hydroxylase activity accumulated on the proximal side of a ligation nearly three times as fast as aromatic l -amino acid decarboxylase activity. Distally dopamine β-hydroxylase activity remained essentially constant for 24 h, whereas aromatic l -amino acid decarboxylase activity fell precipitously. Evidence was obtained to rule out the possibility that differences in the rate of inactivation of the two enzymes could account for the different rates of accumulations observed. The conclusion, that aromatic L-amino acid decarboxylase and dopamine β-hydroxylase are transported in sympathetic nerve at different rates is discussed in relation to the biosynthesis of norepinephrine.     

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.     

BRAIN DOPAMINE-β-HYDROXYLASE: REGIONAL DISTRIBUTION AND EFFECTS OF LESIONS AND 6-HYDROXY-DOPAMINE ON ACTIVITY

Abstract— A modification of a specific and sensitive radioassay was used to measure dopamine-β-hydroxylase (DBH) (EC 1.14.2.1) in various regions of the rat CNS. Highest activity was found in the hypothalamus. Relative to activity in the hypothalamus (= 100 per cent), activity in brainstem was 80 per cent, in sensory motor cortex 55 per cent, in caudate nucleus 32 per cent, and in cervical spinal cord 30 per cent. Two to three weeks after a unilateral electrolytic lesion of the lateral hypothalamus, activity of DBH in the ipsilateral cerebral cortex fell to 17 per cent of control values without changes in activity ipsi- or contra-laterally in the brainstem. Thalamic lesions did not affect DBH activity. In cerebral cortex contralateral to the hypothalamic lesion, enzymic activity rose 30 per cent. After intracisternal administration of 6-hydroxy-dopamine (6-OH-DA), cortical DBH activity fell to 20 per cent of control values. Reserpine (3 mg/kg subcutaneously for 3 days) did not increase the activity of DBH in brain regions but did increase the activity of DBH in adrenal gland 200 per cent. Our results suggest that: (a) DBH is widely distributed in neurons in CNS with a regional pattern of activities that appears to parallel the Jevels of norepinephrine; (b) DBH activity in the cerebral cortex depends on the integrity of structures (e.g. medial forebrain bundle) in lateral hypothalamus; (c) DBH in brain areas lacking cell bodies of nore- pinephrine-neurons (e.g. cerebral cortex) is contained in norepinephrine-containing axon terminals and (d) the activity of DBH in brain is not increased by reserpine under conditions that provoke marked increase of DBH activity in the adrenal gland.     

SUBCELLULAR DISTRIBUTION OF PYRUVATE KINASE (EC 2.7.1.40) IN CEREBRAL CORTEX

—The subcellular distribution of pyruvate kinase (EC 2.7.1.40) in the cerebral cortex of the rat was studied. The enzyme, which had been previously reported in the cytoplasm, was found to be present in synaptosomal, microsomal and mitochondrial fractions as well. The activity of the enzyme in the synaptosomal fraction was localized predominantly in the synaptosomal membrane and was not dissociated by repeated washing or recentri-fuging in a sucrose gradient. Some kinetic parameters of the membrane-associated pyruvate kinase were measured.     

INCREASE IN THE RELATIVE RATE OF SYNTHESIS OF DOPAMINE-β-HYDROXYLASE IN IN THE NUCLEUS LOCUS COERULEUS ELICITED BY RESERPINE

Abstract— Three days following a single injection of reserpine (10 mg/kg, i.p.) the activity and amount of dopamine-β-hydroxylase (DBH) are increased nearly 2-fold in the noradrenergic cell bodies of the nucleus locus coeruleus of rat. To determine if this increased accumulation of DBH is due to an increased rate of enzyme synthesis, [³H]amino acids were infused into the IVth ventricle of reserpine-and saline-injected rats. This method was 35 times more effective than intracisternal infusion and 600 times more effective than intravenous infusion. DBH protein was isolated from the locus coeruleus by immunoprecipitation and SDS-electrophoresis. These steps proved crucial for the complete isolation of DBH from other labelled proteins. Indeed, only 10–15% of the immunoprecipitate was finally identified as labelled DBH protein. The rate of incorporation of [³H]leucine into DBH protein of locus coeruleus was increased to 181%, of control following reserpine, whereas that into TCA-precipitable protein was unchanged. A similar result was obtained using [³H]lysine. In contrast, the apparent half-life of the enzyme did not change following reserpine. The relative rate of synthesis of DBH ([³H]DBH/³H-total protein), denoting selectivity of response, was increased in the locus coeruleus of reserpine-treated rats to 154% of control ( P < 0.01). These findings indicate that increased synthesis accounts for the observed increase in DBH protein in the locus coeruleus following reserpine administration.     

BLOCKADE OF FAST AXONAL TRANSPORT BY DIPHTHERITIC DEMYELINATION IN THE CHICKEN SCIATIC NERVE

Chicken sciatic nerves undergo demyelination following intraneural injection of diphtheria toxin due to a lesion at the site of injection. Paresis occurs after 1 week and lasts for approx 3 weeks; at the height of the lesion we injected [¹⁴C]Ieucine into the ventral horn cells of the spinal cord and followed the axonal transport of fast flowing labelled proteins down the sciatic nerve fibres making measurements of flow rates at two different times. The results showed the fast flowing labelled proteins were blocked at the demyelination site. We measured total protein in the nerves and examined them histologically to confirm the lesion. Further studies are in progress on the post synaptic muscle cells and the impaired nerves.     

ON THE DEVELOPMENT OF SYMPATHETIC NERVE TRUNK VESICLES DURING AXONAL TRANSPORT: DENSITY GRADIENT ANALYSIS OF DOPAMINE β-HYDROXYLASE IN BOVINE SPLENIC NERVE

—Dopamine β-hydroxylase was used as a marker enzyme for sympathetic nerve vesicles which were studied by density gradient technique in bovine splenic nerves. The enzyme analyses were complicated by the occurrence of inhibitors which had to be carefully neutralized with copper. The inhibitor was mainly found in the soluble fraction and no evidence for the occurrence of endogenous inhibitors in the nerve vesicles was obtained. A great variation in density of the dopamine β-hydroxylase containing particles was observed. This was probably mainly due to the variation in vesicle maturation since dopamine β-hydroxylase was distributed more towards the lighter gradient fractions in the proximal nerve segment preparations compared with intrasplenic nerve segment preparations. Noradrenaline/protein and noradrenaline/dopaminc β-hydroxylase ratios were found to be increased about 1·7-fold in the vesicle fraction isolated from the proximal nerve segments to those from the intrasplenic segments. A further increase of the noradrenaline/dopamine β-hydroxylase ratio was observed in a fraction with the same density isolated from the spleen. On the basis of these findings the noradrenaline/protein ratio was calculated to be about 500-600 nmol/mg in the nerve terminal vesicles.     

STOP-FLOW ANALYSIS OF THE AXONAL TRANSPORT OF DOPA DECARBOXYLASE (EC 4.1.1.26) IN RABBIT SCIATIC NERVES

The axonal transport of DOPA-decarboxylase (EC 4.1.1.26) was investigated in rabbit sciatic nerves by means of in vitro stop-flow techniques. Enzyme activity accumulated just proximal to a region that was locally cooled to 5°C in nerves that were elsewhere incubated at 37°C. The accumulation of enzyme activity was linear with time and corresponded to an average orthograde transport velocity of 11 mm/day. Retrograde transport was not detected. When nerves that had been locally cooled for 3 h were rewarmed, the accumulated enzyme activity moved distally along them as a wave with a narrow range of velocities. The front of this wave traveled at a rate of about 150mm/day, and the mean velocity of the wave was about 120 mm/day. These values are much lower than those previously obtained for tyrosine hydroxylase (EC 1.14.16.2), dopamine-β-hydroxylase (EC 1.14.2.1) and norepinephrine in similarly designed experiments. Thus DOPA-decarboxylase appeared to be transported at intermediate velocities, and, since the mean velocity of the moving fraction was about 11 times the average velocity, it is ljkely that only 9% of the enzyme was undergoing transport at any given moment.     

INCREASED SYNTHESIS OF DOPAMINE β-HYDROXYLASE IN CULTURED RAT ADRENAL EDULLAE AFTER IN VIVO ADMINISTRATION OF RESERPINE

Abstract— The present experiments were designed to provide direct evidence that the increase in dopamine β-hydroxylase (DBH) activity after treatment with reserpme results from an augmented synthesis of new enzyme protein. After in vivo experiments had shown that DBH could not be labelled to a sufficient extent even after administration of high doses (8.3 mCi/kg) of [³H]leucine we took advantage of earlier observations that neuronally-mediated enzyme induction initiated in vivo progresses in organ cultures of adrenal medullae in a similar manner as in vivo. With that system it was possible to achieve a sufficient labelling of DBH molecules and to provide evidence that the increase in DBH activity produced by reserpine was really due to a specific increase in DBH synthesis. The fact that the half-life of DBH was identical in organ cultures of controls and reserpine-treated animals eliminated the possibility that the increased labelling of DBH after reserpine results from a slower rate of degradation.
Both the experiments in vivo and in organ culture showed very clearly that the immunoprecipitation of labelled molecules has to be followed by additional purification by gel electrophoresis since in spite of the use of monospecific antibodies and careful washing of the immunoprecipitates contaminating coprecipitated labelled molecules accounted for up to 96% of the total precipitated radioactivity in whole adrenals ( in vivo experiments) and up to 80% in adrenal medulla (organ culture experiments). The coprecipitation of contaminants most probably results from the addition of carrier proteins, used in order to make the precipitation of the small amounts of labelled proteins visible.     

THE SUBCELLULAR DISTRIBUTION OF β-PHENYLETHYLAMINE, p-TYRAMINE AND TRYPTAMINE IN RAT BRAIN

—The quantitative subcellular distribution of β-phenylethylamine, p-tyramine and tryptamine in rat brain was investigated using the mass spectrometric integrated ion current technique. More of the total cellular tryptamine was found to be associated with paniculate fractions than was the case for phenyiethylamine and p-tyramine but a significant amount of this tryptamine was found to be labile. Analysis of the particulate fractions indicated that each of the amines was localized predominantly in the crude P₂ pellet and that the bulk of this was associated with the synaptosomal (P₂B) fraction. Inhibition of monoamine oxidase systems with pargyline caused an increase in the level of all three amines in all fractions, but the increase was greater in the supernatant than in the combined particulate fractions. This treatment produced changes in the distribution of β-phenylethylamine and p-tyramine between the various particulate subcellular fractions but did not markedly alter the distribution of tryptamine between the same fractions.     

SUBCELLULAR DISTRIBUTION AND TRANSPORT OF ACETYLCHOLINE IN CAT VENTRAL ROOTS AND SCIATIC NERVE

Abstract— The axonal transport of radioactive ACh, which was labelled by injecting radioactive choline into the ventral horn of the cat, was studied. Radioactivity analysed as ACh was transported in the nerves at a rate of 20–25 cm/24 h from the place of injection. Morphological and biochemical analysis after density gradient centrifugation revealed that endogenous ACh was principally distributed in three fractions-(a) a soluble fraction (provided homogenization was carried out in the presence of eserine), (b) a vesicular fraction (diameter 600–1600 A) in the 0.4M-sucrose layer of the density gradient, and (c) a fraction containing very small structures (size 90–100 A) in the 0.8–1.0 M-sucrose interphase. The radioactive ACh, however, was exclusively found in the soluble fraction (supernatant) after density gradient centrifugation. Analysis of ACh metabolites showed that radioactive ACh may have been formed locally in the nerves after transport of its precursor. Thus the morphologic and metabolic results do not support the hypothesis that ACh is transported in vesicles.