Elevated plasma dopamine beta hydroxylase activity in rats with neurogenic hypertension.

Increased plasma dopamine beta hydroxylase, DBH, activity has been cited as evidence of increased sympathetic function in essential hypertension. Here-to-fore, experimental hypertension in animals has been associated with normal plasma DBH activity. This study shows that rats with neurogenic hypertension, induced by sinoaortic denervation, SAD, have elevated DBH activity; the mean increase in plasma DBH measured 3 days to 11 weeks after operation was 74% higher in the SAD group than in the sham-operated, control group. DBH activity showed a positive correlation with arterial pressure. Mesentery DBH activity was inversely related to plasma enzyme activity in SAD rats, indicating sympathetic nerve terminals in mesentery are a source of plasma DBH. We conclude that plasma DBH activity is an index of increased sympathetic function since it is consistently elevated in rats with neurogenic hypertension resulting from sustained central activation of the sympathetic nervous system.     

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.     

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.     

Orthograde, Retrograde, and Turnaround Axonal Transport of Dopamine-β-Hydroxylase: Response to Axonal Injury

Reversal of the direction (turnaround) of orthograde axonal transport of dopamine-beta-hydroxylase (DBH) activity was studied at a ligature placed on rat sciatic nerve. DBH was allowed to accumulate at a ligature in vivo for selected intervals, at which time a second ligature was placed proximal to the first and turnaround transport measured just distal to the second tie after incubation in vivo or in vitro. Orthograde accumulation of DBH activity proximal to a ligature peaked at 2 days, and then rapidly decreased as a result of turnaround transport and injury-induced reduction of orthograde transport. Destruction of postganglionic sympathetic axon terminals in vivo with 6 hydroxydopamine resulted in a decrease in orthograde transport similar to that seen after axotomy and turnaround at or proximal to the site of chemical injury. Turnaround transport of DBH in vitro was blocked by incubation in the cold and in the presence of NaCN and vinblastine. Orthograde transport of DBH appeared to reverse direction within a few millimeters of a ligature.     

The effects of electrical inactivity and denervation on the distribution of acetylcholine receptors in developing rat muscle

Explants of thoracic body wall from rat embryos, including intercostal muscles, ribs, and the adjacent segments of spinal cord, were maintained in organ culture. Nerve-muscle differentiation proceeded in culture with a pattern and time course similar to that of the same synapses developing in utero. To understand further the factors that regulate acetylcholine sensitivity in developing rat myotubes, we studied the effects of electrical inactivity and denervation on the distribution of acetylcholine receptors. When muscle and spinal cord were explanted at 15 days of gestation, prior to the appearance of junctional receptor clusters, intact nerve terminals were required to initiate receptor aggregation at the site of nerve-muscle junction. Electrical activity was not necessary for induction of these primary junctional clusters. Inactivity resulted, however, in the appearance of secondary multiple receptor clusters at random sites along the fibers. In the presence of tetrodotoxin, the electrically inactive nerve terminals sprouted; this was accompanied by the enlargement of the junctional receptor clusters, at the end plate, but there was no correlation between nerve sprouting and the location of extrajunctional receptor aggregates. Later in development, at a time when the junctional receptors are metabolically more stable, terminal sprouting failed to induce the increase in size of junctional receptor aggregates.     

Changes in Sympathetic Nerve Terminals in the Heart of Cold-Exposed Rats

Abstract: Changes in sympathetic nerve terminals of the heart after varying periods of exposure of rats to 4°C were investigated. Two indices were used for changes in the number of noradrenaline storage vesicles, i.e., vesicular dopamine β-hydroxylase (DBH) activity and noradrenaline storage capacity. The latter was obtained after uptake of [³H]noradrenaline; endogenous content, uptake of exogenous noradrenaline, and degree of saturation of the vesicles were calculated using the specific activity of the [³H]noradrenaline. As a measure of tyrosine hydroxylase activity, whole ventricular noradrenaline, dopamine, and dihydroxyphenylacetic acid content were used. After 4 h of cold exposure there was an increase in vesicular endogenous noradrenaline content, uptake, storage capacity, and DBH activity as well as a large increase in whole ventricular dopamine. After 6 h in the cold, vesicular endogenous noradrenaline content, storage capacity, and DBH activity were decreased. The results suggest that during cold exposure there is an initial increase followed by a decrease in the number of functional vesicles in the nerve terminal, which could explain the fluctuations in the rate of noradrenaline release.     

TYROSINE HYDROXYLASE IN RAT BRAIN: DEVELOPMENTAL CHARACTERISTICS

Abstract— The development of tyrosine hydroxylase (tyrosine 3-hydroxylase, EC 1.14.3.a) activity has been examined in whole rat brain and in various regions and subcellular fractions thereof. The specific activity of tyrosine hydroxylase increased almost 15-fold from 15 days of gestation to adulthood. With maturation, those regions of the brain that contain only terminals of the catecholaminergic neurons showed the greatest increases in enzyme activity. There was a shift in the subcellular distribution of tyrosine hydroxylase from the soluble fraction in the fetal brain to the synaptosomal fraction in the adult brain. Tyrosine hydroxylase, dopamine hydroxylase (EC 1.14.2.1) and the specific uptake mechanism for norepinephrine appear to develop in a coordinated fashion.     

Blood dopamine beta-hydroxylase activity and noradrenaline levels in the developing white rat.

Dopamine beta-hydroxylase (DBH) (3,4-dihydroxyphenylethylamine, ascorbate:oxygen oxidoreductase (beta-hydroxylating) (EC 1.14.17.1) activity in serum of blood obtained by decapitation of white rats at 19, 20, and 21 days in utero, immediately after birth, and postnatally to 70 days, was measured. Noradrenaline (NA) and DBH in plasma from undisturbed, cannulated, postweaning rats were also assayed. During the last few days in utero and the first 2 postnatal days serum DBH activity tripled and then remained elevated during the suckling period. Upon weaning, serum DBH activity declined at first precipitously and then more slowly, until the adult level was reached around 70 days of age. This postweaning decrease in DBH activity was also observed with the cannulated animals. In contrast, plasma NA levels remained low and constant throughout the postweaning period. In suckling rats treated with 6-hydroxydopamine from 2 to 12 days of age, serum DBH activity decreased to less than half its initial value by day 8. It is suggested that the observed changes in serum DBH activity in fetal and postnatal rats reflect ontogenetic changes in sympathetic nerve terminals and that they are probably not correlated with release of NA.     

Elevation of Serum Dopamine-β-hydroxylase Activity with Forced Immobilization

THE formation of the neurotransmitter noradrenaline from 3,4-dihydroxyphenylethylamine (dopamine) is catalysed by the enzyme dopamine-β-hydroxylase (DBH)¹. This enzyme is associated both with the catecholamine-containing chromaffin granules in the adrenal medulla^2,3 and with the vesicular structures in sympathetic nerve terminals which contain catecholamines⁴. Furthermore, DBH activity is released with catecholamines into the perfusate after stimulation of either the isolated perfused adrenal gland⁵ or the isolated perfused spleen^6–8. DBH activity has been reported in the serum of both man and the rat^9,10. This activity is similar to adrenal and sympathetic nerve DBH activity with regard to cofactor requirements, oxygen requirement and kinetic characteristics^9,10. It has been suggested that serum DBH activity might be present as a result of release of enzyme with catecholamines from the adrenal glands and sympathetic nerves. If this is the case, serum DBH activity might be a useful and convenient index of sympathetic-adrenal activity. The work described here was undertaken to investigate both the source of the serum DBH and the effect on this activity of forced immobilization, a procedure which has been used as a model of stress and which has been shown to release catecholamines from the adrenal gland and increase catecholamine excretion¹¹.     

ACTIVITY OF ALDEHYDE DEHYDROGENASE, ALDEHYDE REDUCTASE, AND ACETYLCHOLINE ESTERASE IN STRITATUM OF RATS BEARING ELECTROLYTIC LESIONS OF THE MEDIAL FOREBRAIN BUNDLE

A number of enzymes have been measured in the striatum of rats in which the dopamine-containing nerve terminals had been unilaterally destroyed by means of unipolar electrolytic lesions of the medial fore-brain bundle. Fourteen and 28 days after such lesions the tyrosine hydroxylase activity of the striatum was reduced to immeasurably low values, but neither aldehyde dehydrogenase, aldehyde reductase, nor acetylcholine esterase was affected when compared with the striatum from the intact side of the same rat or with those from control rats. These results indicate that in the rat the 3 enzymes are not localized with tyrosine hydroxylase, in the dopaminergic nerve terminals of the striatum. This conclusion is supported by a study of the subcellular localization of aldehyde dehydrogenase in rat brain. This enzyme is distributed between the cytosol and the particulate fraction of brain homogenates separated by centrifugal techniques. with no exceptionally high concentration of the enzyme in the synaptosomal fraction. Because neither of the enzymes of post-deaminative catabolism of dopamine is concentrated in the dopaminergic nerve terminals of the striatum of the rat, it is proposed that in this species the amine is not necessarily taken up by the nerve terminals prior to catabolism.     

LOCAL CHANGES IN SUBCELLULAR DISTRIBUTION OF DOPAMINE-β-HYDROXYLASE (EC 1.14.2.1) AFTER BLOCKADE OF AXONAL TRANSPORT

Abstract— The distribution of DBH activity between soluble and sedimentable fractions of hypotonic homogenates was examined in rat sympathetic ganglia and nerves after interruption of axonal transport. Local application of colchicine to superior cervical ganglia caused an increase mainly in particulate DBH activity, which was presumably bound to membranes. Likewise, in sciatic nerves, particulate DBH activity accumulated on both sides of a ligature and disappeared from a region well below a ligature much faster than did soluble activity. On the other hand, 18 h after simultaneous application of two ligatures to the nerve, neither total DBH activity nor subcellular distribution of this activity changed in the isolated nerve region. More detailed analysis showed that ligation affected the distribution of DBH activity within a fraction that sedimented at 140,000 g after homogenization of nerves in isotonic sucrose. Just above a ligature, osmotically releasable DBH activity was a smaller proportion of the sedimentable activity than in control nerves. However, as compared to controls, osmotically releasable DBH activity was a larger proportion of the activity in the sedimentable fraction from a region well below a ligature. A model was developed which accounts for some of these results by postulating that DBH is associated with different compartments in sciatic nerve which have different rates of transport and different proportions of soluble and bound enzyme.     

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.     

Ecto-5''-Nucleotidase Is Associated with Cholinergic Nerve Terminals in the Hippocampus but Not in the Cerebral Cortex of the Rat

The extracellular catabolism of exogenously added AMP was studied in immunopurified cholinergic nerve terminals and in slices of the hippocampus and cerebral cortex of the rat. AMP (10 microM) was catabolized into adenosine and inosine in hippocampal cholinergic nerve terminals and in hippocampal slices, as well as in cortical slices. IMP formation from extracellular AMP was not detected. alpha, beta-Methylene ADP (100 microM) inhibited almost completely the extracellular catabolism of AMP in these preparations. The relative rate of catabolism of AMP was greater in hippocampal slices than in cortical slices. AMP was virtually not catabolized when added to immunopurified cortical cholinergic nerve terminals, although ATP could be catabolized extracellularly under identical conditions. The comparison of the relative rates of catabolism of exogenously added AMP, calculated from the amount of AMP catabolized after 5 min, in hippocampal cholinergic nerve terminals and in hippocampal slices revealed a nearly 50-fold enrichment in the specific activity of ecto-5'-nucleotidase upon immunopurification of the cholinergic nerve terminals from the hippocampus. The results suggest that there is a regional variation in the subcellular distribution of ecto-5'-nucleotidase activity in the rat brain, the ecto-5'-nucleotidase in the hippocampus being closely associated with the cholinergic nerve terminals, whereas in the cerebral cortex ecto-5'-nucleotidase activity seems to be located preferentially outside the cholinergic nerve terminals.     

A comparative study of tyrosine 3-monooxygenase from rat adrenal and brainstem

Spleen cells from a CBF1 (BALB/c X C57BL/6) mouse immunized with rat tyrosine 3-monooxygenase were fused with NS-1 mouse myeloma cells. From 188 hybrid cells, 2 stable clones secreting anti-tyrosine 3-monooxygenase antibody were obtained. Antibody from one clone was coupled to CNBr-activated Sepharose 4B and the monoclonal antibody-Sepharose was shown to be very useful for isolating rat tyrosine 3-monooxygenase from crude preparations. Analyses by monoclonal antibody chromatography followed by SDS-polyacrylamide gel electrophoresis and by gel filtration revealed that tyrosine 3-monooxygenases from nerve cell bodies, nerve terminals, and adrenal medullae were indistinguishable with respect to their molecular structures. However, there were serious differences in the catalytic properties between the enzymes from the brain tissues and adrenal medullae, although there appeared to be no significant difference between the enzymes from nerve cell bodies and nerve terminals. The possibility that the activity of the enzyme may be strongly suppressed especially at the physiological pH in brain tissues is also discussed.     

Serotonin Synthesis Increased in Terminals Four Days after Reserpine Treatment: An Autoradiographic Study in Rat Brain

The rate of 5-HT synthesis was determined in discrete rat brain regions 4 days after a single dose of reserpine (10 mg/kg) or reserpine carrier (controls), using an autoradiographic method with labelled -methyl-L-tryptophan as a tracer. The results show that the rate of 5-HT synthesis was unchanged in the dorsal and median raphe, significantly decreased in the raphe magnus, and significantly increased in areas rich in serotonergic nerve terminals (i.e., hypothalamus, hippocampus, median geniculate body, parietal and visual cortices). An increase in tryptophan hydroxylase activity could account for the increase in the rate of serotonin synthesis seen in some regions. Since the 5-HT synthesis rate showed regional variability there seems to be a need for regional studies of the effect of drugs on the 5-HT synthesis. In addition, the 5-HT synthesis rate was not significantly different from that in controls in many of the brain regions.     

THE POSTNATAL DEVELOPMENT OF CATECHOL-O-METHYL TRANSFERASE IN THE RAT BRAIN

Abstract— The postnatal development of three enzymes in the rat forebrain was studied. When expressed per tissue weight the catechol- O -methyl transferase (COMT) increased 2-fold from birth to adult age, the lactate dehydrogenase (LDH) 4-fold and the monoamine oxidase (MAO) 12-fold. Expressed per mg protein the increase in the enzyme activities in the subcellular fractions which contained the main part of the different enzymes was still 2–4-fold for COMT and LDH while for MAO it was 4-fold.
There was a relative increase in the COMT activity in the P₂ fraction (synaptosomes and mitochondria). This increase was identical with a corresponding increase in LDH activity and protein and was probably due to growth of nerve terminals. The COMT in the cytoplasm of the synaptosomes showed the same increase relative to the proteins as did the 'free' cytoplasmic enzyme.
The conclusion is drawn that the enzymes in the rat brain show a certain degree of development during brain growth. An additional increase of some enzymes is due to the development of specialized structures such as mitochondria and nerve terminals with synapses. COMT is not related to any such specialized structure.     

DEVELOPMENT OF THE UPTAKE AND STORAGE OF L-[3H]NOREPINEPHRINE IN THE RAT BRAIN

The uptake and storage of L-[³H]norepinephrine at various stages of development was examined in homogenates of rat brain. For the adult animal, active uptake accounted for 80 per cent of the total uptake. At 14 days of gestation, no active uptake was demonstrable At 18 days of gestation, saturable uptake of L-[³H]norepinephrine with a K_m of 3 × 10 ^?7m was first demonstrable; the K_m value did not vary during subsequent development. The V_max. of uptake increased five-fold between 18 days of gestation and 28 days postnatally, at which stage it was the same as the adult value. The development of saturable uptake paralleled but preceded the increase in endogenous norepinephrine. When homogenates were incubated with l -[³H]norepinephrine and subjected to centrifugation on linear sucrose gradients, there was a peak of tritium in the synaptosomal fractions; the magnitude of the peak increased with maturation of the brain. The increase in the peak of tritium paralleled the increase in particulate LDH activity and was distinct from the peak of MAO activity. Desipramine, a compound that blocks the initial uptake of norepinephrine, first exhibited inhibition of uptake at 19 days of gestation; the degree of inhibition did not vary during subsequent development. In contrast, reserpine, a compound which inhibits the intra-neuronal storage of norepinephrine, exhibited a progressive increase of inhibition with maturation of the brain at and subsequent to 19 days of gestation.     

APPEARANCE OF GAMMA AMINOBUTYRATE TRANSAMINASE ACTIVITY IN DEVELOPING RAT BRAIN

—The distribution, localization and changes in intensity of γ-aminobutyrate transaminase (4-aminobutyrate: 2-oxoglutarate aminotransferase, EC 2.6.1.19) in rat brain have been studied during the first 20 days of postnatal life by a histochemical technique. Enzyme activity at birth was seen only in Purkinje cells of the cerebellar cortex where it increased markedly during the first 20 days. A rapid increase in enzyme activity was seen in regions of the hind-brain after 3 days but a slower increase was apparent in areas of the fore- and mid-brain. The results indicated that by 10 days post-partum nerve cell GABA-T activity had developed in the majority of brain areas studied, while glial cell GABA-T activity developed between 10 and 15 days post-partum. Evidence is presented which indicates that there is a discontinuous function of GABA-T in the developing brain.     

Subcellular and Perisynaptic Distribution of Rat Brain Aldehyde Dehydrogenase Activity

Abstract: The nuclear mitochondrial and synaptosomal fractions of rat brain were each found to contain some 25–30% of the total aldehyde dehydrogenase activity. The cytoplasmic fraction had a very low total aldehyde dehydrogenase activity. There were differences in the distribution of the activity when different aldehydes were used as substrates, suggesting the presence of isoenzymes in the various subcellular compartments. When rats were treated intra-cisternally with 6-hydroxydopamine there was no change in brain aldehyde dehydrogenase activity, although the noradrenaline content and the activities of tyrosine hydroxylase and dopamine-β-hydroxylase were markedly decreased. Treatment with 6-hydroxydopamine also had no significant effect on the aldehyde dehydrogenase activity in retinal homogenates. The results suggest that the aldehyde dehydrogenase activity in rat brain is predominantly outside the catecholaminergic nerve terminals.     

CHARACTERIZATION OF THE BASIS FOR DIFFERENCES IN SERUM DBH ACTIVITY IN IMMATURE AND ADULT RATS BY USE OF HOMOLOGOUS ANTIBODY

Abstract– Rat serum dopamine-β-hydroxylase (DBH) activity decreased 5-7-fold between 15 and 60 days of age. Immunoprecipitation performed with homologous antibody (guinea-pig anti-rat adrenal DBH) showed that during this time period the quantity of antibody necessary to precipitate 50% of the enzymatic activity (AD₅₀) decreased 5-fold from 0.25 to 0.05 μl/ml. The biochemical properties of rat serum DBH at 15 and 60 days of age were compared to test the hypothesis that there might be different biochemical forms of the enzyme in the blood of immature and adult rats. Thermal stability, apparent K_m for tyramine, electrophoretic mobility, pH optima and elution profile on gel filtratioh chromatography were all found to be similar for rat serum DBH at both ages. On the basis of homospecific activity and multiple similarities in biochemical characteristics, it appears that differences in serum activity at the two ages reflect differences in the steady-state levels of enzyme. To determine the turnover of serum DBH in the two age groups, the recovery of enzyme activity was monitored after acute clearance of the circulating pool of DBH by treatment with the homologous antiserum. Immunotitration of DBH activity in vivo indicated that the total pool of serum enzyme was 4-fold greater in the mature rat than in 4-day-olds. After treatment of adult rats with 2μl of homologous antiserum, serum DBH activity was reduced by 85% with a half-life of recovery of 3.0 ± 0.6 days; the estimated fractional rate of degradation was 0.23 ± 0.06 day^?1 and the rate of entrance was 2.3 ± 0.2 units/ml/day. After treatment of 4-day-old rats with 1 μl of homologous antiserum, serum DBH activity was reduced by 95% with a half-life of recovery of 3.3 ± 0.5 days: the estimated average fractional rate of degradation was 0.22 ± 0.06 day^?1 and the average rate of entrance was 10.7 ± 1.6 units/ml/day. Thus, the several-fold difference in steady-state levels of serum DBH in rat pups as compared to adult rats appears to be due to greatly increased rates of entrance of the enzyme in the immature rats.