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.     

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.     

EFFECT OF DIBUTYRYL-CYCLIC AMP and DEXAMETHASONE ON NORADRENALINE SYNTHESIS IN ISOLATED SUPERIOR CERVICAL GANGLIA

Abstract— Incubation with dibutyryl-cyclic AMP increased levels of both noradrenaline and dopamine- β -hydroxylase in isolated rat superior cervical ganglia. Dexamethasone also increased the dopamine- β -hydroxylase content but did not affect noradrenaline levels. Cycloheximide blocked the effect of dibutyryl-cyclic AMP on ganglion dopamine- β -hydroxylase but did not affect the rise in noradrenaline content.
Dibutyryl-cyclic AMP increased the synthesis of noradrenaline from [¹⁴C]tyrosine but not from [³H]DOPA.
The results are discussed in terms of a possible role for cyclic AMP in the control of noradrenaline synthesis in sympathetic ganglia.     

Effects of fasting and food restriction on sympathetic activity in brown adipose tissue in mice

Summary The activity of the sympathetic nervous system in mice that were either fed ad libitum, food restricted or fasted was estimated by measuring the accumulation of dopamine following the inhibition of dopamine -hydroxylase activity. Mice in each group were injected with the dopamine -hydroxylase inhibitor 1-cyclohexyl-2-mercaptoimidazole and were exposed to either 30°C (warm) or 4°C (cold). Mice were killed 1 h after the injection. Both heart and brown adipose tissue were then quickly removed and homogenized in ice-cold perchloric acid. Dopamine and noradrenaline were determined using high performance liquid chromatography. Regardless of whether mice were warm or cold exposed, both content and concentration of brown adipose tissue and dopamine were predictably higher in 1-cyclohexyl-2-mercaptoimidazole-injected mice than in non-injected animals. In mice fed ad libitum, post-injection content and concentration of dopamine in both brown adipose tissue and heart were higher in cold-exposed mice than in warm-exposed animals. In food-restricted and fasted mice, post-injection concentrations of dopamine in brown adipose tissue were higher in cold-exposed mice than in warm-exposed animals. In food-restricted and fasted mice there was no difference between warm- and cold-exposed animals with respect to post-injection contents and concentrations of dopamine in heart tissue. In fasted mice there was no difference between warm- and cold-exposed animals in post-injection content of dopamine in brown adipose tissue. This study provides further evidence that fasting, in contrast to food restriction, may blunt the tissue sympathetic nervous system response in brown adipose tissue of cold-exposed mice.Abbreviations BAT brown adipose tissue - CHMI 1-cyclohexyl-2-mercaptoimidazole - DA dopamine - DHBA dihydroxybenzylamine - EDTA ethylenediaminetetra-acetic acid - HPLC high performance liquid chromatography - NA noradrenaline - PCA perchloric acid - SNS sympathetic nervous system     

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.     

DOPAMINERGIC PROPERTIES OF A SOMATIC CELL HYBRID LINE OF MOUSE NEUROBLASTOMA X SYMPATHETIC GANGLION CELLS

Abstract— Studies were made on the regulation of dopamine metabolism in a cell line derived by hydridization of a non-tyrosine-hydroxylase-containing line of murine neuroblastoma cells with a neur-onally-enriched population of murine embryonic sympathetic ganglion cells. Hybrid subclones with tyrosine hydroxylase activity were selected by exposure to tyrosine-free medium. The cells also exhibited DOPA decarboxylase activity and the subclone (named T28) with the highest specific activities of both enzymes was further characterized. The hybrid T28 line did not contain dopamine-β-hydroxylase activity. The specific activity of tyrosine hydroxylase as well as of DOPA decarboxylase increased significantly in T28 cultures when the cells entered the stationary phase of growth. Both of these enzymes were also induced after several days of exposure to 1 m m -dibutyryl cyclic AMP in culture medium containing either 5% or 0.8% serum. However, maintenance in medium containing 0.8% serum alone, which inhibited cell multiplication, did not induce either enzyme. The dopamine content of T28 cells was also regulated as a function of cell density. High density (stationary phase) cultures of T28 cells contained about 300 pmol dopamine per mg protein and at least half of this endogenous amine appeared to he stored in vesicles or granules (as judged by depletion with reserpine or α-methyl- m -tyramine). The T28 and other neuronal hybrid lines appear to be useful model systems for neuro-chemical studies.     

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.     

On the soluble phase of adrenergic nerve vesicles: correlation of matrix density and biochemical composition.

Highly purified sympathetic nerve vesicles isolated from bovine splenic nerves were treated by hypo-osmotic shocks, freeze-thawing or incubation in the absence or presence of ATP and MgCl. The vesicle preparations were then studied morphologically by electron microscopy and their content of noradrenaline (NA), and soluble proteins analyzed biochemically with special regard to dopamine beta-hydroxylase (DBH). Hypo-osmotic shocks released about 25 per cent of the NA and protein content and about 8 per cent of the DBH activity. This treatment induced swelling of the vesicles but their membranes remained unruptured and they still contained dense cores. Freeze-thawing released about 35 per cent of the NA, 25 per cent of the proteins and 11 per cent of the DBH. After the latter treatment some matrix material still remained in most vesicles but many were less stainable than the intact vesicles in cold control preparations. During incubation at 30 degrees C in an isotonic sucrose-phosphate medium for 30 min the vesicles released most of their NA and soluble DBH activity as well as much of their matrix density. After incubation at 37 degrees C for 30 min most vesicles appeared translucent. After incubation at 30 degrees C for 30 min in the presence of ATP and MgCl the vesicles lost most of their original NA content but retained their DBH activity and most of their matrix density. The results indicate that there is not always a correlation between NA content and retention of matrix density which suggests that DBH might be a component of a macro-molecular complex responsible for the staining reaction taking place in the maxtrix of NA depleted vesicles. This hypothesis is further supported by the finding of striking similarities between DBH isolated from chromaffin granules and the granular and fibrillar material surrounding the nerve vesicles after depletion.     

Dopamine β-Hydroxylase and Other Glycoproteins from the Soluble Content and the Membranes of Adrenal Chromaffin Granules: Isolation and Carbohydrate Analysis

Abstract: Chromogranin A and two other proteins (A₁ and A₂) of the soluble proteins of bovine chromaffin granules were isolated by extraction from polyacrylamide gels after electrophoresis. The carbohydrate content of these proteins was 5%, with galactose, N -acetylgalactosamine, and sialic acid as the main sugars. Membranes of chromaffin granules were solubilized with sodium dodecyl sulphate (SDS) and three glycoproteins were isolated by sequential affinity chromatography on Concanavalin A (Con A) and wheat germ lectin (WGL) Sepharose columns. Two glycoproteins, designated GP II and III, were found to have a high carbohydrate content of about 30%. Mannose, galactose, N -acetylgalactosamine, and sialic acid were the main sugars. In addition membrane-bound dopamine β-hydroxylase was isolated by this procedure. No significant differences between the carbohydrate composition of the membrane-bound and the soluble enzyme were revealed. It was shown that all four subunits of dopamine β-hydroxylase possess carbohydrate chains with an affinity for Con A. The isolation methods established in this study will be useful for immunological studies on these glycoproteins.     

Noradrenaline Storing Vesicles in Sympathetic Neurons and Their Putative Role in Neurotransmitter Release: An Historical Overview of Controversial Issues

More than 25 years have passed since the original demonstration that proteins such as chromogranin A and dopamine--hydroxylase, which are co-stored together with noradrenaline in large dense cored vesicles in adrenergic nerves, are released by exocytosis. Despite much evidence in favour, it was for a long time thought that large dense cored vesicles were not eminently involved in the release of noradrenaline. The present review attempts to demonstrate, making use of evidence from different approaches, that the release of noradrenaline from sympathetic neurons occurs ultimately from large dense cored vesicles. A model of the secretory cycle is proposed.     

Catecholamines Are Present in a Synaptic-Like Microvesicle-Enriched Fraction from Bovine Adrenal Medulla

Abstract: "Synaptic-like microvesicles" are present in all neuroendocrine cells and cell lines. Despite their resemblance to small synaptic vesicles of the CNS. a thorough biochemical characterization is lacking. Moreover, the subcellular distribution of synaptophysin, the most abundant integral membrane protein of small synaptic vesicles, in adrenal medulla is still controversial. Using gradient centrifugation. we were able to compare the distribution of several markers for small synaptic vesicles and chromaffin granules. Synaptophysin was found at a high density (1.16 g/ml), purifying away from dopamine β-hydroxylase and cytochrome b₅₆₁. Both noradrenaline and adrenaline showed a parallel distribution with synaptophysin, suggesting their presence in synaptic-like microvesicles. Experiments in the presence of tetrabenazine did not influence the catecholamine content. Additionally, tetrabenazine binding showed a consistent shoulder in the region of synaptophysin. [³H]-Noradrenaline uptake was blocked by tetrabenazine, but not by desipramine. Also chromogranin A parallels the distribution of synaptophysin: however, a localization in the Golgi cannot be ruled out. Synaptophysin was shown to undergo very fast phosphorylation, together with another triplet protein of ∼ 18 kDa. In contrast, the latter showed a rather bimodal distribution coinciding with synaptophysin and dopamine β-hydroxylase. Immunoelectron microscopy of synaptic-like microvesicle fractions showed an intense labeling for synaptophysin on 60-90-nm organelles. Whereas abundant gold labeling for cytochrome b₅₆₁ was found over the entire surface of chromaffin granules, synaptophysin labeling was encountered mostly on vesicles adsorbed to granules. We conclude that catecholamines might be stored in synaptic-like microvesicles of the chromaffin cell.     

Uptake of [3H]Dopamine into Dopaminergic and Noradrenergic Neurones of the Isolated Neurointermediate Lobe of the Rat Hypophysis. Effects of Desipramine and Nomifensine

Abstract: The isolated neurointermediate lobe (NIL) of the rat hypophysis accumulates [³H]dopamine from the incubation medium. Column chromatographic analysis showed that 92% of the tissue radioactivity was contained in the catecholamine fraction. [³H]Dopamine represented 70% and [³H]noradrenaline 30% of the [³H]catecholamines. Desipramine (1 μM) prevented the formation of [³H]noradrenaline without affecting the storage of [³H]dopamine. Nomifensine (10 μM) blocked the storage of [³H]dopamine and [³H]noradrenaline. Thus, in the NIL, [³H]dopamine is taken up into dopaminergic and noradrenergic neurones. In the latter, [³H]dopamine is converted to [³H]noradrenaline, indicating a significant dopamine β-hydroxylase activity in the NIL tissue. A selective labeling of the dopamine stores with [³H]dopamine can be achieved in the presence of desipramine.     

Some parasympathetic neurons in the guinea-pig heart express aspects of the catecholaminergic phenotype in vivo

Summary In a histochemical study of intrinsic cardiac ganglia of the guinea-pig in whole-mount preparations, it was found that some 70–80% of the neurons express aspects of the catecholaminergic phenotype. These neurons have an uptake mechanism for L-DOPA, and contain the enzymes for converting L-DOPA, (but not D-DOPA) to dopamine and noradrenaline, i.e. aromatic L-aminoacid decarboxylase and dopamine -hydroxylase. Monoamine oxidase is also present within some of the neurons. In these respects, the neurons resemble noradrenergic neurons of sympathetic ganglia, so we refer to them as intrinsic cardiac amine-handling neurons. However, these neurons do not contain tyrosine hydroxylase and show little or no histochemically detectable uptake of -methyldopa, dopamine or noradrenaline, even after depletion of endogenous stores of amines by pre-treatment with reserpine. Noradrenergic fibres from the sympathetic chain form pericellular baskets around nerve cell bodies. The uptake of L-DOPA into nerve cell bodies is not prevented by treatment with 6-hydroxydopamine sufficient to cause transmitter-depletion or degeneration of the extrinsic noradrenergic fibres. Such degeneration experiments suggest that axons of the amine-handling neurons project to cardiac muscle, blood vessels and other intrinsic neurons. The cardiac neurons do not show any immunohistochemically detectable serotonergic characteristics; there is no evidence for uptake of the precursors L-tryptophan and 5-hydroxytryptophan or 5-HT itself, whereas the extrinsic noradrer ergic nerve fibres within the ganglia can take up 5-HT when it is applied in high concentrations.Abbreviations AChE acetylcholinesterase - DBH-IR dopamine -hydroxylase-like immunoreactivity - L-DOPA L-dihydroxyphenylalamine - 5-HT-IR 5-hydroxytryptamine-like immunoreactivity - 6-OHDA 6-hydroxydopamine - methyldopa L--methyl-dihydroxyphenylalanine - MAO monoamine oxidase - NPY neuropeptide Y - SIF small intensely fluorescent cells - TH-IR tyrosine hydroxylase-like immunoreactivity - VIP vasoactive intestinal polypeptide     

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.     

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.     

PRELIMINARY ESTIMATES OF THE DOPAMINE β-HYDROXYLASE CONTENT AND ACTIVITY IN PURIFIED NORADRENERGIC VESICLES1

The dopamine β-hydroxylase (DβH) content and activity of large dense-core noradrenergic vesicles purified from bovine splenic nerve were determined using two assay procedures : enzymic activity expressed in Units per mg protein and homospecific activity based on radioimmunoassay expressed in Units per mg DβH antigen. Approximately two-thirds of the total enzyme activity is latent in these vesicles, even after various treatments designed to compromise vesicle integrity. DβH can be completely unmasked by brief treatment with 0.01-0.05% Triton X-100 and activity increases from 0.20 to 0.64 Units per mg vesicle protein. Calculations based on both assay methods suggested that an average of 7% (range 3-15%) of the total vesicle protein was DβH and that the average vesicle contained about 4 molecules of enzyme (range 2-9 molecules). The estimated homospecific activities indicated an average of 25 and 50% (range 18-72%) of the vesicle enzyme was inactive in the various samples using the two antibodies. The vesicle can synthesize up to 30 molecules of noradrenaline/s per molecule of DβH at near optimal substrate concentration, and 60-270 molecules of norepinephrine/s per vesicle. The assumptions used in the various calculations were critically analyzed and, based on the methods employed, it is tentatively considered to be unlikely that there could be more than 5-12 molecules of DβH per vesicle. The possibility that circulating DβH originates primarily, if not exclusively, from the large dense-core vesicle type is considered and the functional implications of the data support the concept of vesicle reuse during several cycles of exocytosis involving a quantal size equal to a fraction of the vesicle transmitter content.     

In Vitro Synthesis of Dopamine and Noradrenaline in the Isolated Rat Pineal Gland: Day-Night Variations and Effects of Electrical Stimulation

Isolated rat pineal glands were incubated in vitro in a medium containing [14C]dopamine or [14C]tyrosine, and the tissue contents of 14C-labelled and total dopamine and noradrenaline were determined by HPLC followed by electrochemical detection and scintillation spectrometry. During incubation with [14C]dopamine, the labelled amine accumulated in pineal glands and was partially converted into [14C]noradrenaline. Nomifensine, a neuronal amine uptake blocker, largely inhibited the accumulation of [14C]dopamine and the formation of [14C]noradrenaline. These experiments demonstrated dopamine beta-hydroxylase activity in the sympathetic nerves of the pineal gland. During incubation with [14C]tyrosine, formation of [14C]dopamine and [14C]noradrenaline was observed in the pineal tissue, indicating that noradrenaline can also be synthesized from dopamine, endogenously formed in the gland. Electrical stimulation of the stalk region of the pineal gland during incubation with [14C]dopamine enhanced the accumulation of [14C]dopamine and synthesis of [14C]noradrenaline. Electrical stimulation also enhanced the formation of [14C]dopamine during incubation with [14C]tyrosine. Compared to that at midday, the tissue content of endogenous noradrenaline at midnight was enhanced by 50% and that of dopamine by 450%. The in vitro accumulation of [14C]dopamine, as well as the synthesis of [14C]dopamine and [14C]noradrenaline, was also increased at midnight. In conclusion, sympathetic nerves in the rat pineal gland contain tyrosine hydroxylase and dopamine beta-hydroxylase, the two enzymes required for the synthesis of noradrenaline.(ABSTRACT TRUNCATED AT 250 WORDS)     

RELATIONSHIP BETWEEN THE RATE OF AXOPLASMIC TRANSPORT AND SUBCELLULAR DISTRIBUTION OF ENZYMES INVOLVED IN THE SYNTHESIS OF NOREPINEPHRINE

The net rate of proximo-distal transport of tyrosine hydroxylase, dopamine β-hydroxylase, DOPA decarboxylase and choline acetyltransferase was determined by measuring the accumulation of these enzymes proximal to a ligature of the rat sciatic nerve. The rate of accumulation was constant for at least 12 h. For the enzymes involved in the biosynthesis of norepinephrine the rate of transport was correlated to their subcellular distribution and a close correlation between these two parameters was found. Dopamine β-hydroxylase, an enzyme mainly localized in the particulate fraction of the sciatic nerve, showed the fastest rate of transport (1·94 mm/h) whereas DOPA decarboxylase, exclusively located in the high-speed supernatant fluid, gave the slowest (0·63 mm/h) rate of transport. Tyrosine hydroxylase, predominantly located in the non-particulate fraction of the sciatic nerve was transported much slower (0·75 mm/h) than dopamine β-hydroxylase but still significantly (P < 0.005) faster than DOPA decarboxylase. The subcellular distribution of dopamine β-hydroxylase in ganglia did not differ significantly (0·45 > P > 0·40) from that in the sciatic nerve, but in nerve endings a greater proportion of dopamine β-hydroxylase was localized in particulate fractions. Tyrosine hydroxylase and DOPA decarboxylase were found exclusively in the non-particulate fractions of ganglia. In the nerve endings of the effector organs a small but consistent portion of tyrosine hydroxylase was found in particulate fractions, whereas DOPA decarboxylase was exclusively localized in the high-speed supernatant fluid.     

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.     

Alteration of Catecholamine Phenotype in Transgenic Mice Influences Expression of Adrenergic Receptor Subtypes

Abstract: Agonist-induced regulation of adrenergic receptors (ARs) has an important role in controlling physiological functions in response to changes in catecholamine stimulation. We previously generated transgenic mice expressing phenylethanolamine N -methyltransferase (PNMT) under the control of a human dopamine β-hydroxylase gene promoter to switch catecholamine specificity from the norepinephrine phenotype to the epinephrine phenotype. In the present study, we first examined changes in catecholamine metabolism in peripheral tissues innervated by sympathetic neurons of the transgenic mice. In the transgenic target tissues, a high-level expression of PNMT led to a dramatic increase in the epinephrine levels, whereas the norepinephrine levels were decreased to 48.6–87.9% of the nontransgenic control levels. Analysis of plasma catecholamines in adrenalectomized mice showed large amounts of epinephrine derived from sympathetic neurons in the transgenic mice. Subsequently, we performed radioligand binding assays with (−)-[¹²⁵I]iodocyanopindolol to determine changes in binding sites of β-AR subtypes. In transgenic mice, the number of β2-AR binding sites was 56.4–74.9% of their nontransgenic values in the lung, spleen, submaxillary gland, and kidney, whereas the β1-AR binding sites were regulated in a different fashion among these tissues. Moreover, northern blot analysis of total RNA from the lung tissues showed that down-regulation of β2 binding sites was accompanied by a significant decrease in steady-state levels of the receptor mRNA. These results strongly suggest that alteration of catecholamine specificity in the transgenic sympathetic neurons leads to regulated expression of the β-AR subtypes in their target tissues.