Immunofluorescent and biochemical studies on tyrosine hydroxylase and dopamine-β-hydroxylase of the bullfrog sciatic nerves

Summary Immunofluorescence specific for tyrosine hydroxylase (TH) or dopamine--hydroxylase (DBH) was accentuated in both proximal and distal segments of the sciatic nerve after ligation. Estimations of the enzyme activities confirmed the above results. Mean axoplasmic flow rates of TH and DBH in bullfrog sciatic nerve were found to be 8 and 123 mm/day, respectively. They were decreased by colchicine or by cold temperatures (4° C).     

Storage and fast transport of noradrenaline, dopamine beta-hydroxylase and neuropeptide Y in dog sciatic nerve axons.

The axonal transport and subcellular distribution of noradrenaline (NA), dopamine beta-hydroxylase (DBH) and neuropeptide Y (NPY) were determined in dog sciatic nerve using an accumulation technique. The results were compared with those obtained by application of the same procedures and methods on the splenic nerve in the same animal species. Evidence was found for the coexistence of NA and NPY in large dense-cored vesicles in dog sciatic nerve axons. After differential centrifugation and isopyenic sucrose density gradient centrifugation of 24 h ligated sciatic nerve pieces NA and NPY equilibrated around 1M sucrose. The DBH activity was dispersed broadly on the gradient. Subsequently, the accumulation of NA, DBH and NPY was studied in proximal and sital segments of 8, 12 and 24 h dog ligated sciatic nerve and inferences were made concerning the axonal transport of these compounds. NA, DBH and NPY displayed a divergent accumulation proximal to the ligation. After 12 h of ligation a transport rate was calculated of 4.8 +/- 1.8 mm/h for NA, of 5.9 +/- 1.5 mm/h for DBH and of 4.9 +/- 2.0 mm/h for NPY. With a correction for the stationary fractions, a similar fast transport rate of approximately 10 to 12 mm/h was proposed for NA, DBH and NPY. The occurrence was shown of a limited retrograde transport of DBH and possibly NPY, but not of NA.     

Immunocytochemical study of tyrosine hydroxylase and dopamine β-hydroxylase immunoreactivities in the rat pancreas

An immunohistochemical and immunoelectron microscopic study was used to demonstrate tyrosine hydroxylase (TH) and dopamine -hydroxylase (DBH) immunoreactivities in the rat pancreas. Small TH immunoreactive cells were found in close contact with large TH immunonegative ganglion cells among the exocrine glands and were occasionally found in some islets. Some of these TH immunoreactive cells were also DBH immunopositive. The immunoreaction product was seen diffusely in the cytoplasm and in the granule cores of TH immunoreactive cells. All intra-pancreatic ganglion cells were immunoreactive for DBH, but not for TH. The TH immunoreactive cells were identified as small intensely fluorescent (SIF) cells due to their localization and morphological characteristics and showed no insulin, glucagon, somatostatin or pancreatic polypeptide immunoreactivities. These results indicate that SIF cells may release dopamine or noradrenaline to adequate stimuli while the intra-pancreatic ganglion cells with only DBH may not synthesize catecholamines in a normal biosynthetic pathway. TH immunoreactive nerve bundles without varicosities and fibers with varicosities, associated or unassociated with blood vessels, were found in both the exocrine and endocrine pancreas. Close apposition of TH immunoreactive nerve fibers to the smooth muscle and endothelial cells of the blood vessels was observed. A close apposition between TH immunoreactive nerve fibers and exocrine acinar cells and islet endocrine cells was sometimes found in the pancreas. The immunoreaction product was seen diffusely in the axoplasm and in the granular vesicles of the immunoreactive nerve fibers. Since no TH immunoreactive ganglion cells were present in the rat pancreas, the present study suggests that noradrenergic nerve fibers in the pancreas may be extrinsic in origin, and may exert an effect on the regulation of blood flow and on the secretory acitivity of the acinar cells, duct cells and endocrine cells.     

Ganglion cells immunoreactive for catecholamine-synthesizing enzymes,neuropeptide Y and vasoactive intestinal polypeptide in the rat adrenal gland

Immunohistochemistry has been used to demonstrate tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) immunoreactivities, and acetylcholinesterase (AChE) activity was demonstrated in rat adrenal glands. The TH, DBH, NPY and VIP immunoreactivities and AChE activity were observed in both the large ganglion cells and the small chromaffin cells whereas PNMT immunoreactivity was found only in chromaffin cells, and not in ganglion cells. Most intraadrenal ganglion cells showed NPY immunoreactivity and a few were VIP immunoreactive. Numerous NPY-immunoreactive ganglion cells were also immunoreactive for TH and DBH; these cells were localized as single cells or groups of several cells in the adrenal cortex and medulla. Use of serial sections, or double and triple staining techniques, showed that all TH- and DBH-immunoreactive ganglion cells also showed NPY immunoreactivity, whereas some NPY-immunoreactive ganglion cells were TH and DBH immunonegative. NPY-immunoreactive ganglion cells showed no VIP immunoreactivity. AChE activity was seen in VIP-immunopositive and VIP-immunonegative ganglion cells. These results suggest that ganglion cells containing noradrenaline and NPY, or NPY only, or VIP and acetylcholine occur in the rat adrenal gland; they may project within the adrenal gland or to other target organs. TH, DBH, NPY, and VIP were colocalized in numerous immunoreactive nerve fibres, which were distributed in the superficial adrenal cortex, while TH-, DBH- and NPY-immunoreactive ganglion cells and nerve fibres were different from VIP-immunoreactive ganglion cells and nerve fibres in the medulla. This suggests that the immunoreactive nerve fibres in the superficial cortex may be mainly extrinsic in origin and may be different from those in the medulla.     

Increased gut-derived norepinephrine release in sepsis: up-regulation of intestinal tyrosine hydroxylase

Studies have shown that increased gut-derived norepinephrine (NE) release plays an important role in producing hepatocellular dysfunction at the early stage of sepsis. Although the gut has been demonstrated to be the major source of NE in sepsis, it remains unknown whether the increased NE is associated with up-regulation of intestinal NE biosynthesis enzymes such as tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). To determine this, adult male rats were subjected to sepsis by cecal ligation and puncture (CLP) followed by fluid resuscitation. Small intestinal samples were harvested at 2 h (i.e., early sepsis) or 20 h (late sepsis) after CLP or sham-operation. Protein levels of TH and DBH were determined by Western blot analysis and immunohistochemistry. Their gene expression was assessed by RT-PCR technique. The results indicate that intestinal TH protein levels increased significantly at 2 and 20 h after CLP, while DBH was not altered under such conditions. Immunohistochemical examination shows that both TH and DBH were located in intestinal sympathetic nerve fibers and TH staining was markedly increased in septic animals. TH gene expression increased significantly at 2 h but not at 20 h after CLP, while DBH gene expression was not altered in sepsis. Thus, the increased TH gene and protein expression appears to be responsible for the increased gut-derived NE in sepsis.     

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.     

AXONAL TRANSPORT OF PHENYLETHANOLAMINE-N-METHYLTRANSFERASE IN TOAD SCIATIC NERVE

—The presence of phenylethanolamine-N-methyltransferase (EC 2.1.1.-) and dopamine-β-hydroxylase (EC 1.14.2.1) activities was demonstrated in the sciatic nerve of the toad, Bufo marinus. The rates of accumulation of phenylethanolamine-N-methyltransferase (PNMT) and dopamine-β-hydroxylase (DBH) proximal to a ligation of the sciatic nerve were studied. DBH accumulated proximal to the ligation at a more than 10-fold faster rate than PNMT. By measuring the rate of loss of enzyme activity distal to a ligation, an estimate of per cent clearance of each enzyme was made. Based on the per cent of enzyme activity free to move, the absolute transport rates for each enzyme were estimated to be: PNMT, 3.6 mm/24 h; DBH, 102 mm/24 h. PNMT activity (89 per cent) was recovered in the soluble fraction of sciatic nerve homogenates with no change occurring in the subcellular distribution of the enzyme proximal to ligations. In contrast, 43 per cent of DBH activity was found in the soluble fraction of sciatic nerve homogenates; but a disproportionate increase in paniculate DBH activity was found proximal to sciatic nerve ligations. Reduction of toad body temperature to 4°C resulted in a complete but totally reversible block of the axonal transport of both PNMT and DBH.     

Adrenergic nerve fibers in the human fetal sciatic nerve.

The adrenergic innervation was studied in the human sciatic nerve at the gestational age of 16, 17, 18 and 21 weeks. Formaldehyde-induced catecholamine fluorescence, tyrosine hydroxylase (TH) and neuropeptide Y (NPY) peroxidase-antiperoxidase immunohistochemistry methods were used. At the gestational age of 16, 17 and 18 weeks no adrenergic or NPY-positive nerve fibers were seen. At 21 weeks both fluorescence microscopy and TH immunohistochemistry showed adrenergic nerve fibers around arterioles in the epiperineurium and single nerve fibers in the endoneurium not related to blood vessels. The number of adrenergic nerve fibers appeared to be higher in the sciatic than in the tibial segment of the nerve. At this age, as at earlier stages of gestation, no NPY-positive nerve fibers were seen either in the epiperineurium or in the endoneurium. The results suggest that adrenergic nerve fibers may be associated with the epiperineurial blood vessels in the human sciatic nerve, and that the innervation starts to develop between 18 and 21 weeks of gestational age.     

Intrinsic choroidal neurons in the duck eye receive sympathetic input: anatomical evidence for adrenergic modulation of nitrergic functions in the choroid

Intrinsic choroidal neurons (ICN) in the duck eye form an intramural ganglionic plexus that may subserve complex integrative functions. A key feature of such ganglia is an innervation by sympathetic postganglionic neurons. The present study was thus aimed at determining the sympathetic postganglionic innervation of ICN. Choroids were processed for double immunofluorescence labelling with the following markers: tyrosine-hydroxylase (TH)/nitric oxide synthase (nNOS), TH/galanin (GAL), dopamine-beta-hydroxylase (DBH)/vasoactive intestinal polypeptide (VIP), TH/DBH and DBH/alpha-smooth-muscle actin (alphaSMA), and for triple immunofluorescence labelling with VIP/DBH/TH. Epifluorescence and confocal laser scanning microscopy were used for evaluation. Immunoperoxidase staining for TH or DBH in combination with NADPH-diaphorase histochemistry was applied for electron microscopy. ICN spread over the entire choroid but were concentrated in an equatorial zone passing obliquely from naso-cranial to temporocaudal. More than 80% of nNOS-positive ICN showed close appositions of TH/DBH-immunoreactive varicose nerve fibres at the light-microscopic level, as could be confirmed by confocal laser scanning microscopy. Ultrastructurally, these appositions could be defined as both synapses or close contacts without synaptic specialisation. Vascular and non-vascular smooth muscle fibres also received TH/DBH-immunopositive innervation. Our findings suggest that most ICN receive a sympathetic input that might modulate their nitrergic effects upon vascular and non-vascular smooth muscle fibres in the choroid and that they may have more complex functions than merely being a simple parasympathetic relay.     

Monoamine-synthesizing enzymes in central dopaminergic, noradrenergic and serotonergic neurons. Immunocytochemical localization by light and electron microscopy.

The monoamine-synthesizing enzymes tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and tryptophan hydroxylase (TrH) were immunocytochemical localized in dopaminergic, noradrenergic and serotonergic neurons of rat brain by light and electron microscopy. In dopaminergic and serotonergic neurons, the respective synthesizing enzymes. TH and TrH, were distributed throughout the cytoplasm of the neuronal perikarya, dendrites, axons and terminals. The most selective accumulation of reaction product for the specific enzyme was associated: (a) in perikarya with endoplasmic reticulum, Golgi apparatus and microtubules, (b) in processes with microtubules, and (c) in terminals with dense granules or clear vesicles. The labeled terminals were characterized by their content of labeled organelles and the absence of synaptic junctions. In noradrenergic neurons, both TH and DBH were localized in the perikarya, similar to TH in dopamine neurons. TH and DBH differed in their localization within proximal axons and dendrites in that TH was associated with microtubules but DBH was not. These results provide ultrastructural evidence to suggest that monoamines may be: (a) synthesized by enzymes which are associated with different organelles depending on the portion of the neuron and the type of enzyme; (b) synthesized in both axons and dendrites and (c) released from terminals without postsynaptic membrane specializations.     

Partial Isolation of Two Classes of Dopamine β-Hydroxylase-Containing Particles Undergoing Rapid Axonal Transport in Rat Sciatic Nerve

The rapid bidirectional transport of dopamine beta-hydroxylase (DBH) in adrenergic axons provides a means of analyzing the life cycle of adrenergic storage vesicles. We compared the physical characteristics of DBH-containing particles traveling to or returning from the terminal varicosities of ligated rat sciatic nerves. Density gradient centrifugation and Sephacryl S1000 gel-permeation chromatography were used to fractionate extracts from nerve segments proximal or distal to the ligatures. A series of experiments indicated the existence of at least two populations of rapidly transported DBH-containing particles, a "light" 85-nm particle and a larger "dense" 120-nm particle. The 85-nm particles were prevalent in unligated nerve, but accounted for only one-third of the total anterogradely transported DBH activity accumulated after 18 h. The 120-nm particles were barely detectable in the unligated nerve, but they accumulated at twice the rate of the 85-nm particles and accounted for the rest of the anterogradely transported particulate DBH activity. These two populations of particles were readily isolated from proximal nerve extracts by sucrose density gradient centrifugation. Similar-appearing dense and light peaks of particulate DBH activity were obtained from distal nerve extracts. Much of the retrogradely transported DBH of the extracts, however, was associated with large particles (greater than 300 nm) not resolved by Sephacryl S1000. Retrogradely transported exogenous NGF was found only in the dense sucrose gradient peak. We propose that the 85-nm DBH-containing particles correspond to "large dense-cored vesicles," and that the 120-nm particles are derived from the dense tubules visualized in adrenergic nerves by the chromaffin reaction.     

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.     

Galanin-, neuropeptide Y- and enkephalin-like immunoreactivities in catecholamine-storing paraganglia of the fetal guinea pig and newborn pig

Summary The occurrence and distribution of several neuropeptides and transmitter enzymes have been investigated by means of indirect immunofluorescence histochemistry in preaortal and carotid body-like paraganglia of the fetal guinea pig and the newborn pig. Preaortal paraganglia from the celiac and inferior mesenteric ganglion regions in fetal guinea pigs showed cell bodies immunoreactive (IR) for tyrosine hydroxylase (TH), dopamine -hydroxylase (DBH), neuropeptide Y (NPY), galanin (GAL) and metenkephalin (ENK). Almost all cells were IR for TH and DBH, whereas NPY-like immunoreactivity (-LI), GAL-LI and ENK-LI occurred less frequently. Direct double-labeling revealed the coexistence of NPY/GAL, NPY/ENK and GAL/ENK in paraganglion cells from the celiac and inferior mesenteric region. Nerve fibers and terminals were IR for ENK; fibers IR for calcitonin-gene-related peptide (CGRP) were present in the inferior mesenteric ganglion region. Preaortal paraganglia cells from the newborn pig showed TH-LI, DBH-LI, GAL-LI and ENK-LI, the distribution pattern being similar to that seen in the guinea pig; however, NPY-LI was absent. Carotid-body-like paraganglia from the newborn pig showed cell bodies IR to TH, GAL and ENK. Few cells were seen with DBH-LI. A rich supply of nerve fibers with CGRP-LI was present; some fibers exhibited ENK-LI and CCK-LI. In the adjacent superior cervical ganglion, ganglion cell bodies showed immunoreactivity to TH, DBH and NPY. A small number of cells were positive for GAL, CGRP and vasoactive intestinal polypeptide (VIP). Physiological activation of the paraganglia, leading to release or increase in catecholamines, may also change the content of the neuropeptides present in the paraganglia.     

Catecholamines are present in larval Xenopus laevis: a potential source for cardiac control

Changes in noradrenaline (NA), adrenaline (A), and dopamine (DA) levels in the heart, kidneys, and whole body (without heart and kidneys) during embryonic development were investigated in the frog, Xenopus laevis using high-performance liquid chromatography (HPLC). In addition, the presence of cells immunoreactive to tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and/or phenylethanolamine-N-methyltransferase (PNMT) in the heart of Xenopus larvae was investigated using immunohistochemical techniques. The presence of nerve fibers was visualized using antibodies against acetylated tubulin (AcT). NA and DA concentrations in the heart were low and steady in NF 40-56, showed an increased value at NF 57, and decreased again in froglets. A trend toward higher concentrations of A was observed at NF 43-49 and NF 57. Cells immunoreactive to TH, DBH, and PNMT were found in the heart from NF 40, and the TH immunoreactive cells became more abundant in the whole heart at later stages. The presence of catecholamines in the non-innervated larval heart together with the finding of TH/DBH/PNMT immunoreactive cells suggests that catecholamines are synthesized and stored in the heart and could therefore have a paracrine role in cardiac control in Xenopus larvae. Detectable concentrations of catecholamines were also found in kidneys and whole bodies (except heart and kidneys). Therefore, catecholamine-producing cells outside the heart can be an important source of circulating catecholamines involved in adrenergic cardiac control in Xenopus larvae.     

The synthesis of NPY and DBH is independently regulated in adrenergic nerves after reserpine

A newly developed cytofluorimetric scanning technique was applied in a pharmacological study to investigate the influence of reserpine (10 mg/kg) on the axonal transport of norepinephrine (NE), dopamine--hydroxylase (DBH), tyrosine hydroxylase (TH), and neuropeptide Y (NPY)-like immunoreactivities (LI) in the adrenergic axons of the sciatic nerve of rat. Early after reserpine (18 hr and 24 hr after the reserpine injection) the amounts of NE accumulated proximal to a 12-hr crush werenil or very low, as observed in earlier studies. DBH-LI, TH-LI, and NPY-LI accumulations were also depressed but only to about 50% of control accumulations. This decrease in amounts of transported substances was probably caused by a decrease in protein synthesis and also a lowered velocity of fast axonal transport initially after reserpine, when body temperature is low. The amounts of accumulated NE, DBH-LI, TH-LI, and NPY-LI were normalized around day 2 after reserpine, but on day 4 NE, DBH-LI, and in some rats also TH-LI accumulated in supranormal amounts. However, NPY-LI accumulations were normal, indicating that DBH, butrot NPY, was trans- synaptically induced in rat sympathetic neurons, and that the biochemical composition of axonally transported organelles is altered for some days after reserpine.Dedicated to Dr. Abel Lajtha.     

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.     

Involvement of alpha7 nicotinic acetylcholine receptors in activation of tyrosine hydroxylase and dopamine beta-hydroxylase gene expression in PC12 cells

Nicotine treatment increases intracellular free Ca(2+) concentration [Ca(2+)](i), stimulates catecholamine release, and elevates gene expression for the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). However, the type of nicotinic acetylcholine receptors (nAChRs) mediating these events is unclear. The nAChR receptor antagonists alpha-bungarotoxin (alphaBTX) and methyllycaconitine greatly reduced the nicotine-triggered initial transient rise in [Ca(2+)](i) and prevented the second prolonged elevation of [Ca(2+)](i), suggesting the involvement of alpha7 nAChRs. Two specific alpha7 nicotinic agonists, 3-(2,4-dimethoxybenzilidene)anabaseine (DMXB) and E, E-3-(cinnamylidene)anabaseine (3-CA), were found to elicit a small, delayed increase in [Ca(2+)](i) with kinetics and magnitude similar to the second elevation observed with nicotine. This increase was inhibited by the inositol trisphosphate receptor antagonist xestospongin C. Exposure to 3-CA or DMXB for 6 or 24 h elevated TH and DBH mRNA levels two- to fourfold over control levels. These agonists were more effective than nicotine alone in increasing TH and DBH gene expression and significantly elevated [Ca(2+)](i) for up to 6 h. The increase in [Ca(2+)](i) or the elevation in TH mRNA by 3-CA was completely inhibited by alphaBTX. This study, for the first time, implicates stimulation of alpha7 nAChRs in the activation of TH and DBH gene expression.     

Quantitation of changes in gene expression of norepinephrine biosynthetic enzymes in rat stellate ganglia induced by stress

Enzymes involved in catecholamine synthesis are present in the highest concentration in the adrenal medulla, however they were found also in other, mainly nervous tissues. The aim of our study was to quantify the exact concentration of tyrosine hydroxylase (TH) and dopamine-ss-hydroxylase (DBH) mRNA in rat stellate ganglia under control conditions and at different intervals after exposure to immobilization stress (IMO). In rats immobilized once for 2h, we determined TH and DBH mRNA in different time intervals up to 22 h after the end of the stress stimulus. TH immunoreactive protein levels were also determined in stellate ganglia. TH and DBH mRNA levels were quantified by RT-competitive-PCR.In stellate ganglia, the concentration of TH mRNA was 17+/-1.6 amol/microg of total RNA, which is approximately 30-times lower than in the adrenal medulla. The concentration of DBH mRNA in the stellate ganglia was 2601+/-203 amol/microg of total RNA, which is the concentration similar to adrenal medulla, but is 150-times higher than concentration of TH mRNA in stellate ganglia. After a single 2-h immobilization the highest elevation of TH and DBH mRNA levels was measured 22 h after the termination of the stress stimulus. Repeated immobilization (7 days, 2h daily) did not produce further increase in TH and DBH mRNA levels compared to already elevated levels in adapted control group (immobilized for 6 days, 2h daily and decapitated 22 h later). Levels of TH protein were significantly changed only after the repeated immobilization.This study compared for the first time the precise amounts of TH and DBH mRNA in rat stellate ganglia under control conditions and after immobilization stress, and indicates large differences in their concentration. TH and DBH mRNA concentrations in stellate ganglia are markedly elevated for a prolonged period of time after termination of the stress stimuli.     

Effect of hydrocortisone on catecholamines and the enzymes synthesizing them in the developing sympathetic ganglion

Summary Newborn rats were daily injected with 0.2 mg hydrocortisone acetate for seven days. They were killed 1, 7 or 21 days after the last injection, together with untreated controls. Hydrocortisone caused a great increase in the number of the small, intensely fluorescent (SIF) cells and the appearance of similar small cells with intense immunohistochemical reactions for tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH) and phenylethanolamine (noradrenaline)N-methyltransferase (PNMT) in the superior cervical ganglion. At the same time, the adrenaline content and the PNMT activity of the ganglion greatly increased, while no significant changes were observed in the dopamine or noradrenaline content or TH or DBH activity. All these changes essentially disappeared after a recovery period of seven or 21 days.It is concluded that hydrocortisone causes a temporary increase in the number of SIF cells by causing a synthesis of TH, DBH and PNMT in previously existing small, non-fluorescent cells, which start to synthesize and store adrenaline, thus becoming intensely fluorescent SIF cells. These SIF cells are different from the normal SIF cells of the same ganglion, most of which appear at a later stage of postnatal development when response to hydrocortisone is lost, which contain TH but neither DBH nor PNMT, and which permanently remain in the ganglion.     

In vitro translation of human pheochromocytoma messenger RNAs: characterization of tyrosine-hydroxylase and dopamine-beta-hydroxylase

mRNAs extracted from human pheochromocytoma were translated in vitro in a lysate of a rabbit reticulocytes. Two enzymes of the biosynthetic pathway of the catecholamines, tyrosine-hydroxylase (TH) and dopamine-beta-hydroxylase (DBH), were characterized as translation products after immunoprecipitation by specific antisera and electrophoretic analysis. The precursor of TH is a polypeptide having a molecular mass of 62,000 identical to that found for the mature protein. The molecular mass of the precursor of DBH 73,000 while that of the mature form is 79,000. TH and DBH have been translated from mRNAs having sedimentation coefficients of 22S and 25S, respectively.