Identification of noradrenergic nerve terminals immunoreactive for neuropeptide Y and vasoactive intestinal peptide in the rat kidney

Cryostat- and vibratome-cut sections of rat kidneys were singly or doubly labeled to visualize immunoreactive tyrosine hydroxylase (THI), dopamine beta-hydroxylase (DBHI), vasoactive intestinal peptide (VIPI), and neuropeptide Y (NPYI). Rats were perfusion fixed with 2-4% paraformaldehyde with or without 0.15% picric acid and rinsed in buffer for 18-48 hr. Single antigens were labeled with horseradish peroxidase in vibratome sections, whereas cryostat sections were used to label one antigen with peroxidase and another with a fluorophore in the same tissue section. A dense plexus of DBHI noradrenergic nerves innervates the renal arterial tree, and such nerves innervate the interlobar veins and renal calyx as well. Immunoreactive NPY is colocalized in most of these nerves, but some intrarenal noradrenergic nerves do not contain NPY but do contain VIP immunoreactivity. The distribution of NPYI nerves resembles that of DBHI nerves, whereas most perivascular noradrenergic nerves immunoreactive for VIP innervate selected arcuate and interlobular arteries. A small population of nonadrenergic, VIPI nerves innervates the renal calyx.     

Distribution,pathways and reactions to drug treatment of nerves with neuropeptide Y- and pancreatic polypeptide-like immunoreactivity in the guinea-pig digestive tract

Pancreatic polypeptide-like immunoreactivity (PPLI) has been localized in nerves of the guinea-pig stomach and intestine with the use of antibodies raised against avian, bovine and human pancreatic polypeptide (PP), the C-terminal hexapeptide of mammalian PP, and against the related peptide, NPY. Each of the antibodies revealed the same population of neurones. Reactive cell bodies were found in both myenteric (5% of all neurones) and submucous ganglia (26% of all neurones) of the small intestine, and varicose processes were observed in the myenteric plexus, circular muscle, mucosa and around arterioles. The nerves were unaffected by bilateral subdiaphragmatic truncal vagotomy, but the staining of the periarterial nerves disappeared after treatment of animals with reserpine or 6-hydroxydopamine and was also absent after mesenteric nerves had been cut and allowed to degenerate. Vascular nerves showing immunoreactivity for dopamine beta-hydroxylase and PPLI had the same distribution. It is concluded that PPLI is located in periarterial noradrenergic nerves. However, other noradrenergic nerves in the intestine do not show PPLI, and PPLI also occurs in nerves that are not noradrenergic. Analysis of changes in the distribution of terminals after microsurgical lesions of pathways in the small intestine showed that processes of myenteric PP-nerve cells provide terminals in the underlying circular muscle and in myenteric ganglia up to about 2 mm more anal. Submucous PP-cell bodies provide terminals to the mucosa.     

High concentrations of a novel peptide, neuropeptide Y, in the innervation of mouse and rat heart

A newly discovered bioactive peptide, neuropeptide Y (NPY), has been found in the innervation of the mouse and rat heart by immunocytochemistry, NPY-immuno-reactive nerves were very dense around the nodal tissues. They also surrounded the coronary arteries and arterioles and were found in close association with the cardiac muscle. The distribution of NPY-containing nerves paralleled that of noradrenergic fibers, demonstrated by the use of antibodies to the catecholamine-converting enzymes tyrosine hydroxylase and dopamine beta-hydroxylase. Furthermore, NPY was seen to be present in a proportion of intrinsic neurons mostly found in the atria and in close proximity to the nodal tissue. The concentrations of extractable NPY-immunoreactive material (about 150 pmol/g in whole mouse heart) by far surpasses those of the other peptides so far reported in the cardiac tissue. High performance liquid chromatography demonstrated the NPY immunoreactivity to elute in a single sharp peak, in an identical position to brain NPY.     

A fluorescence histochemical study of the degeneration and regeneration of noradrenergic nerves in the chick following treatment with 6-hydroxydopamine

Summary The fluorescence histochemical method of Falck and Hillarp for the cellular localisation of biogenic amines was used to follow the disappearance and reappearance of peripheral noradrenergic nerves in the chick after treatment with 6-hydroxydopamine. With certain exceptions, it seemed that the disappearance and reappearance of the nerves was due to their degeneration and subsequent regeneration. However, there was not a uniform destruction of the noradrenergic nerves associated with different effector tissues. The extent of degeneration was greater in older than in younger chicks following equivalent doses of the drug, possibly indicating a greater uptake efficiency of the older nerves. It appeared that the noradrenergic nerves in the younger chicks regenerated more rapidly than those of older chicks, but in none of the animals studied were there any obvious morphological modifications of the regenerating nerves.The major part of this work was carried out in the Department of Zoology, Melbourne University, Australia.We are grateful to Professor G. Burnstock for use of laboratory facilities, and to the National Heart Foundation of Australia for financial support.     

Evidence for neurotransmitter plasticity in vivo. II. Immunocytochemical studies of rat sweat gland innervation during development

Previous studies of the cholinergic sympathetic innervation of rat sweat glands provide evidence for a change in neurotransmitter phenotype from noradrenergic to cholinergic during development. To define further the developmental history of cholinergic sympathetic neurons, we have used immunocytochemical techniques to examine developing and mature sweat gland innervation for the presence of the catecholamine synthetic enzymes tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) and for two neuropeptides present in the mature cholinergic innervation, vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP). In 7-day old animals, intensely TH- and DBH-immunoreactive axons were closely associated with the forming glands. The intensity of both the TH and DBH immunofluorescence decreased as the glands and their innervation developed. Neither TH-IR nor DBH-IR disappeared entirely; faint immunoreactivity for both enzymes was reproducibly detected in mature animals. In contrast to noradrenergic properties, the expression of peptide immunoreactivities appeared relatively late. No VIP-IR or CGRP-IR was detectable in the sweat gland innervation at 4 or 7 days. In some glands VIP-IR first appeared in axons at 10 days, and was evident in all glands by 14 days. CGRP-IR was detectable only after 14 days. In addition to VIP-IR and CGRP-IR, we examined the sweat gland innervation for several neuropeptides which have been described in noradrenergic sympathetic neurons including neuropeptide Y, somatostatin, substance P, and leu- and met-enkephalin; these peptides were not evident in either developing or mature sweat gland axons. Our observations provide further evidence for the early expression and subsequent modulation of noradrenergic properties in a population of cholinergic sympathetic neurons in vivo. In addition, the asynchronous appearance during development of the two neuropeptide immunoreactivities raises the possibility that the expression of peptide phenotypes may be controlled independently.     

Vesicular Storage of 3,4-Dihydroxyphenylethylamine and Noradrenaline in Terminal Sympathetic Nerves of Dog Spleen and Kidney

The subcellular distribution of 3,4-dihydroxyphenylethylamine (DA, dopamine) and noradrenaline was examined in preparations of dog spleen and renal cortex following ultracentrifugation on a discontinuous sucrose gradient. In both tissues, only half the total tissue DA was localized to the soluble phase, and 30-50% was found in association with noradrenaline in the large vesicular fraction, suggesting that both catecholamines may be stored together and released by nerve stimulation. The vesicular fraction from renal cortex contained more DA than could be attributed to its presence in noradrenergic axons alone, supporting other evidence for the existence of dopaminergic renal nerves.     

Regulation of dopamine beta-hydroxylase in rat adrenal glands.

Rat adrenal gland levels of dopamine beta-hydroxylase are subject to dual control. Activation of the splanchnic nerves to the adrenal medulla by reserpine induces the synthesis of dopamine beta-hydroxylase without altering the rate of enzyme degradation. In contrast, hypophysectomy causes a decline in steady state dopamine beta-hydroxylase levels by first accelerating the rate of degradation, then by slowing the rate of enzyme synthesis as well. Adrenocorticotropic hormone administration partially reversed the effect of hypophysectomy on dopamin beta-hydroxylase degradation. These findings suggest that the trans-synaptic factors controlling dopamine beta-hydroxylase induction act by a different mechanism (enzyme synthesis) than the hormonal controls regulating steady state levels (enzyme degradation). Thus, active inhibition of enzyme degradation may be an important control in maintenance of steady state enzyme levels.     

PATTERN OF RNA SYNTHESIS IN THE SCIATIC NERVE OF THE HEN DURING WALLERIAN DEGENERATION

The pattern of synthesis of rapidly-labelled RNA of hen sciatic nerve was studied during Wallerian degeneration. At 2,4,8, 16 and 30 days of degeneration the proximal and distal stumps of the severed nerve as well as the intact contralateral sciatic nerve (functional control) were excised and incubated with either [5-³H]uridine or [2-¹⁴C]uridine for 0.5 h. The electrophoretic pattern of RNA from the normal adult sciatic nerve showed that most of the radioactivity was incorporated into RNA species migrating between the 18 S and 4 S components of the bulk RNA. The synthesis of RNA was sensitive to actinomycin-D, an indication that it was directed by a DNA template. The electrophoretic patterns of the rapidly-labelled RNA in the proximal and distal nerve stumps demonstrated a change following nerve section. After 2–4 days of Wallerian degeneration the degenerating distal nerves incorporated more radioactivity in the 4 S region than the corresponding controls, but at 8 and 16-days after degeneration relatively more label appeared in higher molecular weight RNA species. In the intact sciatic nerve of the operated hens progressively more radioactivity was detected in the 4 S region with increasing time after the contralateral nerve section. At each stage of Wallerian degeneration the specific radioactivities of RNA in the control nerves from experimental hens were higher than those of the normal adult sciatic nerve. These results indicated a change of RNA metabolism in increased functional activity and during Wallerian degeneration.     

Dopamine accumulation after dopamine beta-hydroxylase inhibition in rat heart as an index of norepinephrine turnover

Dopamine concentration in rat heart is normally very low, only a few percent of the concentration of norepinephrine. After treatment of rats with a dopamine beta-hydroxylase inhibitor, 1-cyclohexyl-2-mercapto-imidazole (CHMI), there was a rapid increase in dopamine concentration even before norepinephrine concentration had decreased perceptibility. This accumulation of dopamine was readily measured by liquid chromatography with electrochemical detection. Since the percentage change in dopamine was much greater than the percentage change in norepinephrine, especially at early times, measurement of dopamine accumulation rather than norepinephrine decline was considered as a useful measure of norepinephrine turnover. Drugs that act on noradrenergic receptors and are known to alter norepinephrine turnover were found to alter the rate of dopamine accumulation. Clonidine and guanabenz decreased dopamine accumulation after CHMI, whereas piperoxan (but not prazosin) increased dopamine accumulation after CHMI. Pergolide, a dopamine agonist whose lowering of blood pressure and cardiac rate has been suggested to be due to suppression of neurogenic release or norepinephrine, also decreased dopamine accumulation after CHMI. The results suggest that measuring dopamine accumulation may have advantages over measuring norepinephrine disappearance after dopamine beta-hydroxylase inhibition as an indicator of norepinephrine turnover in heart.     

Regulation of Tyrosine Hydroxylase and Dopamine β-Hydroxylase mRNA Levels in Rat Adrenals by a Single and Repeated Immobilization Stress

Adrenal catecholamines are known to mediate many of the physiological consequences of the "fight or flight" response to stress. However, the mechanisms by which the long-term responses to repeated stress are mediated are less well understood and possibly involve alterations in gene expression. In this study the effects of a single and repeated immobilization stress on mRNA levels of the adrenal catecholamine biosynthetic enzymes, tyrosine hydroxylase and dopamine beta-hydroxylase, were examined. A repeated 2-hr daily immobilization for 7 consecutive days markedly elevated both tyrosine hydroxylase and dopamine beta-hydroxylase mRNA levels (about six- and fourfold, respectively). In contrast, tyrosine hydroxylase but not dopamine beta-hydroxylase mRNA levels were elevated immediately following a single immobilization. The elevation in tyrosine hydroxylase mRNA with a single immobilization was as high as with seven daily repeated immobilizations. This elevation was not sustained and returned toward control values 24 hr later. Both tyrosine hydroxylase and dopamine beta-hydroxylase mRNA levels were elevated immediately following two daily immobilizations to levels similar to those observed after seven immobilizations and were maintained 24 hr later. The results indicate that both tyrosine hydroxylase and dopamine beta-hydroxylase mRNA levels are elevated by stress; however, the mechanism and/or timing of their regulation are not identical.     

Immunological Characterization of Secretory Proteins of Chromaffin Granules: Chromogranins A, Chromogranins B, and Enkephalin-Containing Peptides

The soluble proteins of bovine chromaffin granules can be resolved into about 40 proteins by two-dimensional electrophoresis. Use of several antisera enabled us to characterize most of these proteins with the immune replica technique. An antiserum against dopamine beta-hydroxylase reacted with one protein of Mr 75,000. Met-enkephalin antisera labeled eight proteins of Mr 23,000-14,000. A new method was developed to obtain highly purified chromogranin A for immunization. The antiserum reacted with chromogranin A and several smaller proteins of similar pI. This specific antiserum did not react with a second family of hitherto undescribed proteins, which we propose to call chromogranins B. An antiserum against these proteins was raised. It labeled several proteins ranging in Mr from 100,000 to 24,000 and focusing at pH 5.2. Subcellular fractionation established that chromogranins B are specifically localized in chromaffin granules of several species. They are secreted from the adrenal medulla during cholinergic stimulation. We conclude that apart from dopamine beta-hydroxylase chromaffin granules contain three families of immunologically unrelated proteins.     

Synthesis,subcellular distribution and turnover of dopamine beta-hydroxylase in organ cultures of sympathetic ganglia and adrenal medullae

The synthesis, subcellular distribution and turnover of dopamine beta-hydroxylase was studied in organ cultures of rat adrenal medullae and superior cervical ganglia. After exposure to [3H]leucine for 1 or 3 h, the tissues were homogenized at various time intervals and the amount of labelled dopamine beta-hydroxylase in different subcellular fractions (cytosol, soluble and membrane-bound fraction of catecholamine storage vesicles) was determined by immunoprecipitation and subsequent electrophoresis. In cultured adrenal medullae, induction of dopamine beta-hydroxylase initiated in vivo by administration of reserpine affected both soluble and membrane-bound pools of dopamine beta-hydroxylase to a similar extent after pulse-labelling for 1 or 3 h. The half-lives of dopamine beta-hydroxylase, which amounted to 6 h for the cytosol, 7.5 h for the soluble vesicular and 32 h for the membrane-bound vesicular pools were not altered by pretreatment with reserpine. In superior cervical ganglia the half-lives of the soluble pools were 2-3 times longer than in the adrenal medulla, whereas the half-life of the membrane-bound fraction was the same as in the adrenal medulla. In both organs the most heavily labelled fraction (both after a pulse of 1 or 3 h) was always that of the vesicular membrane, suggesting that newly-synthesized dopamine beta-hydroxylase is immediately incorporated into the storage vesicles and not via release into the cytosol from the site of synthesis. The fact that the half-life of membrane-bound dopamine beta-hydroxylase is markedly longer than that of the two soluble pools suggests that the single pools are not only independently supplied by newly-synthesized DBH but there is also no appreciable subsequent exchange between soluble and membrane-bound pools.     

Neuronal and Nonneuronal Contributions to Renal Catecholamine Content in the Dog

Endogenous noradrenaline and 3,4-dihydroxyphenylethylamine (dopamine) levels were measured in different zones of the dog kidney following chronic unilateral renal denervation. In outer and inner renal cortex, and in outer medulla, greater than 95% of the tissue content of both catecholamines was contributed by renal nerves, whereas in inner medulla only nonneuronal catecholamines were found. The amounts of neuronal dopamine present in outer renal cortex were greater than would be expected for a population of solely noradrenergic nerves.     

Developmental pattern of dopamine beta-hydroxylase induction in the adrenals of the rat. Influence of neonatal hypothyroidism

The extent of dopamine beta-hydroxylase induction elicited by reserpine was measured in young rats rendered hypothyroid from birth and in controls. Hypothyroidism impairs adrenal dopamine beta-hydroxylase induction in the young rat up to 50 days of age and also in the adult. In contrast, hypothyroidism has practically no effect on brainstem dopamine beta-hydroxylase induction.     

Subcellular fractionation of splenic nerve: ATP, chromogranin A and dopamine beta-hydroxylase in noradrenergic vesicles

The subcellular particles in axons of the splenic nerve have been studied by centifrugation techniques. By differential centifrugation, five different types of particle could be distinguished and partly separated: noradrenaline-containing particles (noradrenergic vesicles), large and small lysosomes, mitochondria, and microsomal particles. In density gradient centrifugation, only one type of noradrenergic vesicle could he demonstrated. The noradrenergic vesicles and the mitochondria contain ATP. Two proteins (chromogranin A and dopamine beta-hydroxylase) are present in the noradrenergic vesicles.     

An anti-peptide antibody that recognizes the dopamine D2 receptor from bovine striatum.

The bovine dopamine D2 receptor was purified by wheat-germ-agglutinin-Sepharose chromatography and affinity chromatography, using the D2-receptor-specific agonist N-0434. Purification yields a preparation with a major protein band of 95 kDa. In order to ascertain the identity of this protein, polyclonal antibodies against the dopamine D2 receptor have been raised using synthetic peptides based on the predicted amino acid sequence of the cloned D2 receptor. For the initial screening of these antibodies, three fusion proteins consisting of beta-galactosidase and receptor fragments were constructed. One antiserum reacted strongly with the corresponding D2 receptor fusion protein, both on Western blots and in immunoprecipitation experiments. In each case, recognition was inhibited by competition with free peptide. On Western blots of partially purified receptor preparations from bovine striatum, the antiserum specifically recognized a 95-kDa glycoprotein. From similar preparations, the antiserum precipitated a substantial proportion of active D2 receptor, as determined by a decrease in [3H]spiperone binding in the supernatant. Active receptor could be released from the immunoprecipitate by addition of free peptide. Immunocytochemical analysis of cells transiently transfected with DNA coding for the D2 receptor showed specific staining of transfected cells. The antibody raised against a sequence in the third intracellular loop is able to shift the affinity of the receptor for dopamine from high to low, indicating that the antiserum may be interfering with receptor-GTP-binding-protein interactions.     

Sulfation and constitutive secretion of dopamine beta-hydroxylase from rat pheochromocytoma (PC12) cells

The biosynthesis and secretion of dopamine beta-hydroxylase were investigated by radiolabeling rat pheochromocytoma (PC12) cells in culture. Intracellular dopamine beta-hydroxylase from a crude chromaffin vesicle fraction and secreted dopamine beta-hydroxylase from culture medium were immunoprecipitated using antiserum made against purified bovine soluble dopamine beta-hydroxylase. Analysis of the immunoprecipitated enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that: 1) the membrane-bound form of the hydroxylase from crude secretory vesicle membrane extracts contained two nonidentical subunits in approximately stoichiometric amounts (Mr = 77,000 and 73,000); 2) the soluble hydroxylase from the lysate of these secretory vesicles was composed predominantly of a single subunit (Mr = 73,000); and 3) the hydroxylase secreted into the medium under resting conditions was also composed of a single subunit (approximate Mr = 73,000). All subunits of the multiple forms of hydroxylase were glycoproteins. Under resting conditions, the rate of secretion of hydroxylase was approximately 6% of total cellular enzyme/15 min. The secreted form of the hydroxylase incorporated [35S]sulfate, whereas no significant [35S]sulfate was incorporated into the cellular forms of enzyme. We propose that in addition to the dopamine beta-hydroxylase which is found in catecholamine storage vesicles and released during stimulus-coupled exocytosis, PC12 cells also have a constitutive secretory pathway for dopamine beta-hydroxylase and that the enzyme released by this second pathway is sulfated.     

Insulin-like growth factor I enhances proenkephalin synthesis and dopamine beta-hydroxylase activity in adrenal chromaffin cells

Insulin-like growth factor I (IGF-I) increased both the contents of proenkephalin-derived enkephalin-containing peptides and the activity of dopamine beta-hydroxylase in bovine adrenal chromaffin cells. These increases in dopamine beta-hydroxylase and enkephalin-containing peptides continued for at least 8 days. The half-maximal IGF-I concentration for these effects was approximately 1 nM, with maximal effects observed at 10-30 nM. In contrast, insulin was 1000-fold less potent. Pretreatment of chromaffin cells with IGF-I increased the rate of [35S]proenkephalin synthesis 4-fold compared to untreated cells. Total protein synthesis increased only 1.5-fold under these conditions. These results suggest that IGF-I may be a normal regulator of chromaffin cell function.     

Immunofluorescent patterns of clathrin and dopamine beta-hydroxylase in chromaffin cells in culture

Summary Bovine chromaffin cells maintained in culture for eight days were loaded with [³H]noradrenaline and then stimulated by a depolarizing concentration (56 mM) of K⁺. Control and stimulated cells were fixed in 3.7% formaldehyde, treated with acetone or Triton X-100, and then exposed to antibodies raised against dopamine beta-hydroxylase (a secretory granule marker) and clathrin, and purified by affinity chromatography. The cellular distribution of the correspondent antigens was investigated by indirect immunofluorescence. Cells treated with anti-dopamine beta-hydroxylase exhibited a granular pattern of fluorescence in the cytosol of the cell body, neurites, and terminal cones. Chromaffin cells exposed to anti-clathrin also showed a punctate pattern of fluorescence staining. However, in this case, the fluorescent dots were smaller than those observed with anti-dopamine beta-hydroxylase, and they were differently distributed. The speckled anti-clathrin fluorescence was preferentially condensed in the juxtanuclear region of the cell bodies, suggesting the possibility that clathrin was concentrated at the level of the Golgi apparatus.The stimulation of cultured chromaffin cells by 10 pulses of 56 mM K⁺ produced 91±2% (n = 5) depletion in the [³H]noradrenaline cell content and a concomitant displacement of the dopamine beta-hydroxylase fluorescence to the periphery of the cells. Four days after cell stimulation the dopamine beta-hydroxylase fluorescence was similar to that observed in control cells. Under the same conditions of stimulation, the distribution of the clathrin fluorescence was unaltered suggesting either that K⁺induced stimulation of the chromaffin cells does not change the cellular distribution of clathrin, or that the changes in the distribution of clathrin are of such low magnitude that they escape detection by fluorescence microscopy.