Neural uptake of catecholamines and their molecular structures: A histopharmacologic study

Using ultrastructural and histofluorescence methods, we investigated the uptake mechanism of catecholamines by the nerve terminals in the cutaneous smooth muscles of stump-tailed macaques (Macaca arctoides). This in vivo approach ultilized the observed cytotoxic effects of 6-hydroxydopamine on these catecholamine-containing terminals and the protective effects of simultaneous treatment with catecholamines (dopamine, norepinephrine, and epinephrine), their 3–0-methylated derivatives (metanephrine and normetanephrine), and catechol acids (3,4-dihydroxymandelic acid and 2, 4, 5-trihy-droxymandelic acid). Both catecholamines and 3–0-methylated derivatives protected these nerve terminals from destruction by 6-hydroxydopamine, but catechol acids did not. However, the 3–0-methylated derivatives were less effective than the catecholamines. The degree of protection afforded by these amines depended largely on their concentration. Only catecholamines intensified the electron density of the intravesicular mass or the fluorescence in the nerve terminals; therefore, 3–0-methylated derivatives may inhibit 6-hydroxydopamine uptake at axoplasmic membrane sites, but not inside the axon. These observations led to the discovery that there are two sites for the catecholamine uptake process. One site is the axoplasmic membrane. The terminals are protected by catecholamines and their 3–0-methylated derivatives from 6-hydroxydopamine uptake and thus destruction. The other site is the intra-axonal compartments. Here competitive binding between the vesicular protein and both 6-hydroxydopamine and the catecholamines plays a main role.     

The ultrastructure of the sinus gland of the crayfish Astacus leptodactylus (Nordmann)

Summary An ultrastructural study of the sinus gland of the crayfish Astacus leptodactylus demonstrates that this gland is mainly composed of glial cells, axons and axon terminals. On the basis of the size, shape and electron density of the neurosecretory granules, we could distinguish five different types of axon terminals.     

Catecholaminergic innervation of the rat adrenal cortex

Summary The zona glomerulosa of the rat adrenal gland is innervated by catecholaminergic nerves. Using histofluorescence techniques, we observed catecholaminergic plexuses surrounding adrenal capsular and subcapsular blood vessels. Individual varicose nerve fibers that branched off these plexuses were distributed among adrenal glomerulosa cells. This innervation was permanently eliminated after neonatal sympathectomy with guanethidine or 6-hydroxydopamine, but was not affected by ligation of the splanchnic nerve or extirpation of the suprarenal ganglion. At the ultrastructural level, axonal varicosities were commonly observed in close proximity to glomerulosa cells and blood vessels. Nerve fibers and varicosities were found to contain small (30–60 nm) clear vesicles as well as large (60–110 nm) and small (30–60 nm) dense-cored vesicles. In tissue fixed for the dichromate reaction with or without pretreatment with the false transmitter 5-hydroxydopamine, many nerve terminals contained numerous small dense-cored vesicles which are thought to contain catecholamines. These results establish the anatomical substrate for the catecholaminergic innervation of the rat adrenal cortex.     

BDNF promotes target innervation of <Emphasis Type="Italic">Xenopus</Emphasis> mandibular trigeminal axons <Emphasis Type="Italic">in vivo</Emphasis>

Background  

Trigeminal nerves consist of ophthalmic, maxillary, and mandibular branches that project to distinct regions of the facial epidermis. In Xenopus embryos, the mandibular branch of the trigeminal nerve extends toward and innervates the cement gland in the anterior facial epithelium. The cement gland has previously been proposed to provide a short-range chemoattractive signal to promote target innervation by mandibular trigeminal axons. Brain derived neurotrophic factor, BDNF is known to stimulate axon outgrowth and branching. The goal of this study is to determine whether BDNF functions as the proposed target recognition signal in the Xenopus cement gland.     

Adrenergic innervation of pancreatic islets and modulation of insulin secretion by the sympatho-adrenal system

Summary Morphological changes in the adrenergic innervation of pancreatic islets after chemical sympathectomy by use of 6-hydroxydopamine and the influence of the sympatho-adrenal system on insulin secretion were investigated in the mouse and rat.Fluorescence histochemistry revealed a clear-cut reduction in the number of adrenergic nerve fibers in the pancreatic islets 2 days after administration of 6-hydroxydopamine; the reduction was more pronounced in the rat than in the mouse. In the rat, a partial regeneration was seen after 6 weeks. In the pancreas of the mouse, after administration of 6-hydroxydopamine, a severe damage of unmyelinated nerve fibers was revealed electron microscopically. However, no ultrastructural or immunohistochemical alterations could be demonstrated in the endocrine cells of the islets.6-Hydroxydopamine induced a depression of basal plasma insulin concentrations in mice and an elevation in rats. Adrenalectomy depressed basal plasma insulin levels in mice.The -adrenoceptor antagonist phentolamine enhanced insulin secretion in normal mice. The secretory response of insulin to phentolamine was diminished by chemical sympathectomy and almost abolished by adrenalectomy or the combination of chemical sympathectomy and adrenalectomy. Thus, the effect of phentolamine is probably mediated by liberated catecholamines.It is concluded that basal insulin secretion is partially regulated by the sympatho-adrenal system and that species differences exist in this respect. In addition, the results suggest that endogenous catecholamines have the ability to promote insulin secretion.     

A Histochemical Study of the Gas Gland Innervation in the Atlantic God,Gadus morhua

A histochemical study was made of the distribution of catecholamines and cholinesterases in two autonomic ganglia closely associated with the swimbladder of the Atlantic cod, Gadus morhua. The “swimbladder nerve ganglion” comprised large (40 μm) neurones, the majority of which are positive for both catecholamines and acetylcholinesterase. It is argued that these neurones are mainly adrenergic postganglionic elements of sympathetic pathways which pass through the vago-sympathetic trunk. The “gas gland ganglion” comprised small (20 μm) neurones, positive for acetylcholinesterase but showing no catecholamine reaction. It is argued that these neurones are cholinergic postganglionic elements of the parasympathetic vagal innervation of gas gland cells.     

Fine structure of the neurohemal sinus gland of the shore crab,Carcinus maenas,and immuno-electron-microscopic identification of neurosecretory endings according to their neuropeptide contents

Summary The sinus gland of the shore crab, Carcinus maenas, is a compact assembly of interdigitating neurosecretory axon endings abutting upon the thin basal lamina of a central hemolymph lacuna. Four types of axon endings are distinguishable by the size distribution, shape, electron density and core structure of their neurosecretory granules. One additional type of axon ending is characterized by electron-lucent vacuoles and vesicles. The axon profiles are surrounded by astrocyte-like glial cells. Various fixations followed by epoxy- or Lowicrylembedding were compared in order to optimize the preservation of the fine structure of the granule types and the antigenicity of their peptide hormone contents. By use of specific rabbit antisera, the crustacean hyperglycemic, molt-inhibiting, pigment-dispersing, and red-pigment-concentrating hormones were assigned to the four distinct granule types which showed no overlap of immunostaining. Epi-polarization microscopy and ultrathin section analysis of immunogold-stained Lowicrylembedded specimens revealed that immunoreactivity to Leu-enkephalin and proctolin is co-localized with moltinhibiting hormone immunoreactivity in the same type of granule. The size and core structure of the immunocytochemically identified granule types vary little with the different pretreatments but, in some cases, to a statistically significant extent. The present results are compared with those from earlier studies of sinus glands in different crustaceans. The methods of granule identification used in this study supplement the classical approach in granule typing; they are easier to perform and more reliable for the analysis of release phenomena in identified secretory neurons supplying the neurohemal sinus gland.     

Cold prevents the light induced inactivation of pineal N-acetyltransferase in the Djungarian hamster,Phodopus sungorus

Summary In the Djungarian hamster seasonal acclimatization is primarily controlled by photoperiod, but exposure to low ambient temperature amplifies the intensity and duration of short day-induced winter adaptations. The aim of this study was to test, whether the pineal gland is involved in integrating both environmental cues. Exposure of hamsters to cold (0 °C) reduces the sensitivity of the pineal gland to light at night and prevents inactivation of N-acetyltransferase (NAT). The parallel time course of NAT activity and plasma norepinephrine content suggests that circulating catecholamines may stimulate melatonin synthesis under cold load.Abbreviations NAT N-acetyltransferase - NE norepinephrine - T _a ambient temperature     

Excitability changes in the crustacean motor axon following activity

It has been shown experimentally that the crustacean motor axon is supernormally excitable following a train of action potentials (Zucker 1974). Such a phenomenon can lead to recruitment of terminals which are unexcited at low rates of stimulation. Although currents underlying the crustacean motor axon have been characterized (Connor et al. 1977), it is not known whether this membrane model accounts for a supernormal period, what might cause superexcitablity in this model, or how excitability might change during repetitive stimulation. In present study, it is demonstrated that the crustacean motor axon model does predict a supernormal period, that the supernormal period results from slow recovery from inactivation of the transient potassium, or A, current, and that supernormal excitability is enhanced by repetitive stimulation.     

Ultrastructural study of the neurosecretory granules in the sinus gland of the blue crab,Callinectes sapidus

Summary Seven morphologically different types of neurosecretory granules have been found in the axon terminals of the sinus gland of the blue crab, Callinectes sapidus. They differ from each other in size, shape, staining characteristics, solubility characteristics, core matrix characteristics, axon terminal matrix characteristics, presence or absence of space between the granule membrane and granule core matrix, and frequency of occurrence. Five of the types are segregated in different axon terminals and are believed to represent different hormone-protein complexes. Two of the types, which have lost part or all of their granular contents, are thought to be variants of the other five types. The differences in granular morphology are better revealed by some fixation procedures than others. Palade's acetate-veronal buffered osmium tetroxide, in particular, reveals striking differences. The following observations suggest that different hormone-protein complexes are segregated in different axon terminals and that these complexes may be morphologically distinguished at the level of the electron microscope.Supported by USPHS-NIH Training Grant GM-00669 and Grant GB-7595X from the National Science Foundation.     

Microspectrofluorometric study of monoamines in the auricle of the heart of Protopterus aethiopicus

Summary The auricle of the heart of Protopterus aethiopicus contains large numbers of chromaffin cells, often lying immediately adjacent to the endothelium and displaying a bright blue-white fluorescence characteristic for catecholamines after formaldehyde treatment (Falck and Owman 1965). These results combined with X-ray microanalysis after initial fixation with glutaraldehyde and subsequent treatment with dichromate established that these chromaffin cells are the storage site of primary catecholamines (Scheuermann 1978, 1979, 1980; Scheuermann et al. 1980). The aim of the present pilot study was to demonstrate in these cells noradrenaline (NA) or dopamine (DA), or a mixture of both. The evaluation of the excitation spectra of the catecholamine fluorophore transformed by treatment with HCl vapour (excitation maxima at 320 and 370 nm) and the excitation-peak ratio analysis (peak ratio 370/320 nm =1.05–1.5; and 320/280 nm >1.5) identify DA as the primary catecholamine stored in these chromaffin cells. The low fading rate of the monoamine fluorescence after acidification confirms the presence of DA. These microspectrofluorometric findings demonstrate that chromaffin cells in the auricle of the Protopterus heart, which are a part of the medullary homologue of the adrenal gland of higher vertebrates, contain a primary catecholamine, namely DA.     

Histology and ultrastructure of the neurohypophysis of the south american lungfish,Lepidosiren paradoxa

Summary The neurohypophysis of the South American lungfish Lepidosiren paradoxa has been studied with light and electron microscopy, including the Falck-Hillarp technique for catecholamines. The pars nervosa hypophyseos is a well-marked, dorsally located subdivision of the pituitary gland composed of lobes or follicles, each one constituted of a central core of ependymal cells, a subependymal hilar region made up of nerve fibers and a peripheric palisade zone of nerve endings which contact capillary vessels. Four types of neurosecretory axons can be distinguished under the electron microscope. Type I, the most common, contains spherical elementary granules of high electron density, 1500–1800 Å in diameter. The scarce type II axons contain irregularly-shaped elementary granules. Type III contains only small clear vesicles, 400–600 Å in diameter. Type IV, mostly present in regions of the gland contacting the pars intermedia, contain large granulated vesicles, 900–1000 Å in diameter. The Falck-Hillarp technique revealed axons with a positive reaction for catecholamines at sites corresponding approximately to the location of type IV of the electron microscope.Ependymal cells are of large size, linking the cerebrospinal fluid, the nerve endings and the blood vessels. A conspicuous membrane-bound, spherical dense material, 1400–2000 Å in diameter, is observed in both the apical and vascular processes of these cells. The ependymal processes which traverse the hilar and palisade regions contain structures resembling degenerated neurosecretory axons. These results are discussed in relation with the currently available information on the comparative anatomy of the pars nervosa. The possible functional significance of ependymal cells and of each type of axon are also discussed.This study was aided by the following grants: NIH NS 06953 to Prof. De Robertis, Consejo Nacional de Investigaciones Científicas y Técnicas to Prof. Zambrano, Comisión de Investigaciones Científicas de la Provincia de Buenos Aires and Comisión de Investigaciones Cientificas de la Universidad Nacional de la Plata: to Prof. Iturriza.The authors are indebted to Prof. De Robertis for his generosity in granting us his laboratory facilities, and to Dr. F. J. J. Risso and Mr. A. Fernández (Resistencia, Chaco) who provided the specimens used in this study. The able microtechnical assistance of Miss L. Riboldazzi and Mrs. R. Raña and the photographic work of Mr. A. Saenz are much appreciated.Members of the Scientific Career, Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.     

Appearance and innervation of CHH-producing cells in the eyestalk of the crayfish Astacus leptodactylus examined after tracing with Lucifer Yellow

Summary By injection of the fluorescent dye Lucifer Yellow into individual Crustacean Hyperglycemic Hormone (CHH)-producing cells, the shape of these neurosecretory cells in the eyestalk of the crayfish Astacus leptodactylus can be traced. A highly fluorescent perikaryon gives rise to an axon that can be followed by the fluorescent label to the neurohemal region, the sinus gland. The proximal part of that axon sends out extensive branches into the neuropil of the medulla terminalis. Electron-microscopic investigations reveal synaptic input to these axonal ramifications.     

Redox reactions of neurotransmitters possibly involved in the progression of Parkinson's Disease

In Parkinson's Disease the neuromelanin in the substania nigra is known to contain considerably increased amounts of iron suggesting the presence of free, unprotected iron ions during its formation. Iron(II) is known to interact with peroxide via Fenton's reaction producing OH-radicals or ferryl (Fe(IV)) species. This can readily oxidize the neurotransmitter dopamine to the neurotoxic 6-hydroxydopamine (6-OHDA) which is a strong reducing agent. The produced 6-OHDA is, in turn, able to reduce and possibly release iron, as iron(II), from the iron storage protein ferritin. This cycle of events could well explain the development of Parkinson's Disease due to a continuous production of cell damaging species. The contrasting behaviour of 6-OHDA with some other important catecholamines is discussed.     

Effects of stimulation by carbachol on the metabolism of the bovine adrenal medulla

1. A method is described that has made it possible to achieve a great decrease in the catecholamine and adenine nucleotide contents of the perfused bovine adrenal gland by the infusion of carbachol. 2. Although the catecholamines secreted were recovered in the perfusion medium, no evidence was obtained that the nucleotides are secreted by the gland. 3. It is concluded that the secretion of catecholamines is accompanied by extensive chemical alteration of the adenine nucleotides of the chromaffin granules. 4. The secretory response and the spontaneous release of catecholamines depends on the presence of Ca²⁺ in the perfusing Tyrode solution. 5. Anoxia does not have a significant effect on the carbachol-induced secretion of catecholamines. 6. Strips of bovine adrenal medullary tissue perfused with oxygenated Tyrode solution show an increased oxygen consumption when carbachol is added.     

Neuropeptide tyrosine (NPY)-induced potentiation of the pressor activity of catecholamines in conscious rats

IV bolus administration of 2.5-50 micrograms NPY (0.6-12.5 nmol) to conscious rats produced a dose- and time-dependent increase in systolic and diastolic blood pressure. Following priming with 2.5 micrograms NPY, or larger doses, the subsequent administrations of noradrenaline produced pressor responses that were potentiated both in magnitude and duration. The NPY-induced potentiation of the pressor response to noradrenaline was dose-dependent and extended to the pressor action of adrenaline and angiotensin II but not to the hypotensions produced by bradykinin or isoproterenol. The potentiation was not related to the fact that multiple doses of catecholamines were repeated. Reserpine did not substantially modify the NPY-induced potentiation of the pressor activity of the catecholamines. Chemical sympathectomy following 6-hydroxydopamine caused a marked supersensitivity to the catecholamines and NPY but obliterated the NPY-induced potentiation of the pressor effect of adrenaline. Nifedipine reduced the pressor action of the catecholamines and NPY but did not attenuate the NPY-induced potentiation of the pressor action of catecholamines. It is concluded that the acute pressor effect of NPY and of the potentiation of the catecholamine pressor effects involve different mechanisms.     

Secretory and structural effects of 6-hydroxy-dopamine on normal parotid glands of rats, and at different times after surgical sympathectomy.

The effects of i.v. 6-hydroxydopamine (6-OHDA), 100 mg/kg, have been studied on parotid glands of rats at 12, 24, 48, 72 hr and 3 weeks after avulsion of the right superior cervical sympathetic ganglion. The salivary flow from normal left control glands and from right glands 12 hr after ganglionectomy were similar, but at longer times after ganglionectomy the secretory response from the test glands was greatly reduced. Morphological assessment showed that 6-OHDA induced a massive depletion of secretory granules from all control glands and also at 12 hr after ganglionectomy but at 48 and 72 hr there was considerably less depletion of granules on the ganglionectomized side. It is thought that at the longer times after ganglionectomy the secretion from the test glands is caused by circulating catecholamines released by the action of 6-OHDS on adrenergic nerves elsewhere, plus a possible small direct secretogogue effect oomy are thought to be attributable to the release of catecholamines from adrenergic nerves within the gland.     

Innervation of the C cells of chicken ultimobranchial glands studied by immunohistochemistry, fluorescence microscopy, and electron microscopy

Innervation of the ultimobranchial glands in the chicken was investigated by immunohistochemistry, fluorescence microscopy and electron microscopy. The nerve fibers distributed in ultimobranchial glands were clearly visualized by immunoperoxidase staining with antiserum to neurofilament triplet proteins (200K-, 150K- and 68K-dalton) extracted from chicken peripheral nerves. The ultimobranchial glands received numerous nerve fibers originating from both the recurrent laryngeal nerves and direct vagal branches. The left and right sides of the ultimobranchial region were asymmetrical. The left ultimobranchial gland had intimate contact with the vagus nerve trunk, especially with the distal vagal ganglion, but was somewhat separated from the recurrent nerve. The right gland touched the recurrent nerve, the medial edge being frequently penetrated by the nerve, but the gland was separated from the vagal trunk. The left gland was innervated mainly by the branches from the distal vagal ganglion, whereas the right gland received mostly the branches from the recurrent nerve. The carotid body was located cranially near to the ultimobranchial gland. Large nerve bundles in the ultimobranchial gland ran toward and entered into the carotid body. By fluorescence microscopy, nerve fibers in ultimobranchial glands were observed associated with blood vessels. Only a few fluorescent nerve fibers were present in close proximity to C cell groups; the C cells of ultimobranchial glands may receive very few adrenergic sympathetic fibers. By electron microscopy, numerous axons ensheathed with Schwann cell cytoplasm were in close contact with the surfaces of C cells. In addition, naked axons regarded as axon terminals or "en passant" synapses came into direct contact with C cells. The morphology of these axon terminals and synaptic endings suggest that ultimobranchial C cells of chickens are supplied mainly with cholinergic efferent type fibers. In the region where large nerve bundles and complex ramifications of nerve fibers were present, Schwann cell perikarya investing the axons were closely juxtaposed with C cells; long cytoplasmic processes of Schwann cells encompassed large portions of the cell surface. All of these features suggest that C-cell activity, i.e., secretion of hormones and catecholamines, may be regulated by nerve stimuli.     

Ultrastructure and ATPase activity of the rectal gland of the chondrichthyean fish Hydrolagus colliei (Holocephali)

Summary The anatomy, histology, ultrastructure and ATPase activity of the intramural rectal gland of the chondrichthyean Hydrolagus colliei, are described. The cells of the rectal gland of Hydrolagus demonstrate the same well developed lateral and basal cisternae, elongate mitochondria and luminal border as those of their elasmobranch counterparts. ATPase activity within the rectal gland of Hydrolagus is as intense as that in a number of elasmobranchs examined in the course of the study. Despite its primitive intramural location the rectal gland of Hydrolagus respresents a homolog of the more specialized and better known elasmobranch gland and appears as well suited for cation excretion.     

Localization of crustacean hyperglycemic hormone (CHH) in the X-Organ sinus gland complex in the eyestalk of the crayfish,Astacus leptodactylus (Nordmann, 1842)

The eyestalk of Astacus leptodactylus is investigated immunocytochemically by light, fluorescence, and electron microscopy, using an antiserum raised against purified crustacean hyperglycemic hormone (CHH). CHH can be visualized in a group of neurosecretory perikarya on the medualla terminalis (medulla terminalis ganglionic X-organ: MTGX), in fibers forming part of the MTGX-sinus gland tractus, and in a considerable part of the axon terminals composing the sinus gland. Immunocytochemical combined with ultrastructural investigations led to the identification of the CHH-producing cells and the CHH-containing neurosecretory granule type.