The study of adrenal chromaffin of fish, Carassius auratus (Toleostei).

In C. auratus the adrenal chramaffin tissue is situated around the posterior cardinal veins, in the head kidney. Chromaffin tissue consists of two types of cells containing secretory granules, adrenaline and nor adrenaline cells. The cells produced catecholamine hormones. Adrenaline cell contains electron-lucent granules, whereas nor adrenaline cells possesses electron-dense granules. Cholinergic fibers embedded in the head kidney innervated the chromaffin cell. Two types of secretory structures, synaptic vesicles and secretory granules are found within the presynaptic terminal. Secretory granules discharge their contests, as neuropeptide in non synaptic area of nerve terminal by exocytosis, whereas synaptic vesicles discharge their contents as neurotransmitters at the synaptic thickening (active zone) in the presynaptic terminal by exocytosis.

Different adrenal sympathetic preganglionic neurons regulate epinephrine and norepinephrine secretion.

Brain stimulation or activation of certain reflexes can result in differential activation of the two populations of adrenal medullary chromaffin cells: those secreting either epinephrine or norepinephrine, suggesting that they are controlled by different central sympathetic networks. In urethan-chloralose-anesthetized rats, we found that antidromically identified adrenal sympathetic preganglionic neurons (SPNs) were excited by stimulation of the rostral ventrolateral medulla (RVLM) with either a short (mean: 29 ms) or a long (mean: 129 ms) latency. The latter group of adrenal SPNs were remarkably insensitive to baroreceptor reflex activation but strongly activated by the glucopenic agent 2-deoxyglucose (2-DG), indicating their role in regulation of adrenal epinephrine release. In contrast, adrenal SPNs activated by RVLM stimulation at a short latency were completely inhibited by increases in arterial pressure or stimulation of the aortic depressor nerve, were unaffected by 2-DG administration, and are presumed to govern the discharge of adrenal norepinephrine-secreting chromaffin cells. These findings of a functionally distinct preganglionic innervation of epinephrine- and norepinephrine-releasing adrenal chromaffin cells provide a foundation for identifying the different sympathetic networks underlying the differential regulation of epinephrine and norepinephrine secretion from the adrenal medulla in response to physiological challenges and experimental stimuli.

Ultrastructural localization of nerve terminals containing nitric oxide synthase in rat adrenal gland.

Immunoelectron microscopy was performed to localize immunoreactivity for nitric oxide synthase (NOS) in rat adrenal gland. NOS-immunoreactive (NOS-ir) nerve fibers were observed around blood vessels and endocrine cells in the zona glomerulosa of the cortex. Electron microscopy showed that NOS-ir axon varicosities were in close contact with the smooth muscles of blood vessels and with the cytolemma of endocrine cells. In the adrenal medulla, several NOS-ir ganglion cells were found. Synaptic contacts between non-immunoreactive axons and dendrites of NOS-ir neurons were observed. NOS-ir nerve fibers were distributed among chromaffin cells. Positive axon varicosities were in close contact with the catecholamine-storing chromaffin cells. These results suggest that NOS-ir nerve cells control directly the secretion of corticosteroid and catecholamine in addition to the vascular tone.

The innervation of the chromaffin cells in the head kidney of the carp, Cyprinus carpio; regional differences of the connections between nerve endings and chromaffin cells.

Nerve fibres and their connections with chromaffin cells in the carp head kidney were studied by light and electron microscopy. Some nerve bundles entered the head kidney from the dorsal aspect along veins. Many unmyelinated axons emerged from the nerve bundles to invade the clusters of chromaffin cells, the distribution of which was restricted to the neighbourhood of the venous trunks and their tributaries. Most of the nerve endings were attached to a chromaffin cell by synaptic junctions and were generally invaginated into the cell. Some nerve endings were flattened in shape and connected with two chromaffin cells. Occasional exocytotic figures of synaptic vesicles opening into the intercellular space, or synaptic junctions along the course of the nerve fibre were observed. The percentage of the chromaffin cells supplied by nerve endings in the head kidney as a whole was similar to that in primitive amphibians. The distribution of the chromaffin cells and the frequency of their innervation suggest that carp chromaffin cells are phylogenetically similar to those of amphibians. The frequencies of synaptic connections in the carp head kidney showed regional differences. The number in dorsal portion was significantly higher than that in two ventral portions. It is suggested that chromaffin cells in the head kidney are separable into two populations: one (in the dorsal portion) shows closer and the other (in the ventral portions) less contact with nerve fibres. The fine structure of the nerve endings indicates that catecholamine secretion of carp chromaffin cells is partially modulated by nerve fibres (probably preganglionic cholinergic fibres). However, the low frequency of synaptic connections on the chromaffin cells and their distribution suggest that carp chromaffin cells are mainly modulated by the endocrine system via the bloodstream.

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-beta-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 intra-adrenal 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.

Comparative morphology, cytochemistry and innervation of chromaffin tissue in vertebrates.

Chromaffin cells were observed singly or in clusters in the heart and sympathetic cord of 2 genera of dipnoan fish, Protopterus and Lepidosiren. They were invariably found in close association with the autonomic sympathetic nervous system and at sites where chromaffin cells or their precursors are situated in mammals during ontogenetic development. X-ray microanalysis demonstrated that they contained a primary catecholamine which was identified microspectrofluorometrically as dopamine. The chromaffin cells were innervated by efferent axons with typical preganglionic sympathetic terminals which were acetylcholinesterase-positive. Although the general morphology and cytochemistry agree with those of developing intra-adrenal chromaffin cells in mammals, the morphological characteristics implicate them as active secretory gland cells. The dopamine transmitter phenotype seems to be determined by the maintenance throughout life of the separate and distant location of steroidogenic interrenal tissue from suprarenal elements.

Developmental regulation of leucine-enkephalin expression in adrenal chromaffin cells by glucocorticoids and innervation.

Most catecholaminergic cells derived from the sympathoadrenal lineage of the neural crest contain one or more neuropeptides. Although a great deal is known about the development and regulation of catecholaminergic properties in these cells, relatively little is known about the developmental control of their neuropeptidergic properties. We have investigated the possible role of glucocorticoids and preganglionic innervation in the regulation of leucine-enkephalin (L-Enk) expression in cultures of embryonic and neonatal adrenal chromaffin cells and in mature chromaffin cells in vivo. Exposure of embryonic and neonatal chromaffin cells to the synthetic glucocorticoid dexamethasone increases L-Enk content. Neonatal chromaffin cells grown in medium containing elevated levels of potassium to mimic depolarization also exhibited increased L-Enk levels. The depolarization-induced increase in L-Enk was selectively inhibited by treatment with the enkephalin analog [D-Ala, d-Leu]-enkephalin to mimic the enkephalinergic component of the preganglionic innervation. Denervation of the adrenal gland in vivo resulted in a dramatic increase in L-enk expression that could be partially mimicked by selectively blocking enkephalinergic transmission with administration of the opiate receptor antagonist naloxone. Taken together with the developmental time course and pattern of L-Enk expression in vivo, our results suggest that glucocorticoids and the preganglionic innervation regulate the developmental expression of this peptide in adrenal chromaffin cells and therefore participate in the generation of the mature neurochemical phenotypes present in the adrenal medulla. Further, in adult chromaffin cells similar factors appear to regulate the expression of L-Enk, which could in turn participate in physiological responses to stress.

The chromaffin cells of urodele amphibians.

Different conditions in the arrangement of the adrenal gland are observed in urodeles. The gland consists of islets scattered on the ventral surface of the kidneys, the amount, size and position of the islets varying consistently within different families and even within genera. The infraordinal variation also extends to the fine structure of the gland, as observed in 14 species belonging to 6 different families. The ultrastructural characteristics of chromaffin cells and their relationships with interrenal cells appear to be related to the phyletic position. In primitive urodeles (Sirenidae, Proteidae) the chromaffin cells are isolated or in small groups, mostly separated from interrenal cells and often in contact with renal cells. In neourodeles (Amphiumidae, Ambystomidae, Salamandridae, Plethodontidae) the chromaffin cells appear generally grouped and intermingled with steroidogenic cells. Some cytological characteristics of chromaffin cells, such as nerve supply and the shape and electron density of chromaffin granules exhibit a variability related to phyletic position.

The innervation of the adrenal gland. IV. Innervation of the rat adrenal medulla from birth to old age. A descriptive and quantitative morphometric and biochemical study of the innervation of chromaffin cells and adrenal medullary neurons in Wistar rats.

The innervation of the adrenal medulla has been investigated in normal Wistar rats from birth to old age and ultrastructural findings compared with biochemical markers of the cholinergic innervation of the adrenal gland and catecholamine storage. Morphological evidence of the immaturity of the innervation during the first postnatal week is provided and using quantitative morphometry the innervation of chromaffin cells is shown to reach a mean total of 5.4 synapses per chromaffin cell during the period 26 days to 12 weeks of age. The variation in contents of synaptic profiles is discussed in the light of recent work that demonstrates a major sensory as well as visceral efferent innervation of the gland. Adrenal medullary neurons usually occur in closely packed groups, intimately associated with Schwann cells. Axodendritic and axosomatic synapses on these neurons are described and the likely origin of axonal processes innervating the neurons discussed. In old age the density of innervation remains the same as in young adult animals even though the medulla shows evidence of hyperplasia and hypertrophy of individual chromaffin cells.

Catecholamine release by catecholamines in the eel does not require the presence of brain or anterior spinal cord.

The catecholamine-producing chromaffin cells of the American eel are strongly innervated by fibers, which, by ultrastructural criteria, seem to be cholinergic. However, neither removal of the brain nor removal of the brain combined with extirpation of the anterior spinal cord prevents the release of catecholamines into the circulation by catecholamines. It appears that the chromaffin cells are controlled by both nervous and humoral stimuli, and that at least some of the latter do not require the presence of "preganglionic" innervation.

The chromaffin cells of Siren lacertina (Amphibia, Urodela): cytological characteristics and evidence of exocytosis.

In the adrenal gland of Siren lacertina three types of chromaffin cells are described, on the basis of size, shape and electron density of the cytoplasmic granules: adrenaline-secreting cells, noradrenaline-secreting cells and small granulated chromaffin-cells. In A-cells exocytotic profiles are described, in which the granule membrane fuses with the plasma membrane and the granule core is discharged into the intercellular space.

[Innervation of the adrenal chromaffin cells of Anas boscas. Ultrastructural study]. / Sull'innervazione delle cellule cromaffini nell'adrenale di "Anas boscas". Studio ultrastrutturale.

The authors report on ultrastructural study of large nerve trunks, preterminal section of axons and sympatic contacts with chromaffin cells in adrenal gland of "Anas boscas". Only in rare cases, islets of chromaffin cells occur in the deep parts of parenchyma and, in their center, a fray of nerve fibres can be described.

Adrenal paraneurone contractile proteins and stimulus-secretion coupling.

Actin, myosin, and alpha-actinin have been isolated from adrenal chromaffin cells and characterized. Their physicochemical properties have been studied and their cell localization revealed by biochemical, immunocytochemical, and ultrastructural techniques. Alpha-actinin is a component of chromaffin granule membranes and some of the cell actin copurifies with these secretory granules. Myosin is not detected in the granules but is present mainly in the cytosol. Trifluoperazine, a calmodulin antagonist, blocks stimulation-induced hormone release from chromaffin cells at a step distal from Ca2+ entry. High affinity calmodulin binding sites have also been found in chromaffin granule membranes. Furthermore, microinjection of calmodulin antibodies into chromaffin cells blocks hormone output in response to stimulation. In view of the above findings, the possible roles of contractile proteins and calmodulin in chromaffin cell functions is discussed.

Receptors and receptor modulation in cultured chromaffin cells.

The colocalization of acetylcholine (ACh) and neuropeptides (e.g., substance P and enkephalins) in the splanchnic nerve terminals suggests that these compounds might interact to modulate adrenal catecholamine release. Use has been made of primary monolayer and suspension cultures of bovine adrenal chromaffin cells to investigate postsynaptic receptor interactions between acetylcholine and a number of neuropeptides endogenous to the adrenal medulla and splanchnic nerve. The cells have both nicotinic and muscarinic acetylcholine receptors, but only the nicotinic receptors stimulate catecholamine release. Substance P, somatostatin, and the enkephalins all produced an inhibition of the ACh-evoked secretion of catecholamines, but their potency ranged over 100-fold. Substance P was the most potent with a mean inhibitory concentration (IC50) of 10(-6) M and Leu-enkephalin the least potent with an IC50 greater than 10(-4) M. These pharmacological effects were monitored conveniently by measuring the release of [3H]norepinephrine preloaded into the cells or alternatively, "on-line" by measuring ATP released into an incubation medium containing luciferin and firefly tail extract (luciferase). Of interest, the endogenous enkephalin heptapeptide (Met-enkephalin Arg6-Phe7) and "big" Met-enkephalin (BAM- 22P ) were some 100-fold more effective than Leu- or Met-enkephalin at inhibiting the nicotinic secretin of catecholamines, suggesting that a unique opiate receptor may be involved. Substance P had two distinct actions on the nicotinic response: (1) substance P inhibited acetylcholine-induced release of catecholamines; and (2) substance P protected against acetylcholine-induced desensitization of catecholamine release. With regard to (1), substance P inhibited the secretion of catecholamines and ATP evoked by acetylcholine or nicotine but not that evoked by K+ or veratridine, nor did substance P by itself affect secretion. Substance P appeared to interact with a regulatory site on the acetylcholine receptor - ionophore complex. Substance P receptors on chromaffin cells have similar structural requirements for activation as do substance P receptors in other substance P responsive tissues. With regard to (2), substance P (greater than 5 X 10(-6) M) completely protected against desensitization of catecholamine release produced by acetylcholine (greater than 10(-4) M) or nicotine (greater than 2.5 X 10(-6) M) with no effect on K+-induced desensitization.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenal chromaffin cells form functional cholinergic synapses in culture.

Adrenomedullary cells and autonomic ganglion cells originate from the neural crest. Both cell types synthesize, store and release catecholamines; however, their structural and functional properties are distinctly different. Aloe and Levi-Montalcini have shown in vivo that when the adrenal medulla is exposed to exogenous nerve growth factor (NGF) most cells differentiate into neuronal cells substantially similar to sympathetic neurones. Experiments in vitro have also shown that neonatal as well as adult adrenal chromaffin cells and their neoplastic correlate (PC12 cells) undergo neurone-like morphologic differentiation in response to NGF. From these morphological and biochemical studies alone, however, it remains uncertain whether the functional neuronal transformation is also accompanied. We report here that the adrenal chromaffin cells in culture can differentiate into neuronal cells having functional synapses which were found to be cholinergic in nature. Furthermore, the type of synaptic vesicles in the newly formed synapses was apparently dependent upon K+ levels in the culture medium.

Neuro-histochemical study of the adrenal gland of Rattus rattus rufescens (Indian black rat) as revealed by cholinesterase technique.

The present investigation was undertaken to study the innervation and acetylcholinesterase (AChE) distribution in the adrenal gland of Rattus rattus rufescens (Indian black rat) by cholinesterase technique. The percentage of myelinated nerves in the cortical zone (cortex) and medulla zone was high. AChE-positive and multipolar ganglia on the outer medulla region, and the ganglia and nerve cells, arranged in chain-like fashion in the chromaffin tissue, were recorded. AChE activity was marked in the cortical zone (in the form of spots) and in the medulla zone (in the form of white and black grains).

Distribution and morphology of amphibian extra-adrenal chromaffin tissue.

1. The distribution and morphology of chromaffin cells in the para-aortic region and in the ganglia of the paravertebral sympathetic chain was studied with fluorescence histochemistry and electron microscopy. 2. Four types of chromaffin cell were distinguished largely on the basis of their vesicular content: Type I cells contain large, electron-dense vesicles (600-7000 A) and are comparable to noradrenaline-containing cells in the adrenal gland, Type II cells contain large, vesicles (600-7000 A) that are filled with a less electron-dense material than that in Type I cells and are comparable to adrenaline-containing cells in the adrenal gland, Type III cells contain smaller vesicles (1000-3000 A) that are incompletely filled with an electron-dense material and may represent cells that have been depleted of their catecholamines by stimulation, Type IV cells are clearly different from the other three cell types with respect to the size and appearance of the vesicles (1000-1500 A), nuclei and rough endoplasmic reticulum and may represent immature sympathetic neurons. 3. Nerve profiles, identified as cholinergic, were found in close apposition with all four cell types. No examples of a close association between processes of chromaffin cells and sympathetic neurons were found.