Calcitonin gene-related peptide (CGRP)-like immunoreactivity in scattered chromaffin cells and nerve fibers in the adrenal gland of rats

Summary The present immunohistochemical study reveals that a small number of chromaffin cells in the rat adrenal medulla exhibit CGRP-like immunoreactivity. All CGRP-immunoreactive cells were found to be chromaffin cells without noradrenaline fluorescence; from combined immunohistochemistry and fluorescence histochemistry we suggest that these are adrenaline cells. In addition, all CGRP-immunoreactive cells simultaneously exhibited NPY-like immunoreactivity. CGRP-chromaffin cells were characterized by abundant chromaffin granules with round cores in which the immunoreactive material was densely localized. These findings suggest the co-existence of CGRP, NPY and adrenaline within the chromaffin granules in a substantial number of chromaffin cells.Thicker and thinner nerve bundles, which included CGRP-immunoreactive nerve fibers, with or without varicosities, penetrated the adrenal capsule. Most of them passed through the cortex and entered the medulla directly, whereas others were distributed in subcapsular regions and among the cortical cells of the zona glomerulosa. Here the CGRP-fibers were in close contact with cortical cells. A few of the fibers supplying the cortex extended further into the medulla. The CGRP-immunoreactive fibers in the medulla were traced among and within small clusters of chromaffin cells and around ganglion cells. The CGRP-fibers were directly apposed to both CGRP-positive and negative chromaffin cells, as well as to ganglion cells. Immunoreactive fibers, which could not be found close to blood vessels, were characterized by the presence of numerous small clear vesicles mixed with a few large granular vesicles. The immunoreactive material was localized in the large granular vesicles and also in the axoplasm. Since no ganglion cells with CGRP-like immunoreactivity were found in the adrenal gland, the CGRP-fibers are regarded as extrinsic in origin. In double-immunofluorescence staining for CGRP and SP, all the SP-immunoreactive fibers corresponded to CGRP-immunoreactive ones in the adrenal gland. This suggests that CGRP-positive fibers in the adrenal gland may be derived from the spinal ganglia, as has been demonstrated with regard to the SP-nerve fibers.     

Intracellular localization of tyrosine hydroxylase immunoreactivity in the rat adrenal chromaffin and pheochromocytoma cells

The localization of tyrosine hydroxylase (TH) immunoreactivity in rat adrenal chromaffin and pheochromocytoma (PC12) cells was investigated by immunoelectron microscopy using monoclonal and polyclonal antisera against TH purified from rat adrenal medulla. Strong TH immunoreactivity was found uniformly in the granules of the adrenaline cells; the immunoreactivity was visible mainly within the periphery, but not in the clear space of the granules of the noradrenaline cells. In the PC12 cells, strong TH immunoreactivity was also observed uniformly in the granules. In addition, TH immunoreactivity was seen in the cytoplasm, the ribosomes attached to the endoplasmic reticulum and the free ribosomes of both the rat adrenal chromaffin and PC12 cells. These results suggest that TH may be localized in the granules, cytoplasm and ribosomes of rat adrenal chromaffin and PC12 cells.     

Gamma-aminobutyric acid (GABA) immunoreactivity in the mouse adrenal gland

Gamma-aminobutyric acid (GABA) immunoreactivity was revealed by immunocytochemistry in the mouse adrenal gland at the light and electron microscopic levels. Groups of weakly or faintly GABA immunoreactive chromaffin cells were often seen in the adrenal medulla. By means of immunohistochemistry combined with fluorescent microscopy, these GABA immunoreactive chromaffin cells showed noradrenaline fluorescence. The immunoreaction product was seen mainly in the granular cores of these noradrenaline cells. These results suggest the co-existence of GABA and noradrenaline within the chromaffin granules. Sometimes thick or thin bundles of GABA immunoreactive nerve fibers with or without varicosities were found running through the cortex directly into the medulla. In the medulla, GABA immunoreactive varicose nerve fibers were numerous and were often in close contact with small adrenaline cells and large ganglion cells; a few, however, surrounded clusters of the noradrenaline cells, where membrane specializations were formed. Single GABA immunoreactive nerve fibers, and thin or thick bundles of the immunoreactive varicose nerve fibers ran along the blood vessels in the medulla. The immunoreaction deposits were observed diffusely in the axoplasm and in small agranular vesicles of the GABA immunoreactive nerve fibers. Since no ganglion cells with GABA immunoreactivity were found in the adrenal gland, the GABA immunoreactive nerve fibers are regarded as extrinsic in origin.     

Cytochemistry and ultrastructure of the prenatal porcine adrenal medulla

The ultrastructure and cytochemistry of fetal porcine adrenal medullae have been studied at 60, 80, and 100 days of gestation. Adrenal medullae from fetuses at 60 days of pregnancy consisted of norepinephrine cells only. Some cells containing chromaffin granules were seen in the process of mitosis. A few epinephrine cells were present in the outer medullary zone at 80 days at pregnancy, their number increasing by the 100 day of pregnancy. Chromaffin cells containing both norepinephrine and epinephrine storing granules were also present at 80 and 100 days of gestation. Norepinephrine and epinephrine specific granular vesicles in the fetal adrenal medullary cells were smaller than those reported for the adult pig. The general ultrastructural characteristics of the porcine fetal adrenal medulla were similar to those reported for prenatal adrenal medulla of other species.     

On the chromaffin cells in dog adrenal medulla; with special reference to the small granule chromaffin cells (SGC cells)

Small granule chromaffin cells (SGC cells) were identified in the adrenal medulla of adult dogs. They were small in size and usually showed a high nucleo-cytoplasmic ratio. Cytoplasmic projections were occasionally observed in some of these cells. They contained a variable number of small secretory granules with diameters ranging from 70 to 300 nm, but mostly from 100 to 200 nm. The densities of the secretory granules were variable, ranging from highly dense to less dense. These adrenal SGC cells were rich in free ribosomes and polysomes, but were relatively poor in other cell organelles. Chromaffin cells which were intermediate in their characteristics (IM cells) between the SGC cells and the typical A and N cells were also identified. These IM cells contained both highly electron dense and less dense granules in various proportions. The IM cells were classified into two subgroups, according to the proportions of adrenaline type granules and noradrenaline type granules. One group resembled A cells (IM-A cells) and the other resembled N cells (IM-N cells). Light microscopic histochemical studies of A cells stained with the ammoniacal silver solution demonstrated that they contained a small number of darkly stained granules. Electron microscopic cytochemistry revealed that the electron dense granuls in the SGC cells, IM cells and A cells reacted positively with both the potassium dichromate solution at pH 4.1 and the ammoniacal silver solution.     

Growth characteristics of postnatal rat adrenal medulla in culture

Explants and enzyme-dispersed cells of adrenal medulla from 10-12 day old rats were studied in culture for up to 3 weeks. Adrenomedullary chromaffin cells, nerve cells and satellite cells were clearly discernible. The nerve cells were few in number and did not show catecholaminespecific fluorescence. Chromaffin cells stored catecholamines, as judged by the Falck and Hillarp method, in varying amounts decreasing with age of the cultures and the distance from the explants. Exocytosis profiles observed with the electron microscope suggested that cultured chromaffin cells also released catecholamines. Moreover, the cells formed processes and frequently migrated into the outgrowth. After 6 days in culture, the great majority of chromaffin cells stored noradrenaline as revealed by electron microscopy with few adrenaline-storing cells being visible. Granular vesicles (approximately 80-240 nm in diameter) with cores of different electron densities were occasionally present in the same cell suggesting the occurrence of mixtures of primary and secondary amines. Apart from "chromaffin" granules, small clear and dense-cored vesicles (approximately 40-60 nm) were found both in the somata and cell processes. Chromaffin cells and their processes were often closely apposed and occasionally formed specialized attachment zones. As a whole, chromaffin cells in culture resembled small granule-containing cells in sympathetic ganglia. 0.5 mM dbcAMP prevented dedifferentiation of chromaffin cells as judged by the lack of processes, the size and amount of "chromaffin" granules and the high number of adrenaline-storing cells present after 6 days in culture. NGF caused a striking increase in the number of axons growing out from explants.     

Adrenomedullary function is severely impaired in 21-hydroxylase-deficient mice.

Deficiency of 21-hydroxylase (21-OH), one of the most common genetic defects in humans, causes low glucocorticoid and mineralocorticoid production by the adrenal cortex, but the effect of this disorder on the adrenomedullary system is unknown. Therefore, we analyzed the development, structure, and function of the adrenal medulla in 21-OH-deficient mice, an animal model resembling human congenital adrenal hyperplasia. Chromaffin cells of 21-OH-deficient mice exhibited ultrastructural features of neuronal transdifferentiation with reduced granules, increased rough endoplasmic reticulum and small neurite outgrowth. Migration of chromaffin cells in the adrenal to form a central medulla was impaired. Expression of phenylethanolamine-N-methyltransferase (PNMT) was reduced to 27 +/- 9% (P<0.05), as determined by quantitative TaqMan polymerase chain reaction, and there was a significant reduction of cells staining positive for PNMT in the adrenal medulla of the 21-OH-deficient mice. Adrenal contents of epinephrine were decreased to 30 +/- 2% (P<0. 01) whereas norepinephrine and dopamine levels were reduced to 57 +/- 4% (P<0.01) and 50 +/- 9% (P<0.05), respectively. 21-OH-deficient mice demonstrate severe adrenomedullary dysfunction, with alterations in chromaffin cell migration, development, structure, and catecholamine synthesis. This hitherto unrecognized mechanism may contribute to the frequent clinical, mental, and therapeutic problems encountered in humans with this genetic disease.     

Adrenal medullary basophilia in ox,pig and sheep

Summary In ox, pig and sheep the adrenaline storing cells are intensely basophilic compared with the noradrenaline storing cells when aldehyde fixed tissue is stained with toluidine blue at pH 5.0 and above. This has been shown to be due to carboxyl groups from the glutamate rich chromaffin granule soluble protein. In isolated chromaffin granules adenosine nucleotides also bind the dye. Fixation of adrenal medulla in agents not containing aldehydes, or the use of cryostat sections results in equal basophilia in the adrenaline and noradrenaline storing cells. The probable mechanism of the differential basophilia of the two sorts of medullary cells following aldehyde fixation is discussed.     

Immunocytochemical mapping of basic fibroblast growth factor in the developing and adult rat adrenal gland

We studied the spatial and temporal pattern of basic fibroblast growth factor (bFGF) immunoreactivity in the rat adrenal gland during postnatal development. In the cortex the glomerulosa zone reveals a strong anti-bFGF immunoreactivity at all developmental ages studied. In the fasciculata zone the high number of anti-bFGF immunoreactive cells in the first week decreases during the second and third week. The late developing reticularis zone shows only few anti-bFGF labeled cells at all postnatal ages. This distributional pattern of bFGF immunoreactivity matches that of mitotic activity in the rat adrenal cortex strengthening the role of bFGF as an autocrine growth factor for adrenocortical cells. In the medulla anti-bFGF positive chromaffin cells become detectable at postnatal day (P) 8 and increase in number during the second and third week. In the adult rat the staining intensity of the chromaffin cells was higher than at P18. In the adult medulla bFGF colocalizes with noradrenaline suggesting its presence in a chromaffin cell subpopulation. In accordance with previous results the role of the chromaffin cell bFGF as a neurotrophic factor for preganglionic sympathetic neurons is discussed.     

Immunocytochemical mapping of basic fibroblast growth factor in the developing and adult rat adrenal gland

Summary We studied the spatial and temporal pattern of basic fibroblast growth factor (bFGF) immunoreactivity in the rat adrenal gland during postnatal development. In the cortex the glomerulosa zone reveals a strong anti-bFGF immunoreactivity at all developmental ages studied. In the fasciculata zone the high number of anti-bFGF immunoreactive cells in the first week decreases during the second and third week. The late developing reticularis zone shows only few anti-bFGF labeled cells at all postnatal ages. This distributional pattern of bFGF immunoreactivity matches that of mitotic activity in the rat adrenal cortex strengthening the role of bFGF as an autocrine growth factor for adrenocortical cells. In the medulla anti-bFGF positive chromaffin cells become detectable at postnatal day (P) 8 and increase in number during the second and third week. In the adult rat the staining intensity of the chromaffin cells was higher than at P18. In the adult medulla bFGF colocalizes with noradrenaline suggesting its presence in a chromaffin cell subpopulation. In accordance with previous results the role of the chromaffin cell bFGF as a neurotrophic factor for preganglionic sympathetic neurons is discussed.     

Heterogeneous distribution of neurocalcin-immunoreactive nerve terminals in the mouse adrenal medulla

Neurocalcin is a novel calcium-binding protein found in bovine brain tissue. We investigated immunoreactivity for neurocalcin in the mouse adrenal medulla using light and electron microscopy. The immunoreactivity was present in nerve fibers, nerve terminals, and ganglion cells in the adrenal medulla, but chromaffin cells, sustentacular cells, and Schwann cells were negative in reaction. Nerve bundles containing neurocalcin-immunoreactive fibers passed through the adrenal cortex and extended into the medulla. Immunopositive nerve fibers branched off and projected varicose terminals around the chromaffin cells. These varicose terminals contained small and large-cored vesicles and made synapses with the chromaffin cells. We performed paraformaldehyde-induced fluorescence-histochemical studies for catecholamine combined with immunohistochemical studies for neurocalcin. Neurocalcin-immunoreactive nerve terminals were more abundant at noradrenaline (fluorescent) cell-rich regions than at adrenaline (non-fluorescent) cell-rich regions. These results show that neurocalcin-immunoreactive nerves mainly innervate noradrenaline-containing chromaffin cells in the mouse adrenal medulla and that neurocalcin may regulate synaptic function in the nerve terminals. Received: 21 October 1996 / Accepted: 12 February 1997     

An immunohistochemical study of the adrenal medulla of the ox

Summary The adrenal medulla of ox was studied by an indirect immunofluorescent technique using anti-ox chromaffin granule serum. The serum had a weak cross reaction with ox brain stem and splenic nerve. There was a species cross reaction with sheep, pig and horse. Immunoelectrophoresis showed five components in the serum against ox adrenal lysates. The whole adrenal medulla of ox was found to fluoresce by the immunofluorescent technique but not the cortex. The adrenals of sheep, pig and horse behaved similarly using the anti-ox serum. A serum prepared against ox chromogranin-A, the most abundant soluble protein of the chromaffin granules, was also used for immunofluorescence. Again both the adrenaline and noradrenaline storing cells fluoresced, but not the cortex.     

Histological, histochemical and ultrastructural studies on the interrenal and chromaffin cells of the fathead minnow, Pimephales promelas Rafmesque

Light and electron microscopic examination of fathead minnow head kidneys revealed that the interrenal and chromaffin cells were intermingled and always closely associated with the cardinal veins and their tributaries. Histochemical tests for lipids in the interrenal cells were positive, and two types of chromaffin cells were indicated by chromaffin reactions. Interrenal cells contained abundant smooth endoplasmic reticulum and mitochondria with tubulo-vesicular cristae, characteristic of steroid-producing cells. Only one interrenal cell type was found. Two types of chromaffin cells were present with differences in cytoplasmic density and in types of granules. In light cells, adrenaline granules were most common, and in dark cells noradrenaline granules predominated.     

Bombesin-like immunoreactivity in bovine adrenal medulla

The presence of immunoreactive (ir)-bombesin in bovine adrenal medulla, isolated adrenal chromaffin cells and subcellular fractions of the adrenal medulla was demonstrated using a specific antibody to the synthetic peptide. High levels of ir-bombesin were detected in acid (HCl) extracts of the adrenal tissue (27 pmol/g) and isolated cells (0.35 pmol per 10(6) cells). Subpopulations of adrenal chromaffin cells were also obtained by centrifugation of the original cell preparation through a stepwise bovine serum albumin gradient (cell layers I, II and III). The highest concentration of ir-bombesin (0.77 pmol/10(6) cells) was found in a cell population (cell layer I) enriched in noradrenaline (adrenaline/noradrenaline ratio of 0.6). At the subcellular level, ir-bombesin was mainly concentrated in the secretory granules (0.61 pmol/mg protein) along with catecholamines (1097 nmol/mg protein), but a relatively high concentration of ir-bombesin (0.26 pmol/mg protein) was also found in the microsomal fraction. Isolation and high performance liquid chromatography (HPLC) analysis of adrenomedullary ir-bombesin revealed the presence of four molecular forms, one of them corresponding to gastrin releasing peptide (GRP), another one (major peak) eluting closely to synthetic neuromedin B and another one coeluting with GRP-(18-27). HPLC analysis of the molecular forms of ir-bombesin in the microsomes and secretory granules indicated that GRP- and neuromedin B-like materials can be generated between the two fractions.     

Fine structure and innervation of the avian adrenal gland

Summary According to their ultrastructure and histochemistry three types of efferent nerve fibers can be distinguished in the bird's adrenal gland. The main part is made up of cholinergic fibers recognizable by a positive reaction for acetylcholinesterase and two specific populations of granules within the synaptic ending. Synaptic vesicles measuring 300 to 500 Å in diameter and dense-cored vesicles with a diameter of about 1 000 Å are discernible.In the periphery of the gland cholinergic axons for the innervation of adrenal cells form large bundles surrounded by a perineural sheath. The bundles cross the capsule and are situated within the adrenal chromaffin cords or at their periphery. Finally small groups of fibers enter a group of chromaffin cells which are surrounded by a basal lamina and which consist of about a dozen or more cells producing adrenaline and noradrenaline. Synaptic endings occur, above all in passeriform species, in the center of a chromaffin cell complex. They are either attached to the innervated cells or their dendrite-like processes, or embedded into the cells, or connected to short spines of the innervated cells. Synaptic and dense-cored vesicles leave the bouton by exocytosis. One synaptic terminal may innervate up to three A- or NA-cells. The existence of different types of synapses for A- and NA-cells cannot be excluded.Supported by a grant from the Deutsche Forschungsgemeinschaft (Un 34/1).     

Localization of enkephalins in adrenaline cells and the nerves innervating adrenaline cells in rat adrenal medulla

The coexistence of met5- and leu5-enkephalin-like immunoreactivities with catecholamines in the rat adrenal medulla was studied with combined fluorescence microscopy and immunocytochemistry. Both met5- and leu5-enkephalin-like immunoreactivities were localized in few heavily stained adrenaline cells and in a population of nerves innervating adrenaline cells and as well as ganglion cells among the adrenaline cells. Only occasionally single noradrenaline cells exhibited light immunostaining for both enkephalins but no positive fibers could be found around the noradrenaline cells. In electron microscope the immunoreaction was seen in the granules of the adrenaline cells and in the large synaptic vesicles of the nerve terminals around the adrenaline cells. The present findings suggest that enkephalin-like immunoreactivity coexists mainly with adrenaline in rat adrenal medulla and that the enkephalin immunoreactive terminals regulate secretion of adrenaline from rat adrenal medulla.     

Localization of enkephalins in adrenaline cells and the nerves innervating adrenaline cells in rat adrenal medulla

Summary The coexistence of met5- and leu5-enkephalinlike immunoreactivities with catecholamines in the rat adrenal medulla was studied with combined fluorescence microscopy and immunocytochemistry. Both met5- and leu5-enkephalin-like immunoreactivities were localized in few heavily stained adrenaline cells and in a population of nerves innervating adrenaline cells and as well as ganglion cells among the adrenaline cells. Only occasionally single noradrenaline cells exhibited light immunostaining for both enkephalins but no positive fibers could be found around the noradrenaline cells. In electron microscope the immunoreaction was seen in the granules of the adrenaline cells and in the large synaptic vesicles of the nerve terminals around the adrenaline cells. The present findings suggest that enkephalin-like immunoreactivity coexists mainly with adrenaline in rat adrenal medulla and that the enkephalin immunoreactive terminals regulate secretion of adrenaline from rat adrenal medulla.     

Differentiation of embryonic chick sympathetic neurons in vivo: ultrastructure,and quantitative determinations of catecholamines and somatostatin

Summary The ultrastructural and transmitter development of lumbar sympathetic ganglia was studied in embryonic day-6 through-18 chick embryos. At embryonic day 6, ganglia are populated by two morphologically distinct types of neuronal cells and Schwann cell precursors. The neuronal populations basically comprise a granule-containing cell and a developing principal neuron. Granule-containing cells have, an irregularly shaped or oval nucleus with small clumps of chromatin attached to the inner nuclear membrane and numerous large (up to 300 nm) membrane-limited granules. Developing principal neurons display a more rounded vesicular nucleus with evenly distributed chromatin, prominent nucleoli, more developed areas of Golgi complexes, and rough endoplasmic reticulum and large dense-core vesicles up to 120 nm in diameter. There are granule-containing cells with fewer and smaller granules which still display the nucleus typical for granule-containing cells. These granule-containing cells may develop toward developing principal neurons or the resting state of granule-containing cells found in older ganglia. Both granule-containing cells and developing principal neurons proliferate and can undergo degeneration. At embryonic day 9 there are far more developing principal neurons than granule-containing cells. Most granule-containing cells have very few granules. Mitotic figures and signs of cell degeneration are still apparent. Synapse-like terminals are found on both developing principal neurons and granule-containing cells. Ganglionic development from embryonic day 11 through 18 comprises extensive maturation of developing principal neurons and a numerical decline of granule-containing cells. Some granule-containing cells with very few and small granules still persist at embryonic day 18. The mean catecholamine content per neuron increases from 0.044 femtomol at embryonic day 7 to 0.22 femtomol at embryonic day 15. Concomitantly, there is a more than 6-fold increase in tyrosine hydroxylase activity. Adrenaline has a 14% share in total catecholamines at embryonic day 15. Somatostatin levels are relatively high at embryonic day 7 (1.82 attomol per neuron) and are 10-fold reduced by embryonic day 15. Our results suggest the presence of two morphologically distinct sympathetic neuronal precursors at embryonic day 6: one with a binary choice to become a principal neuron or to die, the other one, a granule-containing cell, which alternatively may develop into a principal neuron, acquire a resting state or die.     

The determination of the adrenal medullary cell fate during embryogenesis

One subset of neural crest cells, the sympathoadrenal precursors, undergoes a switch in phenotype expression, when they invade the adrenal anlagen and become associated with adrenocortical cells. To investigate the mechanisms responsible for the conversion of noradrenaline synthesizing precursors to adrenaline producing endocrine chromaffin cells we studied the role of glucocorticoids on the initial induction of adrenaline synthesis in embryonic adrenals and cultures of highly purified chromaffin precursor cells. We could show that in vivo differentiation of rat chromaffin precursors commences between 16.3 and 17.3 days of gestation. While adrenaline and the activity of the enzyme phenylethanolamine N-methyltransferase (PNMT), which converts noradrenaline to adrenaline, were present at Embryonic Day 17.3 (E17.3), they were not detectable in E16.3 adrenals. Small amounts of corticosterone were present in E16.3 adrenals and plasma, but in parallel with the initial induction of adrenaline biosynthesis, a sharp rise in organ and plasma glucocorticoid levels occurred until E17.3. Chromaffin precursor cells, isolated at E16.3 and cultured for 4 days, failed to express PNMT activity and adrenaline. However, 0.1 nM dexamethasone was already sufficient for the initial induction of adrenaline and its synthesizing enzyme. Specific glucocorticoid binding of freshly isolated chromaffin (precursor) cells revealed a developmental increase during embryogenesis, yet no glucocorticoid binding sites were detectable in chromaffin precursor cells at E16.3. They appeared at E17.3 in parallel with the initial induction of adrenaline biosynthesis and the enormous rise of adrenal and plasma corticosterone levels. We therefore conclude that glucocorticoids are essential and sufficient to trigger the differentiation of noradrenergic sympathoadrenal precursors to adrenergic chromaffin cells after a functional glucocorticoid receptor system has been established.     

Structural characteristics and innervation of chromaffin tissue in the adrenal gland of the axolotl

Each adrenal gland of the Axolotl consists of a strip lying all along the medio-lateral edge on the ventral surface of the kidney. The gland is composed of interrenal cells (IC) and chromaffin cells (CC). The IC contained a great number of pleomorphic lipid droplets, smooth endoplasmic reticulum and elongated mitochondria with tubulo-vesicular cristae. Two types of CC, always disposed in clusters and exhibiting long cytoplasmic processes were described according to the electron density, size and shape of granules distributed in their cytoplasm; noradrenaline cells (NA) and adrenaline cells (A). The innervation and ultrastructural differences from the adrenal gland of other Anurans were discussed.