Catecholamine-storing cells in the adrenal medulla of the pre- and postnatal rat

Summary The development of the rat adrenal medulla was studied at the ultrastructural level with particular emphasis placed on early discrimination of different catecholamine-storing cells. The first granule-containing cells, phaeochromoblasts, were seen at day 15 of gestation migrating into the anlage of the cortex. These cells were characterized by a few small granules (80–120 nm in diameter) and a high nuclear to cytoplasmic ratio. Presumably due to differentiation into chromaffin cells, they were no longer present after the eighth postnatal day. Maturation of phaeochromoblasts was indicated by an increase in number and size of their storage granules and a decrease in the nuclear to cytoplasmic ratio. Noradrenaline and adrenaline cell types were first clearly discernible at day 21 of gestation. Another cell type, a giant cell, was also recognized at this stage. In the adult animal, noradrenaline, two morphologically different types of adrenaline, and small granule-containing cells were observed.By applying acetylcholinesterase histochemistry, it was found that at day 17 of gestation a small population of granule-storing cells showed strong positive staining in the endoplasmic reticulum. In the adult animal this cell type was further characterized by small-storage granules. Other chromaffin cells began to show weak staining within the endoplasmic reticulum at day 19 of gestation. This staining appeared more frequently within adrenaline than noradrenaline cells. However, even in the adult animal many cells of both types were completely negative.It is concluded that acetylcholinesterase histochemistry is a useful method for early discrimination of small granule-containing cells in the developing rat adrenal medulla.Supported by grants from the Deutsche Forschungsgemeinschaft     

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.     

Effects of combined hypophysectomy and cyproterone acetate on the adrenal chromaffin cells of the rat. Evidence for postnatal migration of adrenal chromaffin cells

A light and electron-microscopic study was performed concerning the effects of hypophysectomy (HP) followed by cyproterone acetate (CA) treatment on the adrenal chromaffin cells of the rat. The latter drug is reported to interfere with steroid biosynthesis. When given following HP, CA was found to induce degeneration in the inner cells of the zona fasciculata and a marked fall in the blood corticosterone level. The adrenal chromaffin cells which were depleted after the administration of reserpine at the beginning of the experiment failed to recover two weeks later. In addition, some of the depleted chromaffin cells migrated towards the outer cells of the zona fasciculata, had the appearance of pheochromoblasts and showed features of increased synthetic activity. The results are discussed in the light of the generally accepted functional relationship between the cells of the adrenal cortex and the medulla and following a marked fall in the concentration of blood corticosterone, a chemobiotaxis is suggested between the chromaffin and adrenal cortical cells.     

Role of intracellular pH in secretion from adrenal medulla chromaffin cells

The role of intracellular pH in stimulus-secretion coupling was investigated in cultured bovine adrenal medullary chromaffin cells. NH4Cl (1-25 mM) did not affect basal catecholamine or ATP release but markedly inhibited nicotine- or high K+-induced release by up to 60%. The inhibition had a rapid onset (less than 1 min) and was maximal at about 5 mM NH4Cl. The effect of NH4Cl was largely sustained over 20 min and was reversed upon NH4Cl removal. Sodium propionate did not affect secretion but partially reversed the inhibition by NH4Cl in a concentration-dependent manner. Methylamine (10 mM) produced a similar, but slower, inhibition than NH4Cl. Monensin (1-10 microM) inhibited catecholamine secretion by 30-60%, and its effect was reduced in the presence of NH4Cl. Using the fluorescent Ca2+ probe Fura-2, we found that the increase of [Ca2+]i following stimulation was not altered by concentrations of NH4Cl which inhibited secretion maximally. Measurement of cytosolic pH (pHi) with the fluorescent probe 2',7'-bis-carboxyethyl-5(6)-carboxyfluorescein (BCECF) revealed an alkalinization by NH4Cl (2.5-25 mM) of 0.1-0.23 pH units and acidification by sodium propionate (10-20 mM) of 0.2-0.25 pH units, with intermediate combined effects. Monensin (1 microM) caused a cytosolic acidification of 0.26 pH units. All pHi changes were partly recovered in 15 min. Fluorescence quenching measurements using the weakly basic fluorescent probe acridine orange indicated the accumulation of the probe into acidic compartments, presumably the chromaffin granules, which was strongly reduced by both NH4Cl and monensin. From these findings we conclude that the pH of the chromaffin granule modulates secretion by affecting some step in the secretory process unrelated to the rise in [Ca2+]i.     

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.     

Synapses between chromaffin cells of the adrenal medulla of mice transplanted into the brain

Mouse adrenal medullae were transplanted into the brain and studied 4 weeks or more later. In these transplants, some chromaffin cells extend neurite-like processes and form synapses on other chromaffin cells. These synapses exhibit the ultrastructural features of chemical synapses. The possible neoformation of these synapses after transplantation is discussed.     

Characterization of specific insulin binding sites on chromaffin cells from bovine adrenal medulla

1. Insulin receptors were investigated in isolated chromaffin cells from bovine adrenal medulla. 2. The cells were incubated with [125I]insulin in HEPES buffer, pH 7.8 at 15 degrees C for 180 min to obtain steady state binding. Specific binding was linearly related to the number of cells in the range 0.5-10 x 10(6) cells/ml. Insulin and proinsulin caused half maximal displacement of specifically bound tracer in concentrations of 0.18 and 2.46 nM, respectively. 3. Computer analysis of the binding data gave a linear Scatchard plot, consistent with a single class of non-interacting receptors with an affinity constant of 5.6 nM-1, the total number of receptors per cell being 1700. 4. The apparent MW of the insulin binding subunit of the receptor was 135,000, determined by affinity crosslinking and SDS gel electrophoresis under reducing conditions.     

Brefeldin A increases the quantal size and alters the kinetics of catecholamine release from rat adrenal chromaffin cells.

The fungal metabolite, brefeldin A (BFA), is known to inhibit guanine nucleotide exchange on the ADP-ribosylating factors that are involved in vesicle membrane trafficking. Here, we investigated the action of BFA on Ca2+-regulated exocytosis in single rat adrenal chromaffin cells. Incubation of chromaffin cells with BFA (1 or 10 microM) for 2 h effectively disrupted the Golgi membranes but did not affect the pattern of catecholamine release triggered by high extracellular K+, which was monitored with carbon fiber amperometry along with cytosolic Ca2+ measurement. The BFA treatment, however, increased the mean quantal size of catecholamine-containing vesicles and the occurrence of amperometric events with a "foot" or "stand alone" signal (which reflects sluggish or incomplete dilation of the fusion pore). To examine whether BFA altered the Ca2+-dependence of exocytosis, we employed the whole-cell recording technique in conjunction with the capacitance measurement to measure exocytosis evoked from the entire cell during voltage-gated Ca2+ entry. Our results suggested that BFA treatment did not alter either the initial rate of capacitance increase or the total amount of capacitance increase. Therefore, in chromaffin cells, BFA treatment affects Ca2+-regulated exocytosis predominantly by increasing the quantal size and by slowing the fusion kinetics of some vesicles.     

An optical study of isolated rat adrenal chromaffin cells

Isolated rat adrenal chromaffin cells were studied with laser light scattering and Nomarski differential interference contrast microscopy. Evidence of organelle movement and of the fusion of secretory granules with the plasma membrane was obtained. A specially modified microscope was used to collect, coherently, laser light scattered from groups of 1–3 cells. Autocorrelation analysis of intensity fluctuations of the scattered light indicated that relative movements of cytoplasmic particles of about 0.1 μm or larger took seconds or longer, compared with the millisecond time periods that would be expected for free diffusion of the cytoplasmic organelles of the cells in a medium with a viscosity equal to that of water. Cytochalasin B (CB) and reduced temperature were found to reduce the relatively fast component of the decay of the autocorrelation, indicating decreased motion in the cells. Fixation with formaldehyde reduced the autocorrelation amplitude to zero, indicating the absence of motion. It is suggested that the intensity fluctuations were a consequence of organelle motility within the cells. Time-lapse photomicrography with Nomarski differential interference contrast optics indicated that the movements of the cytoplasmic particles which could be distinguished were highly restricted, except for occasional observations of distinct particle saltations at about 0.2 μm/sec. No change was observed in particle motion during stimulation with secretagogues, but microspikes formed on the cell surfaces, presumably due to the addition of secretory granule membranes to the plasma membrane as a consequence of exocytosis.     

Homogeneous Ca2+ stores in rat adrenal chromaffin cells

The localization and function of Ca(2+) stores in isolated chromaffin cells of rat adrenal medulla were investigated using confocal laser microscopy and amperometry. Binding sites for BODIPY-inositol 1,4,5-trisphosphate (IP(3)), -ryanodine (Ry), and -thapsigargin (Thap) were both perinuclear and at the cell periphery. The endoplasmic reticulum (ER), which was identified by ER Tracker dye, took up fluorescent Ry and IP(3), and the majority of BODIPY-Ry-binding area was bound by fluorescent IP(3). Under Ca(2+)-free conditions, the amount of caffeine-induced catecholamine secretion was 33% of that of muscarine-induced secretion, but muscarine induced little or no secretion after exposure to caffeine. Muscarine-induced Ca(2+) increases, as observed with fluo-3, lasted for a few tens of seconds under Ca(2+)-free conditions, whereas a caffeine-induced Ca(2+) transient diminished rapidly with a half decay time of 3s and this spike-like Ca(2+) transient was then followed by a sustained increase with a low level. These results indicate that IP(3) receptors and Ry receptors (RyRs) are present in common ER Ca(2+) storage and the lower potency of caffeine for secretion may be due to a rapid decrease in RyR channel activity to a low level.     

Opossum adrenal medulla: II. Differentiation of the chromaffin cell

The ultrastructure of the opossum adrenal medulla was examined in its postnatal development. Maturation of chromaffin cells and genesis of chromaffin vesicles were of particular interest. The primitive sympathetic cell was seen to contain few organelles with no apparent polarity. Initial pheochromoblasts contained more organelles with some polarity. Endoplasmic reticulum and the Golgi complex increased as the pheochromoblasts matured, which suggested increased synthetic activity. Structures resembling Golgi/endoplasmic reticulum/lysosome (GERL) systems were seen in the pheochromoblasts. It is suggested that some of the components of the chromaffin vesicle may be processed by the GERL while others come directly through the Golgi complex. It is stressed that the developing pheochromoblast in the opossum presents an interesting model in which to study the genesis of the chromaffin vesicle.     

Maternal perinatal undernutrition impairs chromaffin cells proliferation in the postnatal rat.

Numerous data show that malnutrition during early life programs chronic diseases in adulthood. Many of these disorders may result from alterations in the development of neuroendocrine systems, such as the hypothalamo-pituitary-adrenal axis and the sympathoadrenal system. We have previously reported that maternal 50% food restriction during late pregnancy and lactation reduces adrenal weight and impairs chromaffin cell differentiation in male rats at weaning. In addition, maternal undernutrition modifies the expression of several genes involved in proliferation and apoptosis. This study therefore investigated the impact of maternal food restriction on adrenal cell growth in the late postnatal rat. Histological analysis showed that the number of proliferating chromaffin cells assessed by nuclear labelling with BrdU was reduced by 45%, whereas the level of apoptosis visualised by caspase-3 immunoreactivity was increased by 340% in adrenal medulla of offspring from undernourished mothers. In contrast, maternal food restriction did not affect proliferation and apoptosis in cortical cells of rats. These developmental changes were associated with overexpression of TGFbeta2. These data show that perinatal undernutrition impairs the balance between chromaffin cell proliferation and apoptosis. These modifications may lead to "malprogramming" of adrenal medulla development, which could contribute to the pathogenesis of chronic diseases in adulthood.