An electron microscopic study on nerve endings on adrenomedullary adrenaline cells in golden hamsters: position, size and changes due to pinealectomy.

Effects of sham-pinealectomy and pinealectomy on preganglionic nerve endings on adrenomedullary adrenaline cells were investigated electron microscopically. Adult male golden hamsters from the normal, sham-pinealectomy and pinealectomy groups maintained under 24 h light-dark cycle and constant temperature were used at 28 days after surgery. From conventional electron microscopic specimens, montage photographs made of the adrenaline cell region at a magnification of x 11,000 were used for qualitative and quantitative electron microscopic analyses in 14 animals in each experimental group. The preganglionic nerve endings were localized mainly in the following three sites: the basal lamina part, the follicular lumen-junctional intercellular part, and the adrenaline cell-invaginated part. In the latter two parts, nerve endings and fibers had no envelope frequently, and in the former two parts, nerve endings sometimes showed the invagination complex. The frequency of nerve endings was highest in the follicular lumen-intercellular part, next highest in the basal lamina part and lowest in the A cell-invaginated part. The frequency of nerve endings in the basal lamina part was lower in the pinealectomy group than in the sham-pinealectomy group (P < 0.021), and those in the other two parts showed opposite changes, more evidently in the A cell-invaginated part. Nerve ending profiles in the adrenaline cell-invaginated part--which displayed a more rounded shape--increased in size in the pinealectomy group (longer diameter: P < 0.04; shorter diameter: P < 0.05). In conclusion, preganglionic nerve endings in the adrenal medulla of the golden hamster show differential morphological changes following PX depending on the intracellular part of A cells.

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.

Neuropeptide Y immunohistochemistry and ultrastructure of developing chromaffin tissue in the cloudy dogfish, Scyliorhinus torazame (Chondrichthyes, Elasmobranchii).

Ontogenetic changes in neuropeptide Y-like immunoreactivity (NPY-LI) were studied in chromaffin tissue of the cloudy dogfish, Scyliorhinus torazame. In adults and post-hatching juveniles, NPY-LI was demonstrated in chromaffin cells, but not in ganglion cells and supporting cells. Immunoreactive fibers were also found in the axillary body (the major chromaffin tissue) of the adult fish. During the embryonic period, NPY-LI was found at first in chromaffin tissue in the 34-mm stage. In this stage, cells in the periphery of the tissue were positive for NPY. Afterwards, changes were not observed in the topography and relative dominance of labelled cells in the tissue. Transmission electron microscopy of chromaffin tissue of the 26-mm stage showed an early phase of histogenesis in rudimental cell clusters composed of agranular cells and a few granular cells, i.e. pheochromoblasts. In the 43-mm stage, differentiation of the chromaffin tissue enabled ultrastructural classification of adrenalin-producing cells, noradrenalin-producing cells, ganglion cells, supporting cells, and unmyelinated nerve fibers. These results suggest that in the dogfish the appearance of NPY-LI in the developing sympathoadrenal system is related to differentiation of chromaffin cells.

Electron microscopic evidence for multiple types of secretory vesicles in bovine chromaffin cells.

It has been previously shown that the neuron-like chromaffin cells from the bovine adrenal medulla are heterogeneous. Among other differences, the cells also differed in secretory vesicles represented in their cytoplasm. The present study investigates the types of secretory vesicles in bovine chromaffin cells by electron microscopy. Morphometric analysis revealed five types of electron-dense secretory vesicles in chromaffin cells. These were as follows: elementary large catecholamine-storing chromaffin granules of rounded shape, large dense core vesicles of ovoid and rod-like shapes, small dense core vesicles as well as ribosome-coated vesicles of intermediate density. Among the electron-lucent vesicles there were small synaptic-like microvesicles, endocytotic clathrin-coated vesicles, growth cone vesicles, and emptied large light core vesicles. The structural and functional backgrounds of different types of secretory vesicles are described, focusing on their formation and potential role.

Structural and ultrastructural characteristics of interrenal gland and chromaffin cell of matrinxã, Brycon cephalus Gunther 1869 (Teleostei-Characidae).

This work presents the structure and ultrastructure of the interrenal gland and chromaffin cells, as well as the morphology of the head kidney of Brycon cephalus. The head kidney is composed of fused bilateral lobes located anterior to the swim bladder and ventrolateral to the spinal column. The parenchyma revealed lympho-haematopoietic tissue, melano-macrophage centres, interrenal gland and chromaffin cells. The interrenal gland consisted of cords or strands of cells grouped around the posterior cardinal vein and their branches. Chromaffin cells are found in small groups, closely associated with the interrenal gland and/or under the endothelium of the posterior cardinal vein. So far, the ultrastructural analysis has revealed only one interrenal cell type which contained abundant smooth endoplasmic reticulum and numerous mitochondria with tubulo-vesicular cristae, characteristic of steroid-producing cells. Two types of chromaffin cells were observed. The first type was characterized by the presence of vesicles with round, strongly electron-dense granules, which were eccentrically located. Such cells were interpreted as noradrenaline cells. Meanwhile, cells which contained smaller vesicles and electron-lucent granules, with a small halo separating the granule from the vesicular limiting membrane, were identified as adrenaline cells.

Frog chromaffin and adrenocortical cell co-cultures: a model for the study of medullary control of corticosteroidogenesis.

Phylogenetic, physiological and morphological evidence indicates that interactions between chromaffin and adrenocortical cells are involved in the differentiation and maintenance of function of both cell types. Chromaffin-adrenocortical interaction has become recognized as an important component of adrenocortical regulation; however, the mechanisms by which chromaffin cells modulate adrenocortical function are not well understood. To study directly chromaffin-adrenocortical cellular interactions, we developed primary frog (Rana pipiens) adrenal co-cultures. In these co-cultures, chromaffin cells extend processes that project towards or onto adrenocortical cells, mimicking their organization in vivo and indicating a potential for interaction between the two cell types. Cell survival and differentiation were optimized using a combination of NGF, FGF and histamine to enhance neurite outgrowth and fetal calf serum plus 10(-10) M ACTH to maintain steroidogenesis. Characterization of the cells by immunocytochemistry and histochemistry showed that chromaffin cells maintain expression of catecholamine biosynthetic enzymes and that adrenocortical cells maintain expression of steroidogenic enzymes. Furthermore, chromaffin cells release catecholamines upon stimulation with carbamylcholine or potassium while adrenocortical cells sustain a basal secretion rate of aldosterone and corticosterone that is augmented 10-40-fold by 0.1 nM to 10 nM ACTH. We therefore propose that these co-cultures serve as a useful model system to study the cellular and molecular mechanisms by which chromaffin cells modulate adrenocortical cell function.

A morphometric analysis of exocytosis in KCl-stimulated bovine chromaffin cells.

Transmission electron microscopy has been used to morphometrically evaluate exocytosis in bovine adrenal medulla chromaffin cells as the mechanism of catecholamine release. Purified cell suspensions were stimulated with KCl at varying strengths and durations and then conventionally processed for ultrastructural analysis. Quantitation of exocytotic images of dense cored chromaffin granules was a major objective and such images were found in all preparations, attesting to the efficacy of chemical fixation to preserve this event. However, because hundreds of cell profiles had to be screened to find a single granule in the process of release this low frequency precluded any meaningful correlations with estimates of granular involvement based on catecholamine release. Neither KCl molarity nor duration altered this finding nor did these variables significantly affect other parameters linked to exocytotic activity. For example, cell size and numbers of "empty' granules and vesicles remained constant and attempts to label "any' organelle with 30-nm colloidal gold or lanthanum precipitate proved unsuccessful. In short, if exocytosis is responsible for release, it would appear to function without leaving a morphological trace. An alternative hypothesis, therefore, is outlined which better accommodates existing data.

Receptors for atrial natriuretic peptide in adrenal chromaffin cells.

Receptors for atrial natriuretic peptide (ANP) in isolated bovine adrenal chromaffin cells were characterized. 125 I-ANP specifically bound to the cells with a Kd of 103 pM and a Bmax of 5.6 fmol/10(6) cells (16.5 fmol/mg of cell protein). C-ANF, a highly selective ligand for ANP-C receptors of natriuretic peptides, did not compete for 125 I-ANP binding at concentrations up to 10nM. Chemical cross-linking of 125I-ANP to the cells showed a single molecular size of the 120 kDa binding site on SDS gel electrophoresis under reducing conditions. CNP, a specific peptide for the ANP-B receptor, was much less potent than ANP in inhibiting 125 I-ANP binding and in displacing 125 I-ANP from the 120 kDa band. These results suggest that ANP specifically binds to the ANP-A receptor of 120 kDa and that there is no ANP-C receptor in bovine adrenal chromaffin cells.

Comparison of LR White and Unicryl as embedding media for light and electron immunomicroscopy of chromaffin cells.

LR White and Unicryl are members of the same family of acrylic embedding resins and are very suitable for "on grid" postembedding immunogold labeling. We studied the ultrastructure of LR White- and Unicryl-embedded cultured chromaffin cells and the immunolocalization of three chromaffin cell proteins, the enzymes dopamine-beta-hydroxylase (DbetaH) and tyrosine hydroxylase (TH), and the membrane fusion and Ca2+ channel protein synexin (annexin VII). We report here that Unicryl is preferable to LR White as an embedding medium for electron microscopy when osmium tetroxide fixation is omitted. The basis for this distinction is better ultrastructural preservation and improved immunodetection efficiency.

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.

Thapsigargin discriminates strongly between Ca(2+)-ATPase phosphorylated intermediates with different subcellular distributions in bovine adrenal chromaffin cells.

We studied the effects of thapsigargin on the formation of the phosphorylated intermediates (E approximately Ps) of endoplasmic reticulum Ca(2+)-ATPases in microsomes from bovine adrenal medulla. When submicrosomal fractions were separated on a sucrose gradient, two components of 100 kDa Ca(2+)-ATPase E approximately P displaying distinct subcellular distributions were resolved. The first component was defined by Ca(2+)-induced protection against thapsigargin inhibition. The second component did not display such protection, with a 3 orders of magnitude difference in thapsigargin inhibitory potency towards the 2 components. In the absence of Ca2+, both E approximately P components were highly sensitive to thapsigargin inhibition, revealing the presence of high-affinity thapsigargin-binding sites characteristic of SERCA ATPases. These data demonstrate a new level of molecular heterogeneity among Ca(2+)-ATPases of endoplasmic reticulum, and provide the first evidence of differential subcellular localization of individual Ca2+ pump subtypes in cells of neural origin.

Insulin-induced hypoglycemia stimulates both adrenaline and noradrenaline release from adrenal medulla in 21-day-old rats.

It has been well-established that insulin-induced hypoglycemia evokes preferential adrenaline release from the adrenal medulla in fasted adult rats. The present study examined the responsiveness to hypoglycemia in fasted 21-day-old and 8-week-old rats. The recovery of adrenaline in the chromaffin granule fraction prepared from the 8-week-old rat adrenal homogenate decreased 30 min after subcutaneous injection of 3 U/kg insulin, whereas the recovery of both adrenaline and noradrenaline was diminished in 21-day-old rats. In electron microscopy, omega-shaped profiles, indicative of exocytosis, were frequently observed in adrenaline- and noradrenaline-storing cells of 21-day-old rats. These results indicate that the responsiveness of the noradrenaline-storing cells to hypoglycemia in 21-day-old rats is different from that in young adult rats.

Docked granules, the exocytic burst, and the need for ATP hydrolysis in endocrine cells.

Ca(2+)-triggered exocytosis was studied in single rat melanotrophs and bovine chromaffin cells by capacitance measurements. Sustained exocytosis required MgATP, but even in the absence of MgATP, Ca2+ could trigger exocytosis of 2700 granules in a typical melanotroph and of 840 granules in a chromaffin cell. Granules undergoing ATP-independent exocytosis were similar in number to those appearing docked to the plasmalemma in quickly frozen unfixed sections (3300 in a melanotroph and 830 in a chromaffin cell). Most exocytosis required tens of seconds, but a small pool of granules was released in tens of milliseconds. Evidently, only a small subset of docked granules is rapidly releasable. We suggest that, temporally, the last ATP-dependent step in exocytosis is closely associated with docking and that docked granules reach fusion competence only after subsequent steps.

The interchromatin granules.

In the present review, we report on the data obtained on the most prominent elements observed in the interchromatin spaces: the interchromatin granules (IGs). Special emphasis is placed on the recent contributions of immunocytochemical and in situ hybridization methods towards elucidating the composition and the behavior of these nuclear substructures during the cell cycle. The possible roles of IGs in the cell nucleus are also discussed.

Long term stimulation changes the vesicular monoamine transporter content of chromaffin granules.

Bovine chromaffin cells cultured for 5 days in the presence of depolarizing concentrations of K+ ions show a decreased number of secretory (chromaffin) granules per cell. These cells were still capable of exocytosis. Their contents in catecholamine and chromogranin A, components of the granule matrix, and cytochrome b561, a major protein of the granule membrane, were decreased to 35, 30, and 50% of control cells, respectively. However, in the same cells, the number of [3H]dihydrotetrabenazine binding sites, a specific ligand of the vesicular monoamine transporter, was increased to 180% of controls. In situ uptake of noradrenaline in permeabilized cells indicated that [3H]dihydrotetrabenazine binding sites were associated with a functional vesicular monoamine transporter. When analyzed by isopycnic centrifugation, these sites cosedimented with catecholamine, chromogranin A, and cytochrome b561, in a peak with a density lighter than that from controls. The composition of this peak suggests that it contains incompletely matured secretory granules, with a 3-5-fold increase in the vesicular monoamine transporter content of this membrane. This increase might indicate that an adaptative process occurs which allows a faster filling of the granules in continuously secreting cells.

Do caged-Ca2+ compounds mimic the physiological stimulus for secretion?

We measured the exocytotic response induced by a rapid and global increase in the concentration of Ca2+ or GTP gamma S (achieved by flash photolysis of caged compounds) in mast cells and chromaffin cells. Secretion was measured by following both cell membrane capacitance and amperometry. When Ca2+ was used to trigger secretion, we observed an immediate increase in capacitance, however, the first amperometric spike observed was delayed with respect to the change in capacitance (approximately 5 s in mast cells and approximately 0.6 s in chromaffin cells). In contrast, when GTP gamma S was used to trigger secretion, no such discrepancy was seen, both measurements reported a secretory response that was similarly delayed with respect to the stimulus. These data suggest that the capacitance increase, when triggered by a large Ca2+ step, reports events that do not result in the fusion of vesicles containing oxidizable substances. These results contrast with the smaller secretory responses evoked by the transient opening of Ca2+ channels. Under these more physiological conditions, pulsed-laser imaging studies revealed that the Ca2+ influx was localized to discrete areas at the plasma membrane. Thus the Ca2+ stimulus triggered by DM-nitrophen fails to reproduce the small localized changes seen during a physiological stimulation and creates an artifactual non-secretory capacitive response.

Morphological observations of small granule-containing (chromaffin) cells in the celiac ganglion of the guinea pig, with emphasis on cell contacts.

Utilizing electron microscopic observation, several contacts between small, granule-containing cells (SGC) and postganglionic neurons (PGN) in the celiac ganglion of the guinea pig have been observed. A SGC in very close association with a PGN was seen to receive a distinct synaptic contact that contained many vesicles with dense cores. This contact was morphologically unlike cholinergic synapses previously reported on chromaffin cells. Because the SGC and PGN were clearly separated by a thin rim of satellite cell cytoplasm mutual to both cells, it is not known how or if the SGC would possibly exert a synaptic or paracrine effect on the PGN. Also, intraganglion SGC existed as large well-vascularized islands within the celiac ganglion. These intraganglion clusters sometimes contained more than 50 cells and perhaps could be considered to function as localized neuroendocrine components within the ganglion by secreting granule products into the nearby blood vessels for local or distant effects, although this certainly is not known. This work reports a unique synaptic ending upon a single-occurring SGC, which, in turn, closely approximates a ganglion neuron in a soma-somatic relationship. In addition, a very close association (but no actual contact) was observed between granule-containing processes, presumably emanating from the intraganglion clusters, and PGN. Whatever the function of ganglionic SGC may be, the exact relationship between SGC and PGN presumably would be of great interest and potential importance.

Intracellular distribution of kinesin in chromaffin cells.

In this paper we examined the association of the microtubule motor protein kinesin with organelles in chromaffin cells. Approximately 15% of kinesin was associated with membranes as determined by differential and equilibrium centrifugation on sucrose gradients. Kinesin was not enriched in a particular organelle fraction but cofractionated with a variety of organelle markers including markers for early and late endosomes, smooth and rough endoplasmic reticulum (ER) and the Golgi apparatus. Surprisingly, low amounts of kinesin were present in fractions of purified chromaffin granules. The absence of kinesin from the bulk of chromaffin granules was also indicated by immunostaining of tissue sections. A polyclonal antibody that specifically recognized the 120 kDa kinesin heavy chain labeled predominantly a perinuclear region that is typical for most of the kinesin-binding organelles identified by cell fractionation (endosomes, Golgi, ER). Since these organelles are compartments with high membrane turnover, we speculate that kinesin might be involved in certain aspects of trafficking of these membrane systems.

Effects of ACTH and aminoglutethimide on the catecholamine content and chromaffin cell morphology of the adrenal medulla of the neonatal rat.

The rat adrenal medulla is immature at birth, composed of phaeochromoblasts and undifferentiated chromaffin cells, but by 7 d postnatally morphologically distinct adrenaline-storing (A) and noradrenaline-storing (NA) cells can be distinguished in the adult proportions of approximately 80-85% A and 15-20% NA cells. Glucocorticoid hormones are known to play an important role in the initial expression and maintenance of phenylethanolamine N-methyl transferase (PNMT), the enzyme characteristic of A cells. The purpose of the study was to investigate the effects of glucocorticoids on the establishment of the A and NA cell phenotype in vivo during the first postnatal week. Neonatal rats were treated from postnatal d 1 to 7 either with ACTH to increase circulating levels of glucocorticoids or with aminoglutethimide to reduce blood glucocorticoids. On postnatal d 7 the volume fractions of A and NA cells in the adrenal medulla were estimated and the amounts of stored adrenaline and noradrenaline determined by HPLC and compared with untreated controls. Adrenaline levels were increased following ACTH treatment and there was an apparent decrease after aminoglutethimide which was not statistically significant. There was cytological evidence of the effects of ACTH and aminoglutethimide on the adrenal cortex but no resultant effect on medullary cell morphology. A cells remained predominant with NA cells making up approximately 15% of chromaffin cells, suggesting that any effects of altered glucocorticoid levels were confined to a modulation of adrenaline synthesis by a morphologically unchanged chromaffin cell population.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytological and biochemical studies on chromaffin cells in the head kidney of Gasterosteus aculeatus (Teleostei, Gasterosteidae).

Adrenal chromaffin cells in the head kidney of the stickleback Gasterosteus aculeatus were examined by ultrastructural analysis and determination, by HPLC, of the catecholamine content (adrenaline, noradrenaline, and dopamine). Chromaffin cells are characterized by the presence of membrane-bound vesicles and two cell types were identified: (i) those with vesicles containing a strongly electron-dense core; and (ii) those with vesicles that are completely electron lucent or containing smaller and less-dense granules. HPLC analysis revealed the presence of adrenaline and noradrenaline in both right and left head kidneys. Dopamine was not detected. Cytological and biochemical data suggest that the cells with electron-dense granules contain noradrenaline and those with electron-lucent vesicles contain adrenaline.