Search

Enkephalin convertase, an enkephalin synthesizing carboxypeptidase, has been further characterized and purified to virtual homogeneity from brain pituitary and adrenal chromaffin granules. Activity varies more than 10 fold in brain regions, generally reflecting enkephalin distribution. Enkephalin convertase activity is 20 fold higher in the anterior than the posterior pituitary, suggesting a role in the processing of other peptide hormones. Enkephalin convertase activity was not detected in any peripheral tissues.

Summary Biochemical studies have given conflicting interpretations as to the localization of acetylcholinesterase within adrenal chromaffin cells. Previous histochemical studies found no reaction product on the chromaffin vesicles, although some biochemical studies suggest that a form of the enzyme is present. Sensitive histochemical procedures, with the appropriate controls, suggest a small amount of acetylcholinesterase is associated with chromaffin vesicles.

An investigation was carried out using the electron microscope on the chromaffin cells and their innervation in the adrenal gland of Lacerta sicula . Three types of adrenal chromaffin cells were observed: cells containing moderately osmiophilic granules, of diameter approx 2000 A; cells with highly electron dense granules, average diameter 2660 A; and finally, cells with small electron dense granules, of average diameter 900 A. The innervation of these three types of cells appears to be cholinergic. The histochemical technique for acetylcholinesterase (AChE) and for pseudocholinesterases (PsChE) confirmed the cholinergic nature of the nerve endings. Furthermore, AChE was demonstrated in all three types of adrenal cells but with greater constancy and in greater quantity in the small granules cells. The reaction for PsChE was negative.

The field of neural transplantation has moved rapidly forward in the last decade. Initially, fetal cells were used as implants to investigate their potential to ameliorate deficits in animal models of Parkinson's disease. However, because of the moral and legal problems associated with the use of fetal tissues in humans, alternative sources of donor tissue were sought which possessed the structural and functional characteristics needed to improve motor function in Parkinsonian patients. To date, one of the most promising tissues being investigated is the adrenal medulla, whose chromaffin cells possess an inherent plasticity of form and function. Transplanted chromaffin cells currently are being studied by a variety of approaches, including electron microscopy, in mouse, rat, and primate models of Parkinson's disease. An overview of the role of the chromaffin cell in this exciting and clinically important arena is briefly reviewed, with an emphasis on the fine structure of implanted chromaffin cells.

The ability of a cell to carry out many specialized functions is achieved by compartmentation of reactions within the cell. Here we review studies of how the functions of the adrenal chromaffin granules relate to their structure and topography.

1. The positive alkaline phosphatase reacfion, proved by GOMORI'S method, of the adrenal chromaffin cells of the frog after fixation with alcohol (65% or chilled 80%) was thermostable. This positive reaction was also shown without treating with glycero-phosphate substrate medium. Accordingly, it is considered that the positive reaction was caused not by alkaline phosphatase but by the affinity of the cells for cobalt (table 1, fig. 2).2. The adrenal chromaffin cells of the frog showed an intense affinity for cobalt after fixation with alcohol and formalin and a weak affinity for iron and nickel after fixation with formalin.3. The adrenal chromaffin cells of the chicken, after fixation with formalin, had almost the same affinity as those of the frog (table 1, fig. 7).4. Such affinity was not observed in the adrenal medullary cells of the mouse after fixation with formalin or alcohol (table 1, fig. 4).5. In bovine and swine adrenals fixed with formalin, cells of the peripheral portion of the medulla revealed a cobalt affinity weaker than that of the adrenal chromaffin cells of the frog and chicken but stronger than of the other mammals examined (table 1, figs. 11, 12).6. Both chromaffinity and cobalt affinity disappeared from the adrenals of chickens injected with histamine dihydrochloride (figs. 5, 6, 7, 8).

The spectral properties of the noradrenaline storage vesicles of bovine splenic nerves are essentially the same as those recently reported for the adrenal chromaffin granules (T. Flatmark, O. Terland and K.B. Helle, Biochim. Biophys. Acta , 226 (1971) 9) indicating the presence of flavoprotein(s) and a high-potential b -type cytochrome termed cytochrome b 561 . The specific activities of NADH : (acceptor) oxidoreductase (EC 1.6.99.3) were higher than those previously found in the adrenal granules. Based on the assay of these enzymic activities, the flavoprotein(s) also appear to be more stable in the nerve granules than in the adrenal granules upon prolonged storage of the organelles at 0 c .

The presence of tropomyosins in adrenal chromaffin cells was demonstrated by immunoperoxidase staining of cultured chromaffin cells by an anti-tropomyosin antibody. In immunoblotting the antibody labelled polypeptides of Mr 39000, 36500 and 31000 in 100000 × g supernatants and heat-stable fractions of adrenal medulla. The adrenal medullary tropomyosins did not comigrate with smooth muscle tropomyosins. The 39-kDa and 31-kDa polypeptides comigrated with polypeptides labelled by anti-tropomyosin in brain supernatants. Chromaffin granule membranes possessed a 39-kDa polypeptide that was labelled by anti-tropomyosin.

We have investigated the effects of the phorbol ester 12-myristate 13-acetate (PMA) on depolarization-evoked Ca2+ influx and catecholamine secretion in bovine adrenal chromaffin cells. PMA (100 nM) strongly inhibited K(+)-evoked [Ca2+]i transients and Mn2+ quenching of fura-2 fluorescence. In contrast, 4 alpha-phorbol 12,13-didecanoate, a phorbol ester inactive on protein kinase C (PKC), had no effect. Maximal PMA-mediated inhibition occurred at 5-10 min incubations and were variable from cell to cell, ranging from 25 to 65% of controls. The [Ca2+]i transients evoked by the L-type Ca2+ channel activator Bay K 8644 were strongly inhibited by 100 nM PMA. PMA (0.1-10 microM) inhibited K(+)-evoked adrenaline and noradrenaline release by 23-44%. The data indicate that phorbol ester-mediated activation of PKC inhibits voltage-sensitive Ca2+ channels in chromaffin cells, leading to a prominent depression of depolarization-evoked catecholamine secretion.

IT is now a well-established fact that adrenal chromaffin cells of many vertebrates store substantial quantities of both adrenaline and noradrenaline. The relative proportions of the two catecholamines in the normal adult animal is species specific.

Adrenal chromaffin granules incubated with ATP rapidly incorporate the terminal phosphate into diphosphoinositide, resulting in a net increase in the amount of this lipid. The formation of diphosphoinositide has been demonstrated by thin-layer, paper and ionexchange chromatography. The formation of a phosphorylated non-lipid component has been confirmed.

The term chromaffin cells is derived from the properties of the parenchymal cells of the adrenal medulla whereby they are stained from yellow to brown after treatment with chromate- or dichromate-containing fixatives (Bennett 1941; Wood 1963; Coupland 1965a). It was Kohn (1903) who named this phenomenon the chromaffin reaction. The chromaffin reaction was regarded as a specific histochemical test for adrenaline and related substances. Thus, adrenal chromaffin cells have been regarded as occupying a special position among endocrine cells as the only adrenaline-secreting cells.

DEVELOPING neural crest tissues show a certain degree of plasticity with respect to neurotransmitter synthesis. For example, heterotopic transplants of presumptive noradrenergic tissue may develop cholinergic properties and vice versa1,2. Also, monolayer cultures of rat sympathetic neurones which synthesise catecholamines may be influenced to synthesise acetylcholine by exposure to ganglionic non-neuronal cells or to medium conditioned by such cells3,4. Recently, a clonal cell line (designated PC12) was established5 from a transplantable rat adrenal medullary pheochromocytoma6. This line expresses many characteristic properties of neural-crest-derived noradernergic adrenal chromaffin cells and sympathetic neurones, including synthesis, storage, uptake and specific release of catecholamines5,7,8. In addition, within several days after their exposure to nerve growth factor (or NGF, a protein which influences the growth and development of normal sympathetic neurones9), PC12 cells cease mitotic activity and extend long, neuronal processes5. We show here that noradrenergic cultures of PC12 cells can also synthesise, store and release acetylcholine.

Publisher Summary This chapter discusses the biosynthesis of enkephalins by adrenal chromaffin cells. Immunohistochemical studies have shown that bovine adrenal chromaffin cells contain immunoreactive enkephalin. Leu- and Met-enkephalin represent only a part of this immune-reactivity. These cells contain a large amount of a newly characterized enkephalin: met-enkephalin-Arg-Phe. The concentration of this new compound is higher than that of met-enkephalin. The chromaffin granules contain several large Mr peptides containing the aminoacid sequences of the enkephalins. The steady state levels of the enkephalins and their precursors in primary monolayer bovine adrenal chromaffin cells may indicate that they actively synthesize these molecules de-novo.

Autografts of adrenal chromaffin cells from adult guinea pigs to the anterior chamber of the eye were studied electron microscopically and both histo-and biochemically 4, 8 and 12 weeks after transplantation.

Studies on isolated adrenal chromaffin cells in primary cultures may be seriously hampered by the presence of non-chromaffin, mainly fibroblast-like cells, which always occur in dissociates of adrenal medullary tissue and often outnumber the chromaffin cells by the end of the first week of culture, when no measures are taken to control their proliferation. The present study offers a new means to inhibit effectively the proliferation of these accessory cells by treating the cultures with dibutyrylic cyclic AMP (dbcAMP, 0.1 or 0.01 mM) and equimolar amounts of the phosphodiesterase inhibitor theophylline. With this treatment cultures of young rat adrenal chromaffin cells remain virtually free of accessory cells for two weeks of culture. Cultures of bovine adrenomedullary cells retain their initial amounts of non-chromaffin cells, which largely depends upon whether the primary cell suspensions have undergone differential plating prior to seeding. Suppression of accessory cell proliferation with dbcAMP and theophylline is partly due to maintaining differentiation of cortical cells, which otherwise dedifferentiate into rapidly dividing fibroblast-like elements. However, a more direct action of dbcAMP on accessory cells in terms of growth control is also conceivable. DbcAMP and theophylline in the doses applied do not impair the viability, ultrastructure and catecholamine-storing capacity of cultured chromaffin cells.

Effect of hypophysectomy on the disposition of [ 3 H]dopamine-derived radioactivity in the mouse adrenal medulla was examined by autoradiography. Chromaffin cells, both A and NA type, incorporated less radioactivity in the hypophysectomized mice (50 days after operation) than in normal control mice: this decrease in the [ 3 H]dopamine uptake was restored by intraperitoneal injection of ACTH (for 26 days; starting from 24th day after hypophysectomy; 40 μg/gbw/day). In the control mice chromaffin cells near the cortico-medullary junction showed a higher radioactivity than those in the center: hypophysectomy obscured this specific distribution pattern which reappeared after ACTH treatment.

Glucose utilization by cells and tissues can be followed by measuring the release of [ 3 H]H 2 O from added d -[5- 3 H]glucose, and we have developed a method whereby the whole reaction and assay can be performed in a single scintillation vial. The basic principle behind our new assay is that the released tritiated hydrogen ion in water can be quantitatively exchanged with the hydroxyl proton of simple alcohols such as isoamyl alcohol. The radiolabeled alcohol can then be extracted into an organic solvent to which 2,5-diphenyloxazole and p -bis[2-(5-phenyloxazoyl)]benzene have been previously added. Using this new assay we studied isolated chromaffin cells and found them to utilize glucose at a linear rate for at least 30 min. The assay was precise and reproducible enough to allow detailed analysis of various inhibitors of glycolysis and of oxidative phosphorylation. The new method is simple and rapid, can be done in open test tubes, requires no complex equipment, and is intrinsically highly accurate.

Unique histochemical properties of the adrenal medulla were first described in the middle of the nineteenth century. The most important histochemical reaction of this glandular tissue was with chromate salts which became known as the chromaffin reaction. The terms chromaffin cell and chromaffin vesicle stem from this reaction. During the first half of this century it was established that the chromaffin cells of the adrenal medulla are an integral part of the sympathetic nervous system and play a role in the response to acute stress. The chromaffin cell is recognized as a modified postganglionic sympathetic neuron and as such has proven to be an accessible and valuable model. Thirty years ago it was found that the secretory vesicle of the chromaffin cell could be isolated. This has led to the use of the chromaffin vesicle as a model for study of neurosecretory vesicles. This symposium presents an overview of recent work in various areas that have centered on the chromaffin vesicle.

Electron microscopic localization of dopamine-β-hydroxylase (DBH) was carried out in bovine adrenal medullary chromaffin granule fractions and in intact bovine adrenal medulla by means of a peroxidase-antiperoxidase unlabelled antibody technique according to Sternberger , Hardy , Cuculis and Meyer (1970). Adrenal medullary chromaffin granule fractions showed retention of DBH despite successive fractionation, fixation and prolonged washing procedures, indicating that a portion of the DBH in the chromaffin granule is quite tightly bound. Under these circumstances the electron-opaque granular matrix was absent, implying loss of the more soluble constituents of the chromaffin granules, including matrix-associated DBH. Intact bovine adrenal medulla which was fixed, sectioned and subjected to immunocytochemical localization of DBH showed the enzyme to be present throughout the matrix of the chromaffin granule and demonstrated that, under these less vigorous conditions, the electron-opaque granule matrix was not removed. It would therefore appear to be feasible to differentiate directly between the “soluble” and “bound” forms of DBH by immunocytochemical procedures and to apply this technique to elucidate the process of exocytosis in adrenal medulla and adrenergic nerve terminals.

The effects of pro-opiomelanocortin (POMC) peptide fragments on the basal and agonist-induced release of catecholamines (CAs) from monolayer cultures of purified bovine adrenal chromaffin cells were tested. None of the 5 peptides tested, i.e. s -MSH, ACTH 1–39 , γ -MSH 1–13 . γ 3 -MSH and N-terminal POMC fragment, had any effect on basal CA release. However, s -MSH (10 −5 M), γ -MSH 1–13 (10 −6 –10 −5 M), γ 3 -MSH (10 −5 M) and N-terminal POMC fragment (10 −5 M) inhibited the nicotine-induced release of CAs from the chromaffin cells. The possible physiological significance of this inhibitory neuromodulation is discussed.

SGC (Small Granule Chromaffin) cells in the mouse adrenal medulla characterized by the smallness of the secretory granules and by the aggregations of synaptic-type vesicles may represent a third type of chromaffin cell.

Adrenal chromaffin granules in an isoosmotic medium can be induced to release their contents, including epinephrine, in an all or none fashion. Such release is based on osmotic lysis of the granules and is regulated by Mg-ATP and permeant anions. In this paper, we examine the mechanism of this release as it relates to membrane sites involving Mg-ATP and anions. We conclude that osmotic lysis of chromaffin granules in an isoosmotic medium is the result of electrogenic pumping of protons into the granule via a membrane Mg-ATPase and subsequent anion uptake via a selective transport site. Studies involving ATP-independent osmotic release mechanisms and anion transport blocking drugs further suggest that these two granule membrane sites may be physically as well as functionally coupled.

Acidic fibroblast growth factor (aFGF) is a heparin-binding polypeptide that is a mitogen for endothelial cells and glial cells, as well as a differentiation factor for PC12 cells and certain neurons. We show here that aFGF is as potent as nerve growth factor (NGF) in stimulating both neuritic outgrowth and proliferation in adrenal chromaffin cells from young rats, but it fails to support long-term survival. Heparin strongly potentiates aFGF-dependent neuritic outgrowth but not aFGF-dependent proliferation. As is the case with NGF, phorbol myristate acetate depresses aFGF-induced cell division and increases the outgrowth of neurites. On the other hand, dexamethasone antagonizes neuritic outgrowth elicited by both NGF and aFGF but inhibits only proliferation induced by NGF. The effects of basic FGF (bFGF) are similar but not identical to those of aFGF. Thus the regulatory pathways controlled by aFGF, bFGF, and NGF are partially distinct.

Increasing attention has been given in recent years to the dynamic features of the nervous system, the development, maturation and plasticity of neurons and neuronal interactions. Inherent capabilities and environmental cues appear to contribute to the definite expression of the capacities of neurons.

Met-enkephalin-Arg-Gyl-Leu-like immunoreactivity was revealed in many nerve fibers as well as in chromaffin cells in the rat adrenal medulla. The immunoreactive nerve fibers were seen among and around chromaffin cells in the forms of punctate structures or varicose processes and they were characterized by numerous small clear vesicles mixed with several large granular vesicles. The nerve fibers formed synaptic contacts with both Met-enkephalin-Arg-Gly-Leu-like immunoreactive and non-immunoreactive chromaffin cells. The immunoreactive material in the nerve was generally confined to the granular cores. The present findings suggest that preproenkephalin A and its derivatives are produced in adrenal efferents which may be preganglionic cholinergic, and that they may play a significant role in the function of adrenal chromaffin cells.

Exocytosis is initiated by the receptor-mediated influx of calcium that results in fusion of the secretory vesicle with the plasma membrane. We examined the possibility that calcium-dependent exocytosis in mast cells and adrenal chromaffin cells requires metalloendoprotease activity. Metalloendoprotease inhibitors and dipeptide substrates block exocytosis in these cells with the same specificity and dose dependency as that with which they interact with metalloendoproteases. Metalloendoprotease activity is identified in these cells with fluorogenic synthetic substrates, which also blocked exocytosis. Metalloendoprotease activity is highest in the plasma membrane of chromaffin cells. The metalloendoprotease appears to be required in exocytosis at a step dependent on or after calcium entry, since exocytosis initiated by direct calcium introduction in both mast cells and chromaffin cells is blocked by metalloendoprotease inhibitors.

Publisher Summary Implants of both chromaffin and glomus cells reverse amphetamine-induced rotational behavior and appear viable in the unilateral 6-hydroxydopamine (6-OHDA) rodent model. This chapter describes the potential of several alternative sources of donor tissue for neural transplantation, and emphasizes upon two members of the sympathoadrenal cell lineage, the adrenal chromaffin and carotid body glomus cells, as implants in the unilateral 6-hydroxydopamine (6-OHDA) rodent model. The particular focus of the present study is on the fine structure of these paraneuronal grafts 30 days post-implantation into the denervated striatum. Once implanted, the chromaffin cells demonstrate a plasticity of their dense-core vesicles, with the average diameter decreasing in size when compared to normal adrenal medullary cells. However, the study raises some important questions that need to be addressed, such as (1) will these paraneuronal cells survive and continue to exert positive behavioral effects in long-term experiments, and (2) what is the critical mass of implanted tissue needed to achieve functional recovery, and where in the brain are the optimal sites for ransplantation of these cells. The chapter indicates that answers to these questions are essential for designing rationale clinical approaches for the treatment of parkinsonism, and to encourage the acceleration of transplantation research in appropriate animal models.

Uptake of exogenous [3H]dopamine in adrenal chromaffin cells of the mouse was studied by autoradiography under a 12-h light, 12-h dark cycle. The uptake of [3H]dopamine in adrenal chromaffin cells was markedly decreased in the dark period. The decrease was prevented by adrenal denervation. It is postulated that there is a neurally mediated suppression of [3H]dopamine uptake by adrenal chromaffin cells in the dark period. Adrenaline-storing (A) cells are more sensitive to the decreased sympathetic activity than noradrenaline-storing (NA) cells while increased sympathetic activity non-selectively affects both types of adrenal chromaffin cells.

S Our autoradiographic study in the mouse using tritiated leucine, tyrosine, dopa, dopamine and noradrenaline demonstrated that each chromaffin cell possessed special trapping and concentrating systems for catecholamines in addition to those for amino acids. The systems for catecholamines were, under the control of pituitary ACTH, better developed in the chromaffin cells in the periphery of the adrenal medulla than in those in the center of the adrenal medulla.

Freeze-fracture replicas of bovine adrenal chromaffin granules show convex and concave fracture faces. The concave fracture faces are studded with 9 nm diameter particles (on average 950/μm 2 ) while the convex fracture faces are almost devoid of particles (approx 150/μm 2 ). The asymmetry in particle distribution for the membranes of these intracellular organelles is consistent with that observed for plasma membranes. The significance of these results is discussed with reference to the exocytotic release of catecholamines from the adrenal medulla.

The intracellular localization of l-[4,5-3H]leucine in chromaffin cells has been observed using light and electron microscopic autoradiography and the association of the labelled amino acid with particular cell components confirmed by statistical analysis. By making observations at short intervals after a single intravenous pulse of [3H]leucine it has been possible to follow the movement of the isotope from the endoplasmic reticulum through the Golgi complex to the chromaffin granules. No evidence for movement of the label through the Golgi complex was observed in adjacent cortical cells. The time sequence of transport of the amino acid through the various cell organelles was very similar to that observed by previous workers in protein-secreting exocrine cells.

Acetylcholine stimulates a five-fold increase in cyclic GMP within seconds of addition to suspensions of isolated adrenal chromaffin cells. The rapid kinetics and dose-response curve for cyclic GMP accumulation are compared with corresponding results for catecholamine secretion and a possible role of cyclic GMP in stimulus-secretion coupling is considered.

Chromaffin cells in organotypic cultures of adrenal glands from 19 day rat embryos developed a marked increase in the volume of their rough endoplasmic reticulum (RER) and enlargement of nucleoli in response to 1 mM dibutyryl cyclic AMP (dbcAMP) treatment for 17 days in vitro. 0.1 mM dbcAMP had a similar but less dramatic effect. When dbcAMP was removed from the medium after 11 days in vitro, cells became more like control explants treated with no additives, suggesting the effects of dbcAMP were reversible. ACTH which selectively stimulates adrenal cortical cells had no effect on RER morphology of chromaffin cells. The 'neuronal-like' configuration of dbcAMP treated chromaffin cells is discussed.

Enkephalins and enkephalin-containing polypeptides were released from perfused bovine adrenal glands by nicotine and Ba2+, agents that release catecholamines. All the enkephalin-containing polypeptides that are normally found in the adrenal medulla were released by the two secretagogues in approximately the same proportions as are present in adrenal chromaffin granules.