The anatomical distribution and morphology of extraadrenal chromaffin tissue (abdominal paraganglia) in the dog

The anatomical distribution, in situ, and morphology of extraadrenal chromaffin tissue in the retroperitoneum of dogs of various ages was studied by utilizing glutaraldehyde perfusion followed by glutaraldehyde/potassium dichromate immersion. This method of study produced a gross chromaffin reaction that clearly demonstrated abundant catecholamine-containing paraganglionic tissue. The procedure also provided excellent tissue preservation for subsequent light and electron microscopic study of the dichromate-traced organs. The largest chromaffin organ consistently occurred ventral and lateral to the abdominal aorta in the mid-retroperitoneum. Extensions of this body often reached the adrenal glands, but a specific continuity between intra- and extra-adrenal chromaffin tissues was not observed. Smaller organs, probably corresponding to the Organs of Zuckerkandl in the human, occurred around the inferior mesenteric artery. Light microscopy identified a parenchyma rich in epithelial cells exhibiting cytoplasmic vesicles and prominent nuclei and nucleoli. Connective tissue and numerous blood vessels delineated the chromaffin cells into groups. Electron microscopy showed cellular detail characteristic of chromaffin cells and confirmed the cytoplasmic presence of typical catecholamine granules. The majority of granules exhibited homogeneously dense cores. However, many others appeared less dense and displayed granular cores. This study provides evidence that extraadrenal chromaffin organs in dogs are voluminous, rich in catecholamine granules, and persist into adulthood.     

ATP-dependent uptake of 5-hydroxytryptamine by secretory granules isolated from thyroid parafollicular cells.

The current study was done to test the hypotheses that parafollicular granules contain a vacuolar ATPase (V-ATPase) similar to that found in chromaffin granules, that the transport of H+ into granules mediated by this enzyme drives the granular uptake of 5-hydroxytryptamine (5-HT, serotonin), and that secretagogues stimulate both the acidification of parafollicular granules and their ability to take up 5-HT by opening an anion channel in the granular membrane. Our studies indicate that parafollicular granules contain a V-ATPase that is antigenically similar to that of the V-ATPase of adrenal chromaffin granules; however, the parafollicular granular membrane differs from that of chromaffin granules in permeability to Cl- and K+. The membranes of granules derived from resting parafollicular cells appear to be relatively impermeable to Cl- but permeable to K+. Parafollicular granules (and ghosts derived from them) manifest ATP-dependent transmembrane transport of 5-HT. This transport is more dependent on the pH difference (delta pH) than on the membrane potential component of the proton electrochemical gradient across the granular membrane. Transport of 5-HT is thus inhibited more by exposure of parafollicular granules to agents, such as nigericin, that collapse delta pH than by those, such as valinomycin, that decrease transmembrane difference in potential. ATP-dependent uptake of 5-HT by granules isolated from secretagogue-stimulated parafollicular cells is greater than that into granules isolated from unstimulated cells. Since secretagogues open a Cl- channel in parafollicular granule membranes, which enhances acidification of the granules, the facilitation of 5-HT uptake by secretagogues is probably due to an increase in delta pH.     

Docking of chromaffin granules—A necessary step in exocytosis?

Putative docking of secretory vesicles comprising recognition of and attachment to future fusion sites in the plasma membrane has been investigated in chromaffin cells of the bovine adrenal medulla and in rat phaeochromocytoma (PC 12) cells. Upon permeabilization with digitonin, secretion can be stimulated in both cell types by indreasing the free Ca²⁺-concentration to M levels. Secretory activity can be elicited up to 1 hr after starting permeabilization and despite the loss of soluble cytoplasmic components indicating a stable attachment of granules to the plasma membrane awaiting the trigger for fusion. Docked granules can be observed in the electron microscope in permeabilized PC 12 cells which contain a large proportion of their granules aligned underneath the plasma membrane. The population of putatively docked granules in chromaffin cells cannot be as readily discerned due to the dispersal of granules throughout the cytoplasm. Further experiments comparing PC 12 and chromaffin cells suggest that active docking but not transport of granules can still be performed by permeabilized cells in the presence of Ca²⁺: a short (2 min) pulse of Ca²⁺ in PC 12 cells leads to the secretion of almost all releasable hormone over a 15 min observation period whereas, in chromaffin cells, with only a small proportion of granules docked, withdrawal of Ca²⁺ leads to an immediate halt in secretion. Transport of chromaffin granules from the Golgi to the plasma membrane docking sites seems to depend on a mechanism sensitive to permeabilization. This is shown by the difference in the amount of hormone released from the two permeabilized cell types, reflecting the contrast in the proportion of granules docked to the plasma membrane in PC 12 or chromaffin cells. Neither docking nor the docked state are influenced by cytochalasine B or colchicine. The permeabilized cell system is a valuable technique for thein vitro study of interaction between secretory vesicles and their target membrane.     

Muscarinic and nicotinic receptor-mediated Ca2+ dynamics in rat adrenal chromaffin cells during development

To clarify when the cholinergic receptor-mediated secretion mechanism of developing adrenal chromaffin cells is expressed and becomes functional, morphological changes and intracellular calcium dynamics were studied by immunohistochemistry, electron microscopy, and Fura-2 digital image analysis. From embryonic day 14 to 16, adrenal medullary cells were immunoreactive to noradrenaline-synthesizing enzyme (dopamine β-hydroxylase) but not to adrenaline-synthesizing enzyme (phenylethanolamine N-methyltransferase). These cells contained either no granules or just a few granules of high electron density. Exocytotic figures were rarely observed in cells of the control or in cells after carbamylcholine stimulation. Nerve fibers in the adrenal medulla contained either no clear vesicles or very few. Neither methacholine nor nicotine caused a change of intracellular Ca²⁺ in most chromaffin cells. From embryonic day 18 to 20, chromaffin cells were immunoreactive to both dopamine β-hydroxylase and phenylethanolamine N-methyltransferase and they contained relatively numerous secretory granules. Exocytotic figures were often seen in cells after carbamylcholine stimulation. The intra-adrenal nerve fibers contained numerous clear vesicles and a few dense-cored vesicles. Methacholine caused no rise of intracellular Ca²⁺, but nicotine induced a low to relatively high rise in many cells. From postnatal day 2 or 3 to postnatal week 1, numerous cells were immunoreactive to both dopamine β-hydroxylase and phenylethanolamine N-methyltransferase, whereas some cells were reactive to dopamine β-hydroxylase alone. Chromaffin cells were divisible into noradrenaline cells and adrenaline cells based on the ultrastructural features of their granules. Methacholine induced a moderate rise of intracellular Ca²⁺ and nicotine caused a high rise in many chromaffin cells, whereas, in some chromaffin cells, methacholine induced no rise of intracellular Ca²⁺ and nicotine induced a high rise. These results suggest that morphological changes of the developing cells and the intra-adrenal nerve fibers are related to the expression of a cholinergic receptor-mediated secretion mechanism and that this mechanism via a nicotinic receptor-mediated Ca²⁺ signaling pathway precedes the muscarinic receptor-mediated one during development.     

Peptide and amine producing endocrine-like cells in the chicken thymus

Summary The thymus of the chicken contains at least two types of endocrine-like cells predominating in the juxtacortical medulla. One type stores 5-hydroxytryptamine and is stained by the argentaffin, chromaffin and Schmorl methods. Treatment with reserpine markedly reduces its 5-hydroxytryptamine content. The other cell type is devoid of 5-hydroxytryptamine; if supplied withl-dopa it can produce and store dopamine. Both cell types stain with the argyrophil method of Grimelius and with HCl-basic dye methods believed to reflect the presence of peptides with masked carboxyl groups. In the electron microscope both cell types were found to contain numerous cytoplasmic 2000–3000 ? granules similar to those seen in polypeptide hormone-producing cells elsewhere. The cytoplasmic granules in one of the two endocrine-like cell types are argentaffin and chromaffin, indicating that they are the storage site for 5-hydroxytryptamine. It is suggested that the main secretory products of the two endocrine-like cell types are peptides, possibly regulating lymphatic tissue function.     

Dynamic changes in chromaffin cell cytoskeleton as prelude to exocytosis

Earlier work by us as well as others has demonstrated that filamentous actin is mainly localized in the cortical surface of chromaffin cell. This F-actin network acts as a barrier to the chromaffin granules, impeding their contact with the plasma membrane. Chromaffin granules contain α-actinin, an anchorage protein that mediates F-actin association with these vesicles. Consequently, chromaffin granules crosslink and stabilize F-actin networks. Stimulation of chromaffin cell produces disassembly of F-actin and removal of the barrier. This interpretation is based on: (1) Cytochemical experiments with rhodamine-labeled phalloidin indicated that in resting chromaffin cells, the F-actin network is visualized as a strong cortical fluorescent ring; (2) Nicotinic receptor stimulation produced fragmentation of this fluorescent ring, leaving chromaffin cell cortical areas devoid of fluorescence; and (3) These changes are accompanied by a decrease in F-actin, a concomitant increase in G-actin, and a decrease in the F-actin associated with the chromaffin cell cytoskeleton (DNAse I assay). We also have demonstrated the presence in chromaffin cells of gelsolin and scinderin, two Ca²⁺-dependent actin filament-severing proteins, and suggested that chromaffin cell stimulation activates scinderin with the consequent disruption of F-actin networks. Scinderin, a protein recently isolated in our laboratory, is restricted to secretory cells and is present mainly in the cortical chromaffin cell cytoplasm. Scinderin, which is structurally different from gelsolin (different pI_s, amino acid composition, peptide maps, and so on), decreases the viscosity of actin gels as a result of its F-actin-severing properties, as demonstrated by electron microscopy. Stimulation of chromaffin cells either by nicotine (10 μM) or high K+ (56 mM) produces a redistribution of subplasmalemmal scinderin and actin disassembly, which preceded exocytosis. The redistribution of scinderin and exocytosis is Ca²⁺-dependent and is not mediated by muscarinic receptors. Furthermore, our cytochemical experiments demonstrate that chromaffin cell stimulation produces a concomitant and similar redistribution of scinderin (fluorescein-labeled antibody) and F-actin (rhodamine phalloidin fluorescence), suggesting a functional interaction between these two proteins. Stimulation-induced redistribution of scinderin and F-actin disassembly would produce subplasmalemmal areas of decreased cytoplasmic viscosity and increased mobility for chromaffin granules. Exocytosis sites, evaluated by antidopamine-β-hydroxylase (anti-DβH) surface staining, are preferentially localized in plasma membrane areas devoid of F-actin.     

Studies on the metabolic interactions between 4-methylpyrazole and methanol using the monkey as an animal model

Bovine adrenal chromaffin granules have been shown to contain [Met]enkephalin and [Leu]enkephalin and at least seven other small peptides that exhibit specific binding to opiate receptors. Six of these peptides have been characterized and their structures established as (O)-[Met]enkephalin, [Met]enkephalin-Arg⁶-Arg⁷, [Met]enkephalin-Lys⁶, [Met]enkephalin-Arg⁶, [Leu]enkephalin-Arg⁶, and [Met]enkephalin-Arg⁶-Phe⁷. Many of these hexa- and heptapeptides are also present in bovine and human brain. It is suggested that the presence of these peptides in a tissue is evidence of a common biosynthetic pathway of the enkephalins from a large precursor protein.     

Fluorescence and electron microscopy of amine-storing enterochromaffin-like cells in tracheal epithelium of mouse

Summary The tracheo-bronchial mucosa of the mouse has been found to contain an extensive system of argyrophilic epithelial cells. In the trachea the cells morphologically resemble enterochromaffin cells. Normally, these enterochromaffin-like cells contain no fluorogenic amine, as revealed by the Falck-Hillarp formaldehyde technique. On the other hand the cells have the capacity to take up and decarboxylate 3,4-dihydroxyphenylalanine (DOPA) or 5-hydroxytryptophan (5-HTP); the amine formed is stored in the cytoplasm in a reserpine-sensitive store. This capacity to produce and store amines under experimental conditions may reflect the presence in the tracheal enterochromaffin-like cells of an amine which can not be demonstrated with available fluorescence histochemical techniques. In the electron microscope the tracheal enterochromaffin-like cells were identified by a positive argyrophil reaction and by their capacity to accumulate radioactivity after administration of ³H-DOPA or ³H-5-HTP as revealed by autoradiography. The radioactive labelling was associated with cytoplasmic electron-dense granules (800–1000 Å), suggesting that the amine formed was stored in these granules. Accordingly, the granules stained argentaffin after DOPA-pre-treatment of the animal. It is suggested that, like similar cells in the gastric mucosa, these argyrophilic enterochromaffin-like cells constitute an endocrine system in which amines are of cytophysiological importance.     

ELECTRON MICROSCOPIC RADIOAUTOGRAPHIC STUDIES OF THE CAROTID BODY FOLLOWING INJECTIONS OF LABELED BIOGENIC AMINE PRECURSORS

Adult Syrian hamsters were given a subcutaneous injection of reserpine 3 days before an intraperitoneal injection of ³H-3,4 dihydroxyphenylalanine or ³H-5, hydroxytryptophan and the carotid bodies were subsequently prepared for electron microscopic radioautography. Other Syrian hamsters were given a subcutaneous injection of reserpine and the carotid bodies were subjected to a sensitive cytochemical test for the detection of unsubstituted amines. These studies were made to determine whether the labeled amine precursors were incorporated into the cells and to see whether the parenchymal cells were affected by reserpine treatment. Material from hamsters treated first with reserpine and subsequently injected with ³H-3,4 dihydroxyphenylalanine or ³H-5, hydroxytryptophan exhibited reduced grains of silver over the cells which were associated mainly with the dense cores of the cytoplasmic granules. These studies offer evidence that the granules of the carotid body incorporate catecholamine and indolamine precursors. Material from hamsters incubated for the presence of unsubstituted amines gave a positive reaction (opaque cytoplasmic granules) for catecholamines but not for indolamines. The latter substances may not be present in quantities sufficient to register a positive reaction in the cytochemical test. The opaque granules, indicative of the presence of catecholamines, decreased in density after reserpine treatment. 5 days after one reserpine injection the granules had regained opacity and were comparable to those seen in the control cells.     

ELECTRON MICROSCOPE RADIOAUTOGRAPHIC IDENTIFICATION OF SEROTONIN-SYNTHESIZING CELLS IN THE MOUSE GASTRIC MUCOSA

This study correlates the fine structure of mouse gastric endocrine cells with their ability to synthesize serotonin (5-HT) from 5-hydroxytryptophan (5-HTP). Mice were sacrificed 2 hr after the intravenous injection of 5-HTP-³H or 5-HT-³H. Their stomachs were processed for light- and electron microscope radioautography in a manner which retained labeled 5-HT while washing out other labeled substances. Stomachs from additional mice were incubated in vitro with 5-HT-³H and processed similarly. All morphologic types of mouse gastric endocrine cells exhibited a similar facility to incorporate exogenous 5-HTP and to convert it to 5-HT which was bound intracellularly. Differences in densities of silver grains observed over endocrine cells suggested that individual endocrine cells indeed varied in their ability to synthesize and/or to bind 5-HT; such variations, however, were not reflected by differences in fine structure, with the exception that endocrine cells with few granules always contained little newly synthesized 5-HT. The newly synthesized 5-HT was associated with the intracellular granules. The gastric endocrine cells were not labeled by exogenous 5-HT-³H, whereas mast cells were labeled by either 5-HT-³H or 5-HTP-³H administration. The findings of the present study support the position that the gastric endocrine cells represent a single cell type, at least in respect to serotonin metabolism—that the argyrophil or argentaffin reactivity of these cells merely reflects their amine content at a given time.     

5-HT nerve terminals in the fourth ventricle of the rat brain: Their identification and distribution studied by fluorescence histochemistry and electron microscopy

Summary The occurrence and distribution of supra-ependymal nerve terminals storing serotonin (5-HT) are described for the fourth ventricle of the rat brain. The nerve terminals were identified as monoaminergic 1) fluorescence-histochemically, by the presence of a varicose, formaldehyde-induced fluorescence (FIF) on the free surface of the ependyma, 2) electron microscopically, by the presence of electron dense (chromaffin) cores in small (50 nm) and large (100 nm) vesicles found within the varicose regions of supra-ependymal nerve fibres, and 3) by the absence of both the FIF and chromaffin dense cores after treatment with reserpine. Moreover, the serotonergic nature of these nerve fibres could be concluded from 1) the yellow colour of the FIF, 2) the increased FIF after treatment with nialamide or reserpine+nialamide, 3) the diminished FIF and absence of chromaffin dense cores after treatment with p-CPA, and finally 4) the persistence of the FIF and chromaffin dense cores after treatment with -MPT.A high density of 5-HT nerve terminals occurred throughout the floor of the fourth ventricle and on the floor and roof of the lateral recess. Few 5-HT nerve terminals occurred only on the roof of the fourth ventricle (velum medullare, lamina epithelialis of the tela chorioidea), and the surface of the choroid plexus epithelia was devoid of such nerves. Virtually all nerve terminals in the fourth ventricle appear to be serotonergic.Results presented in part at the Autumn Meeting of the British and Italian Pharmacological Societies, Bristol, 1974 (Lorez et al., 1974). The skilful assistance of Mr. R. Wybrecht, Mr. R. Reese and Mrs. M. Gschwind is gratefully acknowledged.     

Inositol 1,4,5-Trisphosphate Receptor in Chromaffin Secretory Granules and Its Relation to Chromogranins

The inositol 1,4,5-trisphosphate (IP₃)-mediated intracellular Ca²⁺ releases in secretory cells play vital roles in controlling not only the intracellular Ca²⁺ concentrations but also the Ca²⁺-dependent exocytotic processes. Of intracellular organelles that release Ca²⁺ in response to IP₃, secretory granules stand out as the most prominent organelle and are responsible for the majority of IP₃-dependent Ca²⁺ releases in the cytoplasm of chromaffin cells. Bovine chromaffin granules were the first granules that demonstrated the IP₃-mediated Ca²⁺ release as well as the presence of the IP₃ receptor (IP₃R) in granule membranes. Secretory granules contain all three (type 1, 2, and 3) IP₃R isoforms, and 58–69% of total cellular IP₃R isoforms are expressed in bovine chromaffin granules. Moreover, secretory granules contain large amounts (2–4 mM) of chromogranins and secretogranins; chromogranins A and B, and secretogranin II being the major species. Chromogranins A and B, and secretogranin II are high-capacity, low-affinity Ca²⁺ binding proteins, binding 30–93 mol of Ca²⁺/mol of protein with dissociation constants of 1.5–4.0 mM. Due to this high Ca²⁺ storage properties of chromogranins secretory granules contain ~40 mM Ca²⁺. Furthermore, chromogranins A and B directly interact with the IP₃Rs and modulate the IP₃R/Ca²⁺ channels, i.e., increasing the open probability and the mean open time of the channels 8- to 16-fold and 9- to 42-fold, respectively. Coupled chromogranins change the IP₃R/Ca²⁺ channels to a more ordered, release-ready state, whereby making the IP₃R/Ca²⁺ channels significantly more sensitive to IP₃.     

Serotonin Is a Key Factor for Mouse Red Blood Cell Survival

Serotonin (5-HT) is a monoamine originally purified from blood as a vasoactive agent. In nonneuronal tissues, its presence is linked with the expression of tryptophan hydroxylase 1 (TPH1) that catalyzes the rate-limiting step of its synthesis. Targeted disruption in mice of the TPH1 gene results in very low levels of circulating 5-HT. Previous analysis of the TPH1 knockout (TPH1^−/−) mouse revealed that they develop a phenotype of macrocytic anemia with a reduced half-life of their circulating red blood cells (RBC). In this study, to establish whether the observed reduced half-life of TPH1^−/− RBC is an intrinsic or an extrinsic characteristic, we compared their survival to RBC isolated from wild-type mice. Both in vivo and in vitro data converge to demonstrate an extrinsic protective effect of 5-HT since presence of 5-HT in the RBC environment protects RBC from senescence. The protective effect played by 5-HT is not mediated through activation of a classical pharmacological pathway as no 5-HT receptors were detected on isolated RBC. Rather, 5-HT acts as an effective antioxidant since reduction of 5-HT circulating levels are associated with a decrease in the plasma antioxidant capacity. We further demonstrate a link between oxidation and the removal of damaged RBC following transfusion, as supplementation with 5-HT improves RBC post-transfusion survival in a mouse model of blood banking.     

Cytochemical studies on cytoplasmic granular elements in the hamster pineal gland

Summary The pineal gland of adult golden hamsters (Mesocricetus auratus) was studied by various cytochemical methods at the electron microscopic level: (1) the modified chromaffin reaction specific for 5-hydroxytryptamine (5-HT), (2) argentaffin reaction, (3) zinc-iodide-osmium (ZIO) mixture reaction and (4) acid phosphatase reaction. In the pinealocytes, the dense-cored vesicles (80–160 nm in diameter) show both chromaffinity and argentaffinity, while the population of dense bodies (150–400 nm in diameter) is reactive to ammoniacal silver solution and ZIO mixture but not to the modified chromaffin reaction. After incubation for demonstration of acid phosphatase activity, reaction products are localized in some, but not all, of the dense bodies, in some of the small vesicles in the Golgi region and in one or two inner Golgi saccules. In nerve fibers in the pineal gland, small granulated vesicles are also reactive to the modified chromaffin reaction and ZIO mixture. Based upon these cytochemical results the following conclusions have been reached: (1) dense cored vesicles in the pinealocytes and small granulated vesicles in the nerve fibers of the hamster pineal gland contain 5-HT, and (2) the population of dense bodies in the pinealocytes is heterogenous, some are lysosomes and the others are possibly the granules responsible for the secretion of pineal peptides.Supported in part by a grant from the National Science Council, Republic of ChinaDedicated to Professor Doctor Huoyao Wei on the occasion of his 70th birthday     

Intergranular bridges in the anterior pituitary cell and their possible involvement in Ca2+-induced granule-granule fusion

Quick-freeze deep-etch electron microscopy showed the presence of bridge-like structures between adjacent secretory granules in rat anterior pituitary secretory cells. These intergranular bridges were variable in length and thickness. The finest bridges were 7–8 nm in length, while the longest ones were as long as 80 nm. Annexin II, one of the Ca²⁺-dependent phospholipid-binding proteins, is known to interlink between two membranes and induce aggregation of liposomes and chromaffin granules under the presence of Ca²⁺. In anterior pituitary cells, annexin II was detected by immunoelectron microscopy at the contact sites of secretory granules with other granules. The anterior pituitary cells treated under the presence of extracellular Ca²⁺ with Clostridium perfringens enterotoxin which induces Ca²⁺ influx showed multigranular exocytosis, i.e., multiple fusions of secretory granules with each other and with the plasma membrane. The granule-granule fusion in progress could be captured by the quick-freeze deep-etch technique. The membranes of adjacent secretory granules were partially fused at their contact sites where intergranular strands were no longer seen, while there existed intergranular strands between unfused portions of the granule membranes. From these results, we consider that the intergranular bridges, some of which may be composed of annexin II, are involved in Ca²⁺-induced granule-granule fusion in anterior pituitary cells.     

Serotonin Modulates Nicotinic Responses of Adrenal Chromaffin Cells

Abstract: 5-Hydroxytryptamine (5-HT) specifically and reversibly inhibits nicotine-induced currents and catecholamine release in bovine adrenal chromaffin cells in culture. Pharmacological analysis indicates that the inhibition is not mediated by known 5-HT receptor subtypes. The inhibition is noncompetitive over a range of nicotine concentrations between 1 and 100 μM. Preincubation with either 5-HT or substance P significantly protects the response from nicotine-induced desensitization. It is concluded that 5-HT inhibits nicotinic acetylcholine receptors on bovine adrenal chromaffin cells, probably by binding to a noncompetitive site on the receptor itself. Because both blood and the chromaffin cells contain 5-HT, the inhibition provides an opportunity for negative control of catecholamine secretion from the adrenals.     

CHEMICAL, ENZYMATIC AND ULTRASTRUCTURAL CHARACTERIZATION OF 5-HYDROXYTRYPTAMINE-CONTAINING NEURONS FROM THE GANGLIA OF APLYSIA CALIFORNICA AND TRITONIA DIOMEDIA

Abstract— Several identified neurons in Aplysia and Tritonia ganglia were shown to contain measurable quantities (4–6 pmol/cell body) of 5-hydroxytryptamine (5-HT). A metabolic correlate for the limited distribution of 5-HT among the neurons of Tritonia is provided by the finding that the enzyme, aromatic acid decarboxylase (AAD), is 500 times more active in nerve cells containing 5-HT than in neurons devoid of the amine. Although all Aplysia neurons have some AAD activity, 5-HT cell bodies in this species are 10-fold more active than cell bodies which do not contain 5-HT. The cytoplasm of 5-HT cell bodies in Aplysia and Tritonia characteristically contains granules that have minimum diameters of approx. 1000 Å and eccentric opaque cores. This type of granule was not found in somata which did not contain measurable 5-HT. These data illustrate the metabolic and morphological specialization in 5-HT-containing neurons of molluscs.     

A cytochemical study of the calcium-activated adenosinetriphosphatase in hamster adrenal medulla: its occurrence in the golgi region of chromaffin cells

Summary Several different fixation procedures and incubation media were used in order to demonstrate the ultrastructural localisation of Ca²⁺-activated adenosinetriphosphatase (ATPase) in the hamster adrenal medulla. Fixation by perfusion with 2.5% glutaraldehyde gave the best preservation of fine structure without markedly inhibiting the enzymic activity. The localisation of Ca²⁺-activated ATPase was different from that of Mg²⁺-activated ATPase: the Mg²⁺-dependent enzyme was confined to plasma membranes. Ca²⁺-dependent ATPase also occurred on the plasma membranes of neurons and of some chromaffin cells, but the most prominent site of this enzyme was in the Golgi apparatus of chromaffin cells. Most of the reaction product was localised between Golgi lamellae, but some was found in Golgi vesicles and in prosecretory granules. The nucleus, mature chromaffin granules, roughsurfaced endoplasmic reticulum and mitochondria were usually free of reaction product. Rarely, some precipitate was found in the matrix of mitochondria and in lysosomes.Wellcome Research Fellow.J. H. Burn Research Scholar.This work was supported by a grant from the Medical Research Council.     

Incorporation of adenosine into nucleotides of chromaffin cells maintained in primary cultures

Extracellular adenosine was incorporated into nucleotides of bovine chromaffin cells maintained in primary culture. In intact chromaffin tissue, a very low incorporation was found (0.8 pmol/10⁶ cells/h at an adenosine concentration of 11.45 μM), which increased 282 times in freshly isolated chromaffin cells. When maintained in primary culture, this value decreased to a value similar to that of chromaffin tissue, but later on, and in the presence of nerve growth factor (NGF), a time dependent increase of adenosine incorporation was observed which, in 84-h old cells reached up to 54 times more than that found in intact tissue. This incorporation might reflect changes in the adenosine transport at the cell membrane level, furthered by NGF effect. Incorporation, which was time-dependent, was weakly modified by stimulation of cells with 10^?4 M acetylcholine. However, acetylcholine-induced release of labelled nucleotides from chromaffin granules was observed, probably in relation to granule maturation.     

Dopamine β-hydroxylase of bovine adrenal medullae. A rapid purification procedure

1. A rapid purification procedure for dopamine β-hydroxylase from bovine adrenal-medulla chromaffin granules is presented. The homogeneity of the purified enzyme was demonstrated by means of three independent criteria. The specific activity of the enzyme compares favourably with that obtained by more involved procedures. 2. The stability of the enzyme was investigated and storage in polypropylene tubes was found preferable to storage in glass. 3. The soluble and particulate forms of dopamine β-hydroxylase appear to be identical, since membrane-bound and membrane-enclosed forms of the enzyme exhibit similar properties as regards size, charge and amino acid composition. 4. Ca²⁺ was found to stimulate the release of dopamine β-hydroxylase from bovine chromaffin granules in vitro. 5. An endogenous inhibitor of the enzyme was found in the chromaffin granules. This inhibitor was not inactivated either by heating at 100°C or by pretreatment with p-chloromercuribenzoate or Cu²⁺ ions.