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Chromogranin A (CgA) is a prohormone and granulogenic factor in neuroendocrine tissues with a regulated secretory pathway. The impact of CgA depletion on secretory granule formation has been previously demonstrated in cell culture. However, studies linking the structural effects of CgA deficiency with secretory performance and cell metabolism in the adrenomedullary chromaffin cells in vivo have not previously been reported. Adrenomedullary content of the secreted adrenal catecholamines norepinephrine (NE) and epinephrine (EPI) was decreased 30-40 % in Chga-KO mice. Quantification of NE and EPI-storing dense core (DC) vesicles (DCV) revealed decreased DCV numbers in chromaffin cells in Chga-KO mice. For both cell types, the DCV diameter in Chga-KO mice was less (100-200 nm) than in WT mice (200-350 nm). The volume density of the vesicle and vesicle number was also lower in Chga-KO mice. Chga-KO mice showed an ~47 % increase in DCV/DC ratio, implying vesicle swelling due to increased osmotically active free catecholamines. Upon challenge with 2 U/kg insulin, there was a diminution in adrenomedullary EPI, no change in NE and a very large increase in the EPI and NE precursor dopamine (DA), consistent with increased catecholamine biosynthesis during prolonged secretion. We found dilated mitochondrial cristae, endoplasmic reticulum and Golgi complex, as well as increased synaptic mitochondria, synaptic vesicles and glycogen granules in Chga-KO mice compared to WT mice, suggesting that decreased granulogenesis and catecholamine storage in CgA-deficient mouse adrenal medulla is compensated by increased VMAT-dependent catecholamine update into storage vesicles, at the expense of enhanced energy expenditure by the chromaffin cell.

Sphingomyelin derivatives like sphingosine have been shown to enhance secretion in a variety of systems, including neuroendocrine and neuronal cells. By studying the mechanisms underlying this effect, we demonstrate here that sphingomyelin rafts co-localize strongly with synaptosomal-associated protein of 25Kda (SNAP-25) clusters in cultured bovine chromaffin cells and that they appear to be linked in a dynamic manner. In functional terms, when cultured rat chromaffin cells are treated with sphingomyelinase (SMase), producing sphingomyelin derivatives, the secretion elicited by repetitive depolarizations is enhanced. This increase was independent of cell size and it was significant 15min after initiating stimulation. Interestingly, by evaluating the membrane capacitance we found that the events in control untreated cells corresponded to two populations of microvesicles and granules, and the fusion of both these populations is clearly enhanced after treatment with SMase. Furthermore, SMase does not increase the size of chromaffin granules. Together, these results strongly suggest that SNARE-mediated exocytosis is enhanced by the generation of SMase derivatives, reflecting an increase in the frequency of fusion of both microvesicles and chromaffin granules rather than an increase in the size of these vesicles., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background and Aims: N-truncated pyroglutamate (pGlu)-amyloid-β [Aβ(3-40/42)] peptides are key components that promote Aβ peptide accumulation, leading to neurodegeneration and memory loss in Alzheimer's disease. Because Aβ deposition in the brain occurs in an activity-dependent manner, it is important to define the subcellular organelle for pGlu-Aβ(3-40/42) production by glutaminyl cyclase (QC) and their colocalization with full-length Aβ(1-40/42) peptides for activity-dependent, regulated secretion. Therefore, the objective of this study was to investigate the hypothesis that pGlu-Aβ and QC are colocalized with Aβ in dense-core secretory vesicles (DCSV) for activity-dependent secretion with neurotransmitters., Methods: Purified DCSV were assessed for pGlu-Aβ(3-40/42), Aβ(1-40/42), QC, and neurotransmitter secretion. Neuron-like chromaffin cells were analyzed for cosecretion of pGlu-Aβ, QC, Aβ, and neuropeptides. The cells were treated with a QC inhibitor, and pGlu-Aβ production was measured. Human neuroblastoma cells were also examined for pGlu-Aβ and QC secretion., Results: Isolated DCSV contain pGlu-Aβ(3-40/42), QC, and Aβ(1-40/42) with neuropeptide and catecholamine neurotransmitters. Cellular pGlu-Aβ and QC undergo activity-dependent cosecretion with Aβ and enkephalin and galanin neurotransmitters. The QC inhibitor decreased the level of secreted pGlu-Aβ. The human neuroblastoma cells displayed regulated secretion of pGlu-Aβ that was colocalized with QC., Conclusions: pGlu-Aβ and QC are present with Aβ in DCSV and undergo activity-dependent, regulated cosecretion with neurotransmitters., (© 2014 S. Karger AG, Basel.)

Animals living in nontropical climates modify their physiology and behavior to adapt to seasonal environmental changes. Part of this adaptation involves the release of catecholamine from sympathetic nerve endings and the adrenal medulla, which play a major role in regulating energy balance. The aim of this work was to investigate whether adult male viscachas in their natural habitat exhibits structural changes in the adrenal medulla during the annual seasonal cycle. In August-September, chromaffin granules revealed ultrastructural changes suggestive of piecemeal degranulation. Quantitative morphometric analysis by transmission electron microscopy showed a significantly lower percentage of resting chromaffin granules and a higher percentage of altered granules and empty containers in August-September (late winter) compared to February-March (late summer), suggesting an increased secretory process of catecholamines in August-September. The mechanism of piecemeal degranulation might amplify this process, encouraging the adaptive response to winter environmental conditions. Tissue levels of epinephrine, norepinephrine, and dopamine (analyzed by high-performance liquid chromatography) changed throughout the year, reaching maximum values in February-March and minimum values in August-September. These results demonstrate morphological and biochemical seasonal variations of the adrenal medulla, suggesting that epinephrine might promote energy mobilization, which allow the Lagostomus to cope with adverse environmental conditions and thus to survive during winter season., (Copyright © 2013 Wiley Periodicals, Inc.)

Regulated secretion of neurotransmitters and neurohumoral factors from dense core secretory vesicles provides essential neuroeffectors for cell-cell communication in the nervous and endocrine systems. This study provides comprehensive proteomic characterization of the categories of proteins in chromaffin dense core secretory vesicles that participate in cell-cell communication from the adrenal medulla. Proteomic studies were conducted by nano-HPLC Chip MS/MS tandem mass spectrometry. Results demonstrate that these secretory vesicles contain proteins of distinct functional categories consisting of neuropeptides and neurohumoral factors, protease systems, neurotransmitter enzymes and transporters, receptors, enzymes for biochemical processes, reduction/oxidation regulation, ATPases, protein folding, lipid biochemistry, signal transduction, exocytosis, calcium regulation, as well as structural and cell adhesion proteins. The secretory vesicle proteomic data identified 371 proteins in the soluble fraction and 384 membrane proteins, for a total of 686 distinct secretory vesicle proteins. Notably, these proteomic analyses illustrate the presence of several neurological disease-related proteins in these secretory vesicles, including huntingtin interacting protein, cystatin C, ataxin 7, and prion protein. Overall, these findings demonstrate that multiple protein categories participate in dense core secretory vesicles for production, storage, and secretion of bioactive neuroeffectors for cell-cell communication in health and disease.

Analyzing the morphological appearance and the spatial distribution of large dense-core vesicles (granules) in the cell cytoplasm is central to the understanding of regulated exocytosis. This paper is concerned with the automatic detection of granules and the statistical analysis of their spatial locations in different cell groups. We model the locations of granules of a given cell as a realization of a finite spatial point process and the point patterns associated with the cell groups as replicated point patterns of different spatial point processes. First, an algorithm to segment the granules using electron microscopy images is proposed. Second, the relative locations of the granules with respect to the plasma membrane are characterized by two functional descriptors: the empirical cumulative distribution function of the distances from the granules to the plasma membrane and the density of granules within a given distance to the plasma membrane. The descriptors of the different cells for each group are compared using bootstrap procedures. Our results show that these descriptors and the testing procedure allow discriminating between control and treated cells. The application of these novel tools to studies of secretion should help in the analysis of diseases associated with dysfunctional secretion, such as diabetes.

Chromaffin vesicles (CV) are highly sophisticated secretory organelles synthesized in adrenal medullary chromaffin cells. They contain a complex mixture of structural proteins, catecholamine neurotransmitters, peptide hormones, and the relative processing enzymes, as well as protease inhibitors. In addition, CV store ATP, ascorbic acid, and calcium. During the last decades, extensive studies have contributed to increase our understanding of the molecular composition of CV. Yet, the recent development of biochemical and imaging procedures has greatly increased the list of CV-soluble constituents and opened new horizons as to the complexity of CV involvement in acute stress responses. Thus, a coherent picture of CV molecular composition is still to be drawn. This review article will provide a detailed account of the content of CV soluble molecules as it emerges from the most recent analytical studies. Moreover, this review article will attempt at focussing on the physiological and pathophysiological implications of the products released by CV., (Copyright (c) 2008 Wiley-Liss, Inc.)

Our current notions of different granule pools, granule interaction with the plasma membrane, and ultimately granule and plasma membrane soluble N-ethylmaleimide-sensitive-factor attachment protein (SNARE) interactions, result largely from inferences based upon biochemical alterations of secretion kinetics. Another view of events comes from studies using total internal reflection fluorescence microscopy (TIRFM) to investigate granule behaviour immediately adjacent to the plasma membrane. The motions of secretory (chromaffin) granules in bovine chromaffin cells visualized by TIRFM are highly restricted, as if granules are caged or tethered. These small motions are regulated by ATP and Ca2+, two factors that increase priming of the secretory response. There is no evidence that granules decrease their motion immediately before secretion. To the contrary, there is a tendency for granules to increase their motions and travel within a few hundred milliseconds of fusion. Hence, the notion of a long-lived docked state as a prelude to fusion does not encompass the physical reality of molecular scale motions, multiple tethering states and significant travel immediately preceding the exocytotic event. Increased travel may increase the probability of granules interacting productively with the plasma membrane constituents, thereby, increasing the probability of fusion.

This article reviews the current status of research about the histogenesis and morphofunctional characteristics of chromaffin cells in the adrenal medulla. First, this study reports the selective migration, transcription and activation factors, and the morphological events of the chromaffin cell precursors during adrenal medulla development. Subsequently, the morphofunctional characteristics of adrenergic and non-adrenergic cells are considered, with particular reference to the characteristics of chromaffin granules and their biological steps, including their formation, traffic (storage, targeting and docking), exocytosis in the strict sense and recapture. Moreover, the relationship of chromaffin cells with other tissue components of the adrenal medulla is also revised, comprising the ganglion cells, sustentacular cells, nerves and connective-vascular tissue.

Chromaffin cell exocytosis is a fascinating interplay between secretory vesicles and cellular components. One of these components is the cytoskeleton and its associated regulatory proteins. Transport of chromaffin secretory granules from their site of biosynthesis towards the active site of exocytosis requires both F-actin fine remodelling as well as microtubule trails. At least two molecular motors, myosins II and V, seem to play a crucial role in the control of F-actin dynamics and vectorial vesicle displacement respectively. Vesicle movement experiences spatial restrictions as they approach the cell cortical region, where the F-actin meshwork constitutes a barrier-limiting vesicle access to the plasmalemma. During secretion, cortical F-actin is locally disrupted providing access of vesicles to release sites on the plasmalemma. Removal of the stimulus restores cortical F-actin. Two pathways (Ca2+-scinderin and PKC-MARCKS) control F-actin changes during the secretory cycle . Furthermore, GTPases such as RhoA, that controls F-actin network integrity, and Cdc42 signalling which induces the formation of local actin filaments at active sites, provide additional evidence on the importance of F-actin as a key element in vesicle transport and in the exocytotic machinery of chromaffin cells.

Chromogranins and secretogranins have traditionally been known as marker proteins of secretory granules that contain the highest concentrations of cellular calcium, reaching approximately 40 mM. In addition, chromogranin B was also shown to exist in the nucleus, localizing in the putative inositol 1,4,5-trisphosphate (IP3)-sensitive nucleoplasmic Ca2+ store vesicles. Chromogranins A (CGA) and B (CGB) are high-capacity, low-affinity Ca2+ binding proteins, binding 30-90 mol of Ca2+/mol with dissociation constants (Kd) of 1.5-4 mM. Yet the Ca2+-binding property of secretogranins has not been studied. Here, we show the localization of secretogranin II (SgII) in the nucleus, more specifically, in the IP3-sensitive nucleoplasmic Ca2+ store vesicles along with CGB and the IP3 receptors. We have also determined the Ca2+-binding property of SgII and found that SgII binds 61 mol of Ca2+/mol (910 nmol Ca2+/mg) with a Kd of 3.0 mM at the intragranular pH 5.5 and 30 mol of Ca2+/mol (440 nmol Ca2+/mg) with a Kd of 2.2 mM at a near-physiological pH 7.5. Chromogranin B also bound 50 mol of Ca2+/mol (670 nmol Ca2+/mg) with a Kd of 3.1 mM at pH 7.5. Given the high-capacity, low-affinity Ca2+-binding property of SgII and its presence in the IP3-sensitive nucleoplasmic Ca2+ store vesicles, these results suggest that SgII may function in the storage and control of Ca2+ in the nucleus through its interaction with CGB in the nucleoplasmic vesicles.

The secretory pro-hormone chromogranin A (CHGA) is densely packed into storage granules along with catecholamines, playing a catalytic role in granule biogenesis. 3-Dimensional structural data on CHGA are lacking. We found a superfamily structural homology for CHGA in the tropomyosin family of alpha-helical coiled-coils, even in mid-molecule regions where primary sequence identity is only modest. The assignment was confirmed by an independent algorithm, suggesting approximately 6-7 such domains spanning CHGA. We provide additional physiochemical evidence (chromatographic, spectral, microscopic) consistent with this unusual structure. Alpha-helical secondary structure (at up to approximately 45%) was confirmed by circular dichroism. CHGA molecular mass was estimated by MALDI-TOF mass spectrometry at approximately 50 kDa and by denaturing gel filtration at approximately 50-61 kDa, while its native Stokes radius was approximately 84.8 A, as compared to an expected approximately 30 A; the increase gave rise to an apparent native molecular weight of approximately 578 kDa, also consistent with the extended conformation of a coiled-coil. Small-angle X-ray scattering (SAXS) on CHGA in solution best fit an elongated cylindrical conformation in the monodisperse region with a radius of gyration of the rod cross-section (Rt) of approximately 52 A, compatible with a coiled-coil in the hydrated, aqueous state, or a multimeric coiled-coil. Electron microscopy with negative staining revealed an extended, filamentous CHGA structure with a diameter of approximately 94 +/- 4.5 A. Extended, coiled-coil conformation is likely to permit protein "packing" in the secretory granule at approximately 50% higher density than a globular/spherical conformation. Natural allelic variation in the catestatin region was predicted to disrupt the coiled-coil. Chromaffin granule ultrastructure revealed a approximately 108 +/- 6.3 A periodicity of electron density, suggesting nucleation of a binding complex by the CHGA core. Inhibition of CHGA expression, by siRNA, disrupted regulated secretory protein traffic by approximately 65%, while targeted ablation of the CHGA gene in the mouse reduced chromaffin granule cotransmitter concentrations by approximately 40-80%. These results suggest new roles for secretory protein tertiary structure in hormone and transmitter storage, with implications for secretory cargo condensation (or dense core "packing" structure) within the regulated pathway.

Dense vesicles can be observed in live bovine chromaffin cells using fluorescent reflection confocal microscopy. These vesicles display a similar distribution, cytoplasmic density and average size as the chromaffin granules visualized by electron microscopy. In addition, the acidic vesicles labeled with Lysotracker Red comprised a subpopulation of the vesicles that are visualized by reflection fluorescence. A combination of fluorescence reflection and transmitted light images permitted the movements of vesicles in relation to the cortical cytoskeleton to be studied. The movement of vesicles located on the outside of this structure was restricted, with an apparent diffusion coefficient of 1.0+/-0.4 x 10(-4) microm(2)/s. In contrast, vesicles located in the interior moved much more freely and escaped from the visual confocal plane. Lysotracker labeling was more appropriate to study the movement of the faster moving vesicles, whose diffusion coefficient was five times higher. Using this type of labeling we confirmed the restriction on cortical movement and showed a clear relationship between vesicle mobility and the kinetics of cytoskeletal movement on both sides of the cortical cytoskeleton. This relationship was further emphasized by studying cytoskeletal organization and kinetics. Indeed, an estimate of the size of the cytoskeletal polygonal cages present in the cortical region and in the cell interior agreed well with the calculation of the theoretical radius of the cages imprisoning vesicle movement. Therefore, these data suggest that the structure and kinetics of the cytoskeleton governs vesicle movements in different regions of chromaffin cells.

Observation by transmission electron microscopy, coupled with morphometric analysis and estimation procedure, revealed unique ultrastructural features in 25.94% of noradrenaline (NA)-containing granules and 16.85% of adrenaline (A)-containing granules in the rat adrenal medulla. These consisted of evaginations of the granule limiting membrane to form budding structures having different morphology and extension. In 14.8% of NA granules and 12.0% of A granules, outpouches were relatively short, looked like small blebs emerging from the granule surface and generally contained electron-dense material. A proportion of 11.2% of NA granules and 4.9% of A granules revealed the most striking ultrastructural features. These secretory organelles presented thin, elongated, tail-like or stem-like appendages, which were variably filled by chromaffin substance and terminated with spherical expansions of different electron density. A cohort of vesicles of variable size (30-150 nm in diameter) and content was found either close to them or in the intergranular cytosol. Examination of adrenal medullary cells fixed by zinc iodide-osmium tetroxide (ZIO) revealed fine electron dense precipitates in chromaffin granules, budding structures as well as cytoplasmic vesicles. These data indicate that a common constituent is revealed by the ZIO histochemical reaction in chromaffin cells. As catecholic compounds are the main tissue targets of ZIO complexes, catecholamines are good candidates to be responsible for the observed ZIO reactivity. This study adds further to the hypothesis that release of secretory material from chromaffin granules may be accomplished by a vesiclular transport mechanism typical of piecemeal degranulation.

The quantal hypothesis states that neurotransmitter is released in discrete packages, quanta, thought to represent the neurotransmitter content of individual vesicles. If true, then vesicle size should influence quantal size. Although chromaffin cells are generally thought to have a single population of secretory vesicles, our electron microscopy analysis suggested two populations as the size distribution was best described as the sum of two Gaussians. The average volume difference was fivefold. To test whether this difference in volume affected quantal size, neurotransmitter release from permeabilized cells exposed to 100 microM Ca2+ was measured with amperometry. Quantal content was bimodally distributed with both large and small events; the distribution of vesicle sizes predicted by amperometry was extremely similar to those measured with electron microscopy. In addition, each population of events exhibited distinct release kinetics. These results suggest that chromaffin cells have two populations of dense core vesicles (DCV) with unique secretory properties and which may represent two distinct synthetic pathways for DCV biogenesis or alternatively they may represent different stages of biosynthesis.

Piecemeal degranulation (PMD) has been recognized in two cases of human pheochromocytoma from the adrenal medulla, which were studied by transmission electron microscopy. Tumour pheochromocytes presented a highly characteristic cytoplasmic admixture of normal resting granules, swollen granules with eroded matrices and enlarged empty containers. Chromaffin granules that appeared to be normal and altered granules maintained their individual structure, and did not fuse with each other or with the plasma membrane. In accordance with the currently accepted model for granule discharge during PMD, electron-dense or clear vesicles 30-150 nm in diameter were seen either attached to the surface of chromaffin granules and the plasma membrane or free in the cytosol. This is the first description of PMD in human adrenal chromaffin cells and, in addition, is the first report of PMD in tumour secretory cells. These findings add further to the concept that PMD may have a broader spectrum of expression than hitherto recognized.

We identify a new naturally occurring class of inhibitor of vacuolar H+-ATPases (V-ATPases) isolated from vacuolar membranes of Neurospora crassa and from chromaffin granule membranes of Bos taurus. To date, the new class includes six chondropsins and poecillastrin A, large polyketide-derived macrolide lactams with 33-37 membered rings. In the National Cancer Institute's 60-cell screen the chondropsin class showed a tumor cell growth inhibitory fingerprint essentially indistinguishable from that of the bafilomycin/concanamycin and the salicylihalamide/lobatamide classes of well-established V-ATPase inhibitors. Half-maximal inhibition of V-ATPase activity in vitro occurred at 0.04-0.7 microM for the fungal vacuolar V-ATPase and at 0.4 to >10 microM for the chromaffin granule V-ATPase. Thus, the new inhibitors are somewhat less potent than the other two classes, which typically have Ki values of <10 nM for V-ATPases, and the new inhibitors differ from the other two classes in their specificity. The bafilomycin class inhibits all eucaryotic V-ATPases, the salicylihalamide class inhibits mammalian V-ATPases but not fungal V-ATPases, and the new chondropsin class inhibits the N. crassa V-ATPase better than the chromaffin granule V-ATPase. Two mutations in the N. crassa V-ATPase that affect the binding of bafilomycin had small but reproducible effects on the affinity of chondropsins for the V-ATPase, suggesting the possibility of a similar mechanism of inhibition.

The number of transmitter molecules released in a quantal event can be regulated, and recent studies suggest that the modulation of quantal size is associated with corresponding changes in vesicle volume (Colliver et al., 2000; Pothos et al., 2002). If so, this could occur either by distension of the vesicle membrane or by incorporation and removal of vesicle membrane. We performed simultaneous measurements of vesicle membrane area and catecholamine release in individual quantal events from chromaffin cells using cell-attached patch amperometry. Cells were treated with reserpine, a vesicular monoamine transport blocker that decreases quantal size, or l-dopa, a catecholamine precursor that increases quantal size. We show that decrease and increase in quantal size are associated with a respective decrease and increase in vesicle membrane area. These results point to a novel mechanism of vesicle membrane dynamics by which vesicles physically change their membrane area in response to changes in transmitter content such that the intravesicular concentration of transmitter is maintained.

Heterotrimeric G-proteins at the plasma membrane serve as switches between heptahelical receptors and intracellular signal cascades. Likewise endomembrane associated G-proteins may transduce signals from intracellular compartments provided they consist of a functional trimer. Using quantitative immunoelectron microscopy we found heterotrimeric G-protein subunits Galpha2, Galpha(q/11), Gbeta2 and Gbeta5 to reside on secretory granules in chromaffin cells of rat adrenal glands. Thus rat chromaffin granules are equipped with functional G-proteins that consist of a specific alpha-, beta- and probably gamma-subunit combination. Serotonin uptake into a crude rat chromaffin granule preparation was inhibited by activated Galphao2 (10 nM) to nearly the same extent as by GMppNp (50 microM) whereas GDPbetaS was ineffective. The data support the idea that vesicular G-proteins directly regulate the transmitter content of secretory vesicles. In this respect Galphao2 appears to be the main regulator of vesicular momoamine transporter activity.

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.

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., (Copyright 2001 Academic Press.)

Morphometry of numerical density and electron probe X-ray quantitative microanalysis were used to measure the alterations in the number and calcium content of chromaffin granules in adrenal medulla cells during emergency reaction of restrained rats. It was found that both the alterations showed monotonic decrease during the period of restraint, but the rate of decrease of the calcium content was significantly faster than that of the granules. These data are consistent with the hypothesis that calcium release from the granules may increase the cytoplasmic calcium concentration, which again triggers off the granule exocytosis.

Quantitative time-lapse evanescent-wave imaging of individual fluorescently labelled chromaffin granules was used for kinetic analysis of granule trafficking through a approximately 300-nm(1/e2) optical section beneath the plasma membrane. The mean squared displacement (MSD) was used to estimate the three-dimensional diffusion coefficient (D(3)). We calculated the granules' speed, frame-to-frame displacement and direction and their autocorrelation to identify different stages of approach to the membrane. D(3) was about 10,000 times lower than expected for free diffusion. Granules located approximately 60 nm beneath the plasma membrane moved on random tracks (D(3) approximately 10(-10) cm(2)s(-1)) with several reversals in direction before they approached their docking site at angles larger than 45 degrees. Docking was observed as a loss of vesicle mobility by two orders of magnitude within <100 ms. For longer observation times the MSD saturated, as if the granules' movement was confined to a volume only slightly larger than the granule. Rarely, the local random motion was superimposed with a directed movement in a plane beneath the membrane. Stimulation of exocytosis selectively depleted the immobile, near-membrane granule population and caused a recruitment of distant granules to sites at the plasma membrane. Their absolute mobility levels were not significantly altered. Application of latrunculin or jasplakinolide to change F-actin polymerisation caused a change in D(3) of the mobile granule population as well as a reduction of the rate of release, suggesting that granule mobility is constrained by the filamentous actin meshwork and that stimulation-dependent changes in actin viscosity propel granules through the actin cortex.

The biosynthesis of enkephalin opioid neuropeptides as well as numerous peptide hormones and neurotransmitters requires proteolytic processing of the respective prohormone precursors. We previously identified a novel cysteine protease known as prohormone thiol protease (PTP) as the major proenkephalin-processing enzyme in chromaffin granules (secretory vesicles) of bovine adrenal medulla. In this study, colocalization of PTP with (Met)enkephalin in regulated secretory vesicles was assessed by immunochemical approaches. Western blots demonstrated the presence of PTP in chromaffin granules, with equivalent levels of PTP protein in the soluble and membrane components of the vesicle. The presence of PTP in pituitary was also demonstrated by immunoblots. Immunoelectron microscopy demonstrated immunogold-labeled PTP and (Met)enkephalin within isolated chromaffin granules. In primary cultures of chromaffin cells, the discrete pattern of PTP and (Met)enkephalin immunofluorescence staining in neuritic extensions and cytoplasmic (perinuclear) regions of chromaffin cells is consistent with localization to secretory vesicles. Moreover, cosecretion of PTP and (Met)enkephalin from chromaffin cells occurred upon KCl depolarization in a calcium-dependent manner, indicating the localization of PTP and (Met)enkephalin within regulated secretory vesicles. Calcium-dependent secretion is a well known property of regulated secretory vesicle exocytosis. Overall, these results are consistent with the localization of PTP to functional, regulated secretory vesicles that contain (Met)enkephalin.

Rab3a, a small GTP-binding protein, is believed to mediate Ca2+-dependent exocytosis. Consistent with such a role was the previously reported specific association of Rab3a with synaptic vesicles in neurons and secretory granules in adrenal chromaffin cells. Secretory vesicles are believed to be the final point of Rab3a membrane association, as it was shown by several groups that Rab3a dissociates from the secretory vesicle membrane during stimulated exocytosis. In chromaffin cells, Rab3a is not exclusively localized on secretory granules since a fraction is present on a previously unidentified subcellular compartment equilibrating at light sucrose density. This 'light' membraneous structure could be the starting point for reassociation of Rab3a with membranes involved in granule formation, or it could be a structure unrelated to granules. The present study used several subcellular fractionation techniques and immunomicroscopy to unravel the nature of the 'light' Rab3a-containing structures from bovine chromaffin cells in primary culture. After stimulation, amounts of both Rab3a-d and the granule marker dopamine-beta-hydroxylase (DbetaH) increase transiently in sucrose gradient fractions enriched in endosomal markers. A diaminobenzidine-induced density shift of endosomes alters the distribution of DbetaH and Rab3a-d. At the ultrastructural level, subplasmalemmal pleiomorphic organelles were detected by Rab3a-d-immunogold labelling. Taken together our data provide for the first time evidence that internalised secretory granule membranes go through an endosomal stage where Rab3a is present, resembling the neuronal synaptic vesicle cycle. This indicates that the endosome is an important trafficking route in the biogenesis/recycling of secretory vesicles in chromaffin cells, in which Rab3a could have an as yet unknown regulatory function, and could point to the existence of alternative recycling pathways for the chromaffin granule membrane.

We have analyzed ultrathin sections from isolated bovine chromaffin cells grown on plastic support, after fast freezing, by quantitative electron microscopy. We determined the size and intracellular distribution of dense core vesicles (DVs or chromaffin granules) and of clear vesicles (CVs). The average diameter of DVs is 356 nm, and that of CVs varies between 35-195 nm (average 90 nm). DVs appear randomly packed inside cells. When the distance of the center of DVs to the cell membrane (CM) is analyzed, DV density is found to decrease as the CM is approached. According to Monte Carlo simulations performed on the basis of the measured size distribution of DVs, this decay can be assigned to a "wall effect." Any cortical barrier, regardless of its function, seems to not impose a restriction to a random cortical DV packing pattern. The number of DVs closely approaching the CM (docked DVs) is estimated to be between 364 and 629 (average 496), i.e., 0.45 to 0.78 DVs/micron2 CM. Deprivation of Ca2+, priming by increasing [Ca2+]i, or depolarization by high [K+]e for 10 s (the effect of which was controlled electrophysiologically and predicted to change the number of readily releasable granules [RRGs]) does not significantly change the number of peripheral DVs. The reason may be that (a) structural docking implies only in part functional docking (capability of immediate release), and (b) exocytosis is rapidly followed by endocytosis and replenishment of the pool of docked DVs. Whereas the potential contribution of DVs to CM area increase by immediate release can be estimated at 19-33%, that of CVs is expected to be in the range of 5.6-8.0%.

The (annexin II-p11)2 tetramer has been proposed to participate in exocytosis and several other members of the annexin superfamily have been reported to aggregate liposomes in vitro. In this context, the Ca2+-dependent binding of several annexins to chromaffin granules and liposomes was investigated by cryo-electron microscopy. The Ca2+-dependent aggregation of lipid membranes by (annexin II-p11)2 results from the spontaneous self-organization of the protein into two-dimensional plaques, which are visualized in projection as characteristic junctions. The junctions have a constant thickness of 210(+/-10) A and present a symmetrical distribution of electron-dense material arranged into seven stripes. They were observed over a wide range of Ca2+ concentrations, down to 2 microM. The molecular components corresponding to the seven electron-dense stripes were assigned as follows: the two associated membranes give rise to two outer stripes each and the three central stripes correspond to the (annexin II-p11)2 tetramer. Each annexin II molecule interacts with the outer lipid leaflet of one membrane, giving rise to one stripe, while the central stripe is due to the (p11)2 dimer with which both annexin II molecules interact. Both annexin II and annexin I also induced the Ca2+-dependent aggregation of liposomes via junctions that lack the central (p11)2 moiety and present only six high-density stripes. As expected, both annexin V and annexin III bind to liposomes without inducing their aggregation., (Copyright 1997 Academic Press Limited.)

N-ethylmaleimide sensitive fusion protein (NSF) and soluble NSF attachment proteins (SNAPs) are involved in many vesicular transport steps. It has been proposed that SNAPs and NSF associate with their membrane receptors only when vesicles dock on the target membrane. Analysis of NSF and alpha-SNAP distribution in fractionation of organelles from adrenal medulla indicated that a substantial amount of both proteins distributed with chromaffin granules. Further fractionation of intact granules and lysed granule membranes showed exact overlap of NSF and alpha-SNAP distribution with chromaffin granules. These results suggest that NSF and alpha-SNAP are associated with chromaffin granules and support the idea that they function prior to docking of the granules on the plasma membrane.

Excitation-secretion uncoupling peptides (ESUPs) are inhibitors of Ca2+-dependent exocytosis in neural and endocrine cells. Their mechanism of action, however, remains elusive. We report that ESUP-A, a 20-mer peptide patterned after the C terminus of SNAP-25 (synaptosomal associated protein of 25 kDa) and containing the cleavage sequence for botulinum neurotoxin A (BoNT A), abrogates the slow, ATP-dependent component of the exocytotic pathway, without affecting the fast, ATP-independent, Ca2+-mediated fusion event. Ultrastructural analysis indicates that ESUP-A induces a drastic accumulation of dense-core vesicles near the plasma membrane, mimicking the effect of BoNT A. Together, these findings argue in favor of the notion that ESUP-A inhibits ATP-primed exocytosis by blocking vesicle docking. Identification of blocking peptides which mimic sequences that bind to complementary partner domains on interacting proteins of the exocytotic machinery provides new pharmacological tools to dissect the molecular and mechanistic details of neurosecretion. Our findings may assist in developing ESUPs as substitute drugs to BoNTs for the treatment of spasmodic disorders.

Syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa), associated with the neuronal plasmalemma, and synaptobrevin, a membrane protein of synaptic vesicles, are essential components of the exocytotic apparatus of synaptic vesicles. All three can be proteolytically cleaved by tetanus and/or botulinum neurotoxins. As a consequence of their cleavage, exocytosis of neurotransmitters is blocked. In adrenal chromaffin cells botulinum neurotoxin A only incompletely inhibits exocytosis. This incomplete inhibition of exocytosis is associated with only partial cleavage of SNAP-25 by the toxin, indicating that distinct pools of SNAP-25 may exist in chromaffin cells which differ in their sensitivities to botulinum neurotoxin A. In line with this result we localized SNAP-25 by immunogold electron microscopy not only to the plasmalemma but also to the chromaffin vesicle membrane. Moreover, in addition to SNAP-25 monomers, stable SNAP-25/syntaxin heterodimers were found in chromaffin cells. Subfractionation studies revealed the presence of SNAP-25/syntaxin heterodimers in an enriched fraction of chromaffin vesicles. This complex proved to be stable in SDS, and SNAP-25 within heterodimers was resistant to proteolytic attack by botulinum neurotoxin A. We suggest that these preexisting heterodimers may serve as receptors of soluble NSF attachment proteins (SNAP receptors) during chromaffin vesicle exocytosis.

In this work, we have examined the subcellular distribution of cysteine string proteins (Csps) in bovine adrenal medullary chromaffin cells. Csps did not leak from digitonin-permeabilized chromaffin cells, suggesting that there is no cytosolic pool of the protein in these cells. Subcellular fractionation studies confirmed that there was essentially no Csp immunoreactivity in the cytosolic fraction. However, immunoreactivity was detected in the membrane fractions of these cells. Csp immunoreactivity codistributed with dopamine beta-hydroxylase, a granule marker protein, in sucrose gradient-separated granule fractions. Immunofluorescence studies showed that all chromaffin cells in culture were stained with a punctate appearance consistent with a granular localization. These results were confirmed by immunogold labeling, which demonstrated specific labeling of chromaffin granule membranes. In addition to its presence on synaptic vesicles, cysteine string protein is therefore a bona fide chromaffin granule membrane protein.

The release of chromaffin granular content from staphylococcal enterotoxin B (SEB)-treated and -untreated PC12 cells was studied by electron microscopy. The treatment of the cells with SEB at the concentration of 20 micrograms/ml caused marked increase of the chromaffin granules that either bound to the plasma membrane by the characteristic rods, measuring 15 to 20 nm in length and showing a tubular structure, or budded off at the free cell surface, surrounded by a layer of rod-containing cytoplasm and enclosed by the plasma membrane. The binding between the granular and plasma membranes by the rods did not lead to membrane fusion and exocytosis of the granular content. Many of the bound granules showed vesiculation with loss of the electron-dense core material; at the same time, some of the binding rods contained intraluminal electron-dense material similar to the granular core material. These findings suggested that the electron-dense material (i.e., norepinephrine) of the bound granules was released extracellularly through channels within the rods. Although the granules were bound to the plasma membrane with equal frequency at the free and contiguous cell surfaces, the granular budding occurred only at the free cell surface, indicating that it occurred incidentally to some granules bound at the free cell surfaces. On the basis of the morphological observations, it is postulated that the electron-dense material of the bound granule is selectively released extracellularly through the rods, leaving the vesiculated granules behind in the cytoplasm. The same mode of release of the granular content was observed, though less frequently, in the untreated control cells. No morphological evidence that indicated that the granular content was released extracellularly by exocytosis was found in the treated and control cells. The present observations indicated that the SEB treatment of PC12 cells stimulated the binding of chromaffin granules to the plasma membrane by the rods and the budding of the bound granules at the free cell surface.

Transforming growth factors beta (TGF-beta), a family of pleiotropic cytokines, are widely distributed in the developing and adult nervous system. In order to further determine the neural functions of TGF-beta, we have localized the TGF-beta isoforms 1, 2 and 3 in the adult rat adrenal medulla and studied the neuroprotective capacity of one representative family member, TGF-beta 2, for those spinal cord neurons which innervate adrenal chromaffin cells and which die after destruction of the adrenal medulla. Unilateral electrothermal destruction of the adrenal medulla led to the disappearance of 25% of sympathetic preganglionic neurons, which are located in the intermediolateral (IML) column of thoracic spinal cord segments 7-10 and can be selectively marked by NADPH-diaphorase. The neurons which disappeared following adrenomedullectomy constitute the full set of neurons that innervate the adrenal medulla. Implantation of gelfoam soaked with 0.5 micrograms TGF-beta 2 into the adrenal wound cavity rescued all spinal cord neurons in the IML ipsilaterally to the lesioned side. Cytochrome c was not effective. Injections of [125I]TGF-beta 2 into the adrenal medulla did not result in retrograde transport and subsequent labelling of spinal cord neurons, suggesting that TGF-beta may exert its neuroprotective actions by indirect mechanisms. TGF-beta applied to cultured adrenocortical cells did not overtly increase the amount of mRNA for fibroblast growth factor-2, an established trophic molecule for sympathetic preganglionic spinal cord neurons. The mechanisms by which TGF-beta exerts its neurotrophic effect are therefore unclear. Even so, our data provide the first evidence that TGF-beta may play an important role in vivo in the control of maintenance of a population of spinal cord neurons.

Heterotetrameric annexin 2 phosphorylated "in vitro" by rat brain protein kinase C is purified and obtained devoid of unphosphorylated protein; it contains 2 mol of phosphate/mol of heterotetramer. The aggregative and binding properties of the phosphorylated annexin 2 toward purified chromaffin granules are compared with those of the unphosphorylated annexin 2. Annexin 2 binds to chromaffin granules with high affinity. Phosphorylation of annexin 2 decreases the affinity of this binding without affecting the maximum binding capacity. The binding curves are strongly cooperative. It is suggested that a surface oligomerization of the proteins may take place upon binding. Besides, phosphorylation of annexin 2 is followed by a dissociation of the light chains from the heavy chains in the heterotetramer. Whereas annexin 2 induces the aggregation of chromaffin granules at microM calcium concentration, the phosphorylated annexin 2 does not induce aggregation at any concentration of calcium either at pH 6 or 7. The phosphorylation of annexin 2 by protein kinase C, MgATP, and 12-O-tetradecanoylphorbol-13-acetate on chromaffin granules induces a fusion of chromaffin granules membranes observed in electron microscopy. The fusion requires the activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate. Given these results and since annexin 2 is phosphorylated by protein kinase C under stimulation of chromaffin cells, it is suggested that phosphorylated annexin 2 may be implicated in the fusion step during exocytosis of chromaffin granules.

Small GTPases of the rab family control intracellular vesicle traffic in eukaryotic cells. Although the molecular mechanisms underlying the activity of the Rab proteins have not been elucidated yet, it is known that the function of these proteins is dependent on their precise subcellular localization. It has been suggested that Rab3a, which is mainly expressed in neural and endocrine cells, might regulate exocytosis. Recently, direct experimental evidence supporting this hypothesis has been obtained. Consistent with such a role for Rab3a in regulated exocytosis was the previously reported specific association of Rab3a with synaptic vesicles and with secretory granules in adrenal chromaffin cells. Since the latter result, based on subcellular fractionation, has been controversial, we have re-investigated the subcellular localization of this GTP-binding protein by using a combination of morphological techniques. Bovine chromaffin cells were labelled with an affinity-purified polyclonal anti-Rab3a antibody and analyzed by confocal microcopy. Rab3a was found to colocalize partially with dopamine beta-hydroxylase, a chromaffin granule marker. In agreement with this observation, immunoelectron microscopy revealed a specific staining of chromaffin granules. In addition to large dense core vesicles, some small vesicles were labelled. To eliminate the possibility that the staining was due to a Rab3a-related protein, we investigated by immunoelectron microscopy the localization of an epitope-tagged Rab3a expressed in rat PC12 cells. Secretory granules were specifically labelled, whereas clear microvesicles were not. These results provide further evidence supporting a specific association of the GTPase Rab3a with large dense core secretory vesicles.

Morphological, biochemical, and membrane capacitance measurements were used to study the role of cortical filamentous actin (F-actin) in exocytosis. Fluorescence and electron microscopy of resting chromaffin cells revealed a cortical actin network that excluded secretory vesicles from the subplasmalemmal area. Phorbol ester (PMA) treatment disrupted cortical F-actin and increased both the number of vesicles within the 0-50 nm subplasmalemmal zone and the initial rate of stimulated catecholamine release. In PMA-pretreated cells, membrane capacitance studies showed an increased number of vesicles fusing with the plasmalemma during the first two depolarizations of a train. PMA did not affect voltage-dependent Ca2+ influx. The total number of vesicles fused with the plasma membrane correlated well with the number of vesicles occupying the 0-50 nm cortical zone. Therefore, cortical F-actin disassembly allows translocation of vesicles to the plasmalemma in preparation for exocytosis.

Annexin II tetramer (AIIt) is a Ca2+ and phospholipid binding protein that has been shown to reconstitute secretion in permeabilized adrenal medulla cells. In the present study, we have characterized the interactions of AIIt with biological membranes using isolated adrenal medulla secretory granules as a model system. Without added salt, maximal binding of AIIt to chromaffin granules occurred in the absence of AIIt-dependent chromaffin granule aggregation, whereas increasing the osmolality of the reaction mixture with sucrose did not activate AIIt-dependent chromaffin granule aggregation. As the KCl or potassium glutamate concentration of the reaction mixture was increased to between 30 and 50 mM salt, AIIt-dependent chromaffin granule aggregation increased to a maximum, while AIIt binding to chromaffin granules decreased. As the salt concentration was increased from 50 to 150 mM, both AIIt-dependent chromaffin granule aggregation and the binding of AIIt to chromaffin granules were decreased. Furthermore, at optimal salt concentration, KCl and potassium glutamate activated AIIt-dependent aggregation of chromaffin granules to maximum values of about 210% and 195% of control, respectively, whereas potassium phosphate supported AIIt-dependent aggregation of chromaffin granules to only 120% of control. The concentration of AIIt for half-maximal binding to chromaffin granules without added salt or at 50 mM KCl was 0.163 +/- 0.007 (mean +/- SD, n = 3) or 0.173 +/- 0.034 microM AIIt (mean +/- SD, n = 3), respectively, and binding of AIIt to chromaffin granules was not measurable at 150 mM KCl. In contrast, at 50 mM KCl, half-maximal AIIt-dependent chromaffin granule aggregation required 0.171 +/- 0.001 microM AIIt (mean +/- SD, n = 3) and was not measurable without added salt or in the presence of 150 mM KCl. Without added salt, at 50 mM KCl, or at 150 mM KCl, the Ca2+ concentrations for half-maximal aggregation of chromaffin granules and the maximal extent of chromaffin granule aggregation (Amax) were pCa2+ = 3.79 +/- 0.062 (mean +/- SD, n = 3) and Amax = 127% of control, pCa2+ = 6.07 +/- 0.021 (mean +/- SD, n = 3) and Amax = 185% of control, or pCa2+ 4.41 +/- 0.07 (mean +/- SD, n = 3) and Amax = 156% of control, respectively. The stimulation of chromaffin granule aggregation activity and the chromaffin granule binding activity of AIIt was reversible by removal of Ca2+. These results suggest that both ionic strength and salt composition modulate both AIIt-dependent chromaffin granule aggregation and binding to the membranes of these secretory granules.

The role of phosphatidylserine and diacylglycerol in the fusion of chromaffin granules with target membranes was investigated in vitro, monitoring the mixing of membrane lipids with the octadecyl rhodamine B fluorescence dequenching assay. Polylysine was able to induce fusion of chromaffin granule ghosts with plasma membrane vesicles in the absence of Ca2+. This fusion was maximal at 1.6 microM polylysine and the kinetics were dependent on the amount of plasma membrane added. Polylysine lowered the Ca2+ threshold concentration for inducing fusion, increased the extent of fusion at a given Ca2+ concentration and acted synergistically with Ca2+ when added prior to the cation. Removal of membrane proteins by trypsinization of chromaffin granule ghost and/or plasma membranes increased the extent of polylysine induced fusion. Incorporation of diacylglycerol into the plasma membrane promoted Ca(2+)-induced fusion. Chromaffin granule ghosts were induced to fuse with model membranes of different complexities from one resembling the inner monolayer of the erythrocyte membrane to one composed solely of pure phosphatidylserine. The characteristics of the fusion with model membranes were qualitatively similar to that observed with target plasma membrane, although differences in kinetics and stoichiometries were found. Interestingly, considerably low (microM) Ca2+ concentrations were able to induce fusion of chromaffin granule ghosts with diacylglycerol containing phosphatidylserine vesicles in the presence of polylysine. Our results indicate that acidic phospholipid-like phosphatidylserine may have an important role in the process of fusion and suggest that diacylglycerol, as a destabilizing lipid, may have a role by itself in promoting exocytosis in chromaffin cells.

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.

Preparations containing ovine chromaffin granules and lysosomes were obtained by differential centrifugation and applied to density gradients. In isoosmolar linear Metrizamide gradients the granules had a lower density than the major portion of the lysosomes (1.05 compared to 1.15); however, in hyperosmolar linear Metrizamide gradients the granule density increased and they migrated close to the lysosomes. The granules separated into two bands on a discontinuous isoosmolar Metrizamide gradient; however, these two bands were similar in terms of granule and lysosomal markers. On a discontinuous hyperosmolar sucrose gradient the granules were more dense than the lysosomes, the reverse of the situation on the Metrizamide gradient. Separation on a discontinuous isoosmolar Metrizamide gradient followed by a 1.8 M sucrose cushion provided a 54-fold purification of granules from lysosomes and similar separations from other subcellular markers. This procedure also provided a 37-fold purification of bovine granules from lysosomes. It was demonstrated that thimet oligopeptidase (EC 3.4.24.15) occurred in the adrenal medulla but is not principally located in the chromaffin granule.

The presence of a glycoprotein laminin in bovine adrenal chromaffin granules was examined by SDS-PAGE followed by immunoblotting. The two chromaffin granule membrane fractions were obtained by linear sucrose gradient centrifugation followed by freezing and thawing and gel-filtration of the chromaffin granule-rich fraction, respectively. The purity of the granules in these fractions was examined by electron microscopy. These fractions contained laminin B chain-like immunoreactivity as a major immunoreactive component against anti-laminin. Laminin A chain-like immunoreactive protein was undetectable. The soluble fraction of the chromaffin granules contained no immunoreactive peptide. The presence of laminin-like immunoreactivity in the chromaffin granules was confirmed by immunocytochemical study. Laminin B chain-like immunoreactivity was also identified in the rat adrenal chromaffin granule fraction. Laminin A chain was hardly detected, as in the case of bovine adrenals. Structure of laminin in chromaffin granules in bovine and rat adrenals may be different from that of mouse Englebrethe-Holm-Swarm sarcoma laminin. The functional significance of laminin B chain-like protein in the granules is unknown at present.

Behavioral and immunocytochemical techniques were used to study the effects of adrenal medulla grafts implanted in hippocampus--after lesion of this structure--on the memory capacities of rats. Performances of the grafted rats in the radial maze test were significantly improved and, in some aspects, fully restored. On the other hand, grafts had no significant effects on a one-trial spatial recognition test and impaired object recognition. Immunocytochemical identification showed that the grafts contained chromaffin cells with a choline acetyltransferase stainings while, in parallel, phenylethanolamine-N-methyltransferase stainings seemed to be decreased. Cholinergic innervation was established between the graft and the host hippocampus. A likely interpretation of this complex pattern of results is that the functional effects of the grafts depended on the arousal level induced by the behavioral task. At the neurobiological level, these effects probably set into play an interaction between opioid, catecholaminergic and cholinergic factors. Our results may contribute to the clarification of the problem of specificity of functional effects of intracerebral grafts as well as the problem of hippocampal role in learning and memory.

In order to elucidate the mechanism involved in the specialized function of the nerve terminals, we studied the growth cone of cultured chromaffin cells using a video-enhanced Nomarski microscope. At a very high magnification, we found rapid and discrete morphological changes of small granules induced by electrical stimulation. Since they shared properties similar to those of the exocytotic responses found in granules in the cell body region, we conclude that they also reflect exocytosis of transmitter-containing granules. The frequency of the exocytotic responses was highest in the growth cone, lower in the cell body region, and none in the thin shaft of the neurite, suggesting a functional differentiation in the growth cone. Similar differentiation may be instrumental in physiological functions of the neuronal growth cone in vivo.

The graft of chromaffin adrenaline-containing (A) cells of rabbit adrenal medulla implanted to mouse brain and treated with NGF contains more survived cells 1 month after grafting than adrenal medulla alone. The cells developed either an intermediate (e.g. chromaffin cell and neuron) or a neuron-like phenotypes accompanied with a decrease in an immunoreactivity for PNMT (phenyletanolamine-N-methyltransferase). A gap junctions and attached plaques were found between grafted cells. The grafts received a synaptic input. The NGF influence on the fate of chromaffin A-containing cells is discussed.

The chromatin network was revealed in DNA stained cell imprints of developing chick embryo chrondrocytes using the recently developed method of TV image processing. The network consists of two classes of alveoles. The large alveoles, which accompany large chromocentres, are concentrated at the centromeric nuclear pole representing the most stable part of the network. The small alveoles, connected with the small chromocentres and located around the chromosome arms and telomeric ends, are dynamic. These smaller alveoles appear in differentiating chondroblasts and disappear in ageing chondrocytes. Ectopic conjunctions involving constitutive and intercalary heterochromatin are responsible for the network formation.

The transsynaptic induction of the monoamine transporter present on the membrane of chromaffin granules was studied in primary cultures of dissociated bovine adrenomedullary cells submitted to a chronic secretory stimulation. The amount of the vesicular monoamine transporter was assayed by binding of the specific ligand [3H]-dihydrotetrabenazine. After several days of incubation in the presence of high potassium, the concentration of [3H]-dihydrotetrabenazine binding sites was increased by a 1.5-2.5 factor. This increase was smaller in the presence of the cholinergic agonist carbachol. The long-term inductions of the vesicular monoamine transporter, of tyrosine hydroxylase, and of acetylcholinesterase were of similar magnitude. Under the same conditions, we found no variation in either the activities of other catecholamine biosynthetic enzymes (dopamine beta-hydroxylase and DOPA decarboxylase), or in metabolic enzymes such as lactate dehydrogenase and cytochrome c oxidase, and a decrease in the cellular content of chromogranin A and cytochrome b-561. The induction of the vesicular monoamine transporter was inhibited by the calcium channel antagonists, fluspirilene and nifedipine, and was increased by the agonist Bay K 8644. It was abolished by cycloheximide and actinomycin D. These results indicate that calcium entry into chromaffin cells increases the synthesis of the vesicular monoamine transporter, presumably by transcriptional activation. Elevation of intracellular cyclic AMP concentration or activation of protein kinase C also induced an increase in the expression of the vesicular monoamine transporter. Our results confirm that components of storage vesicle membranes are differentially regulated in response to secretory stimulation, as are several cytosolic or intravesicular soluble proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Intact adrenal chromaffin granules and purified granule membrane ghosts were allowed to fuse with acidic phospholipid planar bilayer membranes in the presence of Ca2+ (1 mM). From both preparations, we were able to detect a large conductance potassium channel (ca. 160 pS in symmetrical 400 mM K+), which was highly selective for K+ over Na+ (PK/PNa = 11) as estimated from the reversal potential of the channel current. Channel activity was unaffected by charybdotoxin, a blocker of the [Ca2+]-activated K+ channel of large conductance. Furthermore, this channel proved quite different from the previously described channels from other types of secretory vesicle preparations, not only in its selectivity and conductance, but also in its insensitivity to both calcium and potential across the bilayer. We conclude that the chromaffin granule membrane contains a K(+)-selective channel with large conductance. We suggest that the role of this channel may include ion movement during granule assembly or recycling, and do not rule out events leading to exocytosis.

Annexin I (lipocortin I) binds to secretory granule membranes and promotes their aggregation in a Ca(2+)-dependent manner [Creutz, C. E., et al. (1987) J. Biol. Chem. 262, 1860-1868; Drust, D. S., & Creutz, C. E. (1988) Nature 331, 88-91]. It is also phosphorylated on serine residues when bovine chromaffin cells are stimulated to secrete [Michener, M. L., et al. (1986) J. Biol. Chem. 261, 6548-6555], suggesting phosphorylation may be involved in modulating the function of annexin I. We report here that phosphorylation of the N-terminal tail by protein kinase C strongly inhibits the ability of annexin I to aggregate chromaffin granules by increasing the calcium requirement 4-fold. This inhibition was readily reversed when the protein was dephosphorylated by protein phosphatase 2A. The inhibition was not due to inability of phosphorylated annexin I to bind to chromaffin granules, since the phosphorylated form bound to the granule membrane at slightly lower levels of calcium than the native form. The phosphorylated annexin I also bound to 20% phosphatidylserine/80% phosphatidylcholine vesicles at lower Ca2+ levels than the native form. The inhibitory effect of phosphorylation on the granule aggregating activity of annexin I was found to be amplified by an unusual mechanism: The phosphorylated form inhibited the activity of the unphosphorylated form. The possible importance of the regulation of annexin I activity by phosphorylation in exocytosis is discussed.