Intragranular vesicles: new organelles in the secretory granules of adrenal chromaffin cells

Summary Chromaffin granules from bovine adrenal medullary chromaffin cells have been found to contain small vesicular structures bounded by unit membranes. Detection of these intragranular vesicles within intact cells requires the use of quick-freezing methods. The intragranular vesicles are labile to fixation by aldehydes which explains why they have not been described in intact cells until now. They are found in approximately 60% of the dense-core chromaffin granules in cells and 85% of isolated granules. They are usually clustered in groups of one to as many as five between the core and the inner surface of the granule membrane. The intragranular vesicles are independent vesicles in that they do not appear as simple invaginations of the granule membrane in either serial thin-section or freeze-etch views. Furthermore, they are released from the cell along with granule contents during nicotine-induced secretion of catecholamines. The structural heterogeneity provided by the intragranular vesicles may be related to the functional heterogeneity of granule contents observed in many recent biochemical studies.     

Paradoxical facilitation of exocytosis by inhibition of L-type calcium channels of bovine chromaffin cells

Ca²⁺ entry through the L-subtype (α_1D, Ca_v1,3) of voltage-dependent calcium channels (VDCCs) seems to selectively regulate the endocytotic response after the application of a single depolarizing pulse to voltage-clamped bovine chromaffin cells. Here we have found that L channel blockade with nifedipine transformed the exocytotic responses elicited by a double-pulse protocol, from depression to facilitation. This apparent paradoxical effect was mimicked by pharmacological interventions that directly block endocytosis namely, dynasore, calmidazolium, GTP-γS and GDP-βS. This reinforces our view that Ca²⁺ entry through PQ channels (α_1A; Ca_v2.1) regulates fast exocytosis while Ca²⁺ entry through L channels preferentially controls rapid endocytosis.     

L-type calcium channels are preferentially coupled to endocytosis in bovine chromaffin cells

Exocytosis and endocytosis are Ca(2+)-dependent processes. The contribution of high-voltage activated Ca(2+) channels subtypes to exocytosis has been thoroughly studied in chromaffin cells. However, similar reports concerning endocytosis are unavailable. Thus, we studied here the effects of blockers of L (nifedipine), N (omega-conotoxin GVIA) and P/Q (omega-agatoxin IVA) Ca(2+) channel on Ca(2+) currents (I(Ca)), Ca(2+) entry (Q(Ca)), as well as on the changes in membrane capacitance (C(m)) in perforated-patch voltage-clamped bovine adrenal chromaffin cells. Using 500-ms pulses to 0 or +10 mV, given from a holding potential of -80 mV and 2 mM Ca(2+) we found that omega-conotoxin GVIA affected little the exo-endocytotic responses while omega-agatoxin IVA markedly blocked those responses. However, nifedipine blocked little exocytosis but almost completely inhibited endocytosis. We conclude that L-type Ca(2+) channels seem to be selectively coupled to endocytosis.     

Amine weak bases disrupt vesicular storage and promote exocytosis in chromaffin cells

The vesicular contents in bovine chromaffin cells are maintained at high levels owing to the strong association of its contents, which is promoted by the low vesicular pH. The association is among the catecholamines, Ca2+, ATP, and vesicular proteins. It was found that transient application of a weak base, methylamine (30 mM), amphetamine (10 microM), or tyramine (10 microM), induced exocytotic release. Exposure to these agents was also found to increase both cytosolic catecholamine and intracellular Ca2+ concentration, as measured by amperometry and fura-2 fluorescence. Amphetamine, the most potent amine with respect to evoking exocytosis, was found to be effective even in buffer without external Ca2+; however, the occurrence of spikes was suppressed when BAPTA-acetoxymethyl ester was used to complex intracellular Ca2+. Amphetamine-induced spikes in Ca2+-free medium were not suppressed by thapsigargin or ruthenium red, inhibitors of the sarco(endo)plasmic reticulum Ca2+-ATPase and mitochondrial Ca2+ stores. Atomic absorption measurements of amphetamine- and methylamine-treated vesicles reveal that intravesicular Ca2+ stores are decreased after a 15-min incubation. Taken together, these data indicate that amphetamine and methylamine can disrupt vesicular stores to a sufficient degree that Ca2+ can escape and trigger exocytosis.     

Dynamin I plays dual roles in the activity-dependent shift in exocytic mode in mouse adrenal chromaffin cells

Under low stimulation, adrenal chromaffin cells release freely soluble catecholamines through a restricted granule fusion pore while retaining the large neuropeptide-containing proteinacious granule core. Elevated activity causes dilation of the pore and release of all granule contents. Thus, physiological differential transmitter release is achieved through regulation of fusion pore dilation. We examined the mechanism for pore dilation utilizing a combined approach of peptide transfection, electrophysiology, electrochemistry and quantitative imaging techniques. We report that disruption of dynamin I function alters both fusion modes. Under low stimulation, interference with dynamin I does not affect granule fusion but blocks its re-internalization. In full collapse mode, disruption of dynamin I limits fusion pore dilation, but does not block membrane re-internalization. These data suggest that dynamin I is involved in both modes of exocytosis by regulating contraction or dilation of the fusion pore and thus contributes to activity-dependent differential transmitter release from the adrenal medulla.     

Freeze-fracture study of the chromaffin cell during exocytosis: Evidence for connections between the plasma membrane and secretory granules and for movements of plasma membrane-associated particles

Summary Exocytosis was studied in acetylcholine-stimulated bovine adrenal medulla. During a pre-exocytotic stage, chromaffin granules are found in juxtaposition to the plasma membrane and separated from it by an electron dense space 25–27 Å in width. Freeze-fracture studies show this stage to be characterized by connections between the granules and the plasma membrane. These connections are apparently cytoplasmic but bridge both membranes; they are presumably proteinaceous, but their exact nature remains to be elucidated. Later stages of exocytosis were also studied by the freeze-fracture technique; a typical feature is the lack of intramembrane particles around the fusion site. Both connections and membrane particle movement are discussed in terms of recent biochemical findings.     

Rundown of secretion after depletion of intracellular calcium stores in bovine adrenal chromaffin cells

In this study, the relationship between intracellular calcium stores and depolarization-evoked stimulation was examined in bovine chromaffin cells, using changes in membrane capacitance to monitor both exocytosis and endocytosis. Cells were voltage-clamped using the perforated whole-cell patch configuration to minimize alterations in intracellular constituents. Control cells exhibited reproducible secretory responses each time the cell was stimulated. However, the same stimulation protocol elicited progressively smaller secretory responses in cells where their intracellular calcium store was emptied by thapsigargin. Transient elevation of the intracellular calcium concentration with a brief histamine treatment enhanced subsequent secretory responses in control but not in thapsigargin-treated cells. A series of depolarizations to -20 mV, which allowed small amounts of Ca(2+) influx but which by itself did not trigger catecholamine secretion, enhanced subsequent exocytosis in both control and thapsigargin-treated cells. Caffeine-pretreated cells exhibited a rundown in the secretory response that was similar to that produced by thapsigargin. These results suggest that brief elevations of [Ca(2+)](i) could enhance subsequent secretory responses. In addition, the data suggest that intracellular calcium stores are vital for the maintenance of exocytosis during repetitive stimulation.     

Comparison of apex and bottom secretion efficiency at chromaffin cells as measured by amperometry

In chromaffin cells, the exocytosis of neuromediators involves the fusion between a secretory vesicle and the cell membrane. Many techniques based on electrophysiology, electrochemistry and fluorescence microscopy allow the study of such a complex process at active zones of single immobilized cells. These techniques can provide an effective analysis either at the apex, either at the base of the cell adhering onto a substrate. For instance, patch-clamp (electrophysiology) and amperometry (electrochemistry) deal with detection at the exposed top of the cell, whereas evanescent field microscopy concerns mainly its bottom, i.e., the zone on which the cell rests onto the surface. However, in chromaffin cells, comparison between the two sets of methods remains to be established and whether apex fusion events are comparable or not to those observed at the base of the cell is an open question. In this work, we compare both active zones upon using the same measurement method, viz., by performing electrochemical detection at these both poles (top and bottom) of bovine chromaffin cells. This is performed upon using carbon fiber microelectrodes (apical analysis) and planar ITO transparent (basal analysis) electrodes, respectively. Our results indicate that the processes monitored at each pole differ though the same technique is used.     

Biosynthetic relationship between the major matrix proteins of adrenal chromaffin granules

The matrix of the chromaffin granule contains a family of acidic proteins, collectively known as the chromogranins. It has been suggested that this family results from protease action on the major component, chromogranin A. Evidence for this has now been obtained from in vitro translation of adrenal medullary messenger RNA and immunoprecipitation of translation products using an antiserum directed against chromogranin A, but which also recognises other chromogranins.     

Immunolocalization of synexin (annexin VII) in adrenal chromaffin granules and chromaffin cells: evidence for a dynamic role in the secretory process

Summary Synexin (annexin VII) is a Ca²⁺- and phospholid-binding protein which has been proposed to play a role in Ca²⁺-dependent membrane fusion processes. Using a monoclonal antibody against synexin, Mab 10E7, and immunogold, we carried out a semiquantitative localization study of synexin in bovine adrenal medullary chromaffin granules, and in resting and nicotine-stimulated adrenal chromaffin cells. Isolated chromaffin granules contained very little synexin, whereas chromaffin granules aggregated with synexin (24 g/mg) and Ca²⁺ (1 mM) clearly showed synexin-associated immunogold particles in the vicinity of the granule membrane (1.88 gold particles per granule profile). In isolated, cultured adrenal chromaffin cells, synexin was present in the nucleus (5.5 particles/m²) and in the cytosol (5.3 particles/m²), but mainly around the granule membrane in the granular cell area (11.7 particles/m²). During the active phase of cholinergically stimulated catecholamine secretion, the amount of synexin label was reduced by 33% in the nucleus, by 23% in the cytosol, and by 51% in the granule area. The plasma membrane contained a small amount of synexin, which did not significantly change upon stimulation of the cells. We conclude that synexin is involved in the secretory process in chromaffin cells.     

Acetylcholine-induced calcium signalling in adrenaline- and noradrenaline-containing adrenal chromaffin cells

Adrenal chromaffin cells secrete catecholamines in response to cholinergic receptor activation by acetylcholine (ACh). Characteristics of Ca(2+) transients induced by activation of nicotinic (nAChRs) and muscarinic (mAChRs) receptors were analyzed using Fura-2 fluorescent measurements on rat chromaffin cells. We first found two populations of chromaffin cells, which differently responded on AChR stimulation. In the first group (n-cells), consecutive ACh applications evoked persistent Ca(2+) transients, whereas desensitizing transients were observed in the other group (m-cells). The AChR agonists and antagonists precisely imitated or abolished the ACh action on n- and m-type cells, respectively. Cytochemical staining showed that n-cells contained adrenaline, whereas m-cells-noradrenaline. Thus, for the first time we found that nAChRs and mAChRs are differentially expressed in adrenergic and noradrenergic chromaffin cells, respectively. Our data suppose that chromaffin cells can be differentially regulated by incoming ACh signals and in such way release different substances-adrenaline and noradrenaline.     

Interaction of antimycin with cytochrome b-561: A study in secretory granules and in plasma membrane isolated from chromaffin cells of bovine adrenal medulla

Cytochrome b-561 in chromaffin granules interacts with antimycin and its -peak shifts 1 nm towards red. When chromaffin granules were treated with Triton X-100 antimycin no effect was observed. Cytochrome b-561 is located in the plasma membrane isolated from the chromaffin cells. The plasma membrane b-561 does not seem to interact with antimycin. A number of NADH or NADPH (acceptor) oxidoreductase activity has been observed in isolated plasma membrane providing clues to the origin of plasma membrane dehydrogenase. The possible role of cytochrome b561 in secretory granules other than its accredited energy conserving electron transport property is projected.     

Phospholipase D in calcium-regulated exocytosis: Lessons from chromaffin cells

Membrane fusion remains one of the less well-understood processes in cell biology. A variety of mechanisms have been proposed to explain how the generation of fusogenic lipids at sites of exocytosis facilitates secretion in mammalian cells. Over the last decade, chromaffin cells have served as an important cellular model to demonstrate a key role for phospholipase D1 (PLD1) generated phosphatidic acid in regulated exocytosis. The current model proposes that phosphatidic acid plays a biophysical role, generating a negative curvature and thus promoting fusion of secretory vesicles with the plasma membrane. Moreover, multiple signaling pathways converging on PLD1 regulation have been unraveled in chromaffin cells, suggesting a complex level of regulation dependant on the physiological context.     

On the uptake of exogenous catecholamines by adrenal chromaffin cells and nerve endings

Summary Light-microscopic autoradiography has revealed characteristic labelling patterns in adrenal medullary cells following the intravenous administration of different catecholamines. The uptake patterns for [³H] dopa, [³H] dopamine, [³H] noradrenaline and [³H] adrenaline have been compared. In all cases A cells were more active than NA cells and cells situated in the zone nearest the cortex demonstrated a markedly higher rate of uptake than central cells. It was concluded that adjacent chromaffin cells with very similar morphology may differ as much as 50 fold in their capacities to incorporate exogenous amines. The adrenergic nature of the innervation of the vessels of the adrenal cortex and capsule in the mouse was confirmed.     

Triggered exocytosis and endocytosis have different requirements for calcium and nucleotides in permeabilized bovine chromaffin cells

The intracellular requirements for membrane recapture in permeabilized chromaffin cells were compared to the requirements for exocytosis from the same cells.In permeabilized bovine chromaffin cells, calcium-driven exocytosis also triggers, with a short delay, uptake of extracellular horseradish peroxidase (HRP). This internalized HRP remains compartmentalized within the cell and migrates to a low density band on a Percoll gradient which is distinct from the heavier chromaffin granules.The amount of horseradish peroxidase internalized is similar in intact and leaky cells and is approximately equivalent to the volumes secreted. Endocytosis in both preparations is blocked by botulinum toxin, operates in a collapsed membrane potential, and is inhibited by low temperature. In permeabilized cells, exocytosis and coupled endocytosis are activated by the same concentrations of Ca²⁺ and MgATP. Although secretion requires Ca²⁺ and MgATP, once exocytosis has occurred the subsequent endocytosis can proceed in the virtual absence of Ca²⁺ or MgATP, and is largely unaffected by a variety of nucleotide triphosphates (including nonhydrolyzable analogues), and cyclic nucleotides.These data suggest that endocytosis can proceed, once exocytosis has been triggered, under conditions that are quite different from those necessary to support exocytosis, and that the specific requirements for Ca²⁺ and MgATP in secretion are for the exocytotic limb of the secretory cycle rather than for the associated endocytotic pathway.We are grateful to Mr. John Gibbs for excellent technical assistance, and to the Medical Research Council (UK) for financial support.     

Localization of lysophosphatidylcholine in bovine chromaffin granules

One of the unique features of the chromaffin granule membrane is the presence of about 17 mol% lysophosphatidylcholine. Lysophosphatidylcholine isolated from the granules could be degraded by approx. 94% by lysophospholipase. This result is consistent with chemical analyses data showing that about 9% of this lysophospholipid is 1′-alkenyl glycerophosphocholine.The localization of the acylglycerophosphocholine in the chromaffin granule membrane was studied by using pure bovine liver lysophospholipases. In intact granules only about 10% of the total lysophosphatidylcholine was directly available for enzymic hydrolysis. In contrast, when granule membranes (ghosts) were treated with lysophospholipases approx. 60% of the lysophosphatidylcholine was deacylated. These values did not increase after pre-treatment of intact granules or ghosts with trypsin. Added $\text{1-[1-}{}^{14}\text{C]palmitoyl}\text{-sn-}\text{glycero-3-phosphocholine}$ did not mix with the endogenous lysophosphatidylcholine pool(s) and remained completely accessible to added lysophospholipases.     

Identification of a key domain in annexin and 14-3-3 proteins that stimulate calcium-dependent exocytosis in permeabilized adrenal chromaffin cells

Calcium-dependent secretion in digitonin-permeabilized adrenal chromaffin cells is stimulated by exogenous annexin II and 14-3-3 proteins. These proteins share a conserved domain that has been suggested to be involved in specific protein-protein interactions. We examined whether this domain was involved in secretion by using a synthetic peptide (P16) of sequence KGDYQKALLYLCGGDD corresponding to the C-terminus of annexin II. P16, but not truncated peptides, prevented the stimulation of secretion by 14-3-3 proteins and produced a partial inhibition of control secretion. These data suggest that the shared annexin/14-3-3 domain is important in the mechanisms controlling Ca²⁺-dependent secretion and may play a key role in protein-protein interactions during exocytosis.     

Morphological evidence for a close interaction of chromaffin cells with cortical cells within the adrenal gland

Summary The adrenal medulla appears to exert a regulatory influence on adrenocortical steroidogenesis. We have therefore studied the morphology of rat, porcine and bovine adrenals in order to characterize the contact zones of adrenomedullary and adrenocortical tissues. The distribution of chromaffin cells located within the adrenal cortex and of cortical cells located within the adrenal medulla was investigated. Chromaffin cells were characterized by immunostaining for synaptophysin and chromogranin A, both being considered specific for neuroendocrine cells. Cortical cells were characterized by immunostaining for 17-hydroxylase, an enzyme of the steroid pathway. Cellular contacts of chromaffin cells and cortical cells were examined at the electron microscopical level. In rat and porcine adrenals, rays of chromaffin cells, small cell clusters and single chromaffin cells or small invaginations from the medulla could be detected in all three zones of the cortex. Chromaffin cells often spread in the subcapsular space of the zona glomerulosa. In porcine and bovine adrenals, 17-hydroxylase immunoreactive cells were localized within the medulla. Single cortical cells and small accumulations of cells were spread throughout this region. At the ultrastructural level, the chromaffin cells located within the cortex in pig and rat adrenals formed close cellular contacts with cortical cells in all three zones. Our morphological data provide evidence for a possible paracrine role of chromaffin cells; this may be important for the neuroregulation of the adrenal cortex.     

Exocytotic release of secretory granules from endocrine cells in the midgut of insects

Summary Exocytotic release of the secretory granules of the endocrine cells in the midgut of a cockroach, Periplaneta americana, was studied by means of fixation with tannic acid in combination with glutaraldehyde and osmium tetroxide. A sequence of images indicative of exocytosis suggests the following steps in this process: (1) A delicate connection appears between the granule-limiting membrane and the plasma membrane. (2) The plasma membrane approaches the granule, forming a concave indentation. (3) The granule-limiting membrane fuses with the plasma membrane and opens to give rise to an omega profile. (4) The granule content is voided into extracellular space. Exocytosis occurs not only at the base of the cell but occasionally at its side facing adjacent cells. (5) The exocytotic invagination after release becomes smaller and narrower; sometimes a coated pit with bristles appears. Multiple exocytosis, and exocytosis in the endocrine cells of the nidus, i.e., the regenerative cell mass, are also described.     

Metal Selectivity of Exocytosis in α-Toxin-Permeabilized Bovine Chromaffin Cells

Abstract: Metal selectivity of exocytosis was analyzed by comparing the effects of polyvalent metal cations Ca²⁺, Ba²⁺, Sr²⁺, Pb²⁺, La³⁺, Cd²⁺, Co²⁺, Tb³⁺, Mn²⁺, and Zn²⁺ on the release of norepinephrine (NE) from staphylococcal α-toxin-permeabilized bovine chromaffin cells. Pb²⁺, La³⁺, Cd²⁺, Sr²⁺, and Ba²⁺ activated NE secretion accompanied by the release of intragranular dopamine β-hydroxylase but not cytosolic lactate dehydrogenase, indicating the activation of the mechanism of exocytosis. The release triggered by saturating concentrations of Pb²⁺, La³⁺, Cd²⁺, and Sr²⁺ was nonadditive with Ca²⁺, indicating a common site of action. In contrast, the Ba²⁺-evoked NE release was additive with Ca²⁺ and the Ca²⁺ agonists Pb²⁺, La³⁺, Cd²⁺, and Sr²⁺, suggesting that Ba²⁺ activates secretion at a site distinct from the Ca²⁺ receptor. In distinction to the NE release evoked by Pb²⁺, La³⁺, Cd²⁺, and Ba²⁺, the Sr²⁺-evoked NE release was associated with a significant elevation of Ca²⁺ concentration in the medium and abolished by Ca²⁺ chelation. This indicates that the secretagogue effect of Sr²⁺ was indirect and secondary to the displacement of bound Ca²⁺. Co²⁺ and Mn²⁺ inhibited the NE release evoked by Ca²⁺, Sr²⁺, Pb²⁺, La³⁺, and Cd²⁺ but had no effect on the Ba²⁺-dependent secretion. Tb³⁺ and Zn²⁺ were without effect on exocytosis.