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. 2010 Jan 28;216(4):518–524. doi: 10.1111/j.1469-7580.2009.01198.x

Suggestive evidence of a vesicle-mediated mode of cell degranulation in chromaffin cells. A high-resolution scanning electron microscopy investigation

Enrico Crivellato 1, Paola Solinas 2, Raffaella Isola 2, Domenico Ribatti 3, Alessandro Riva 2
PMCID: PMC2849529  PMID: 20136671

Abstract

In this study we used a modified osmium maceration method for high-resolution scanning electron microscopy to study some ultrastructural details fitting the schema of piecemeal degranulation in chromaffin cells. Piecemeal degranulation refers to a particulate pattern of cell secretion that is accomplished by vesicle-mediated extracellular transport of granule-stored material. We investigated adrenal samples from control and angiotensin II-treated rats, and identified a variable proportion of smooth, 30–60-nm-diameter vesicles in the cytoplasm of chromaffin cells. A percentage of these vesicles were interspersed in the cytosol among chromaffin granules but the majority appeared to be attached to granules. Remarkably, the number of unattached cytoplasmic vesicles was greatly increased in chromaffin cells from angiotensin II-treated animals. Vesicles of the same structure and dimension were detected close to or attached to the cytoplasmic face of the plasma membrane; these, too, were increased in number in chromaffin cells from rats stimulated with angiotensin II. In specimens shaken with a rotating agitator during maceration, the cytoplasmic organelles could be partially removed and the fine structure of the vesicular interaction with the inner side of the plasma membrane emerged most clearly. A proportion of chromaffin granules showed protrusions that we interpreted as vesicular structures budding from the granular envelope. In some instances, the transection plane intersected granules with putative vesicles emerging from the surfaces. In these cases, the protrusions of budding vesicles could be observed from the internal side. This study provides high-resolution scanning electron microscopy images compatible with a vesicle-mediated degranulation mode of cell secretion in adrenal chromaffin cells. The data indicating an increase in the number of vesicles observed in chromaffin cells after stimulation with the chromaffin cell secretagogue angiotensin II suggests that this secretory process may be susceptible to fine regulation.

Keywords: angiotensin II, chromaffin cells, high-resolution scanning electron microscopy, osmium maceration, piecemeal degranulation

Introduction

Chromaffin cells of the adrenal medulla are neuroendocrine cells that play an important role in the physiological adaptation to stress. These cells were first identified in the mid-19th century by Joeston (1864) and Henle (1865). Both authors observed that fixation of adrenal glands with compounds of chromium resulted in the adrenal medulla staining brown (Santer, 1994). It was not until 1902 that the term ‘chromaffin’ was used by Kohn (1902) to describe cells that exhibited such coloration following treatment with chromate compounds. Chromaffin cells are neural crest-derived cells that have been identified in all vertebrate classes (LeDouarin & Kalcheim, 1999; Huber, 2006). They exert both endocrine and paracrine functions through their plentiful complement of cytoplasmic secretory granules. Chromaffin granules represent the main substructural component in chromaffin cells. They are membrane-bound, 150–350-nm-diameter, moderately or strongly electron-dense organelles that originate from the Golgi network and belong to the class of dense-core vesicles, so-called because of the opaque core visible by electron microscopy. They are specialized secretory organelles that contain catecholamines, matrix scaffolding proteins mainly belonging to the granin family, a large array of peptide hormones along with the respective processing enzymes and protease inhibitors, some enzymes of the aminergic biosynthetic pathway, ascorbic acid, ATP and calcium (Crivellato et al. 2008).

There is general consensus that chromaffin cells release their granule constituents by exocytosis, either in the form of ‘full-fusion’ exocytosis or ‘kiss-and-run’ exocytosis (Burgoyne, 1991; Burgoyne & Morgan, 2003). Recent work from our laboratory, however, has questioned this assumption and suggested that a further type of secretory pathway, called piecemeal degranulation (PMD), may affect the release of stored products from chromaffin cell granules. PMD refers to a particulate pattern of cell degranulation, which was formerly described in basophils, mast cells and eosinophils (Dvorak, 2005a,b;). This ultrastructurally defined secretory model implies a discrete release of granule particles from storage granules without granule fusion with the plasma membrane. Secretion occurs by translocation of loaded vesicles or by means of the vesiculotubular structures. In the former case, small vesicles filled with secretory cargoes bud from granules, move to the cell periphery and fuse with the plasma membrane, thus releasing small quanta of secretory material (Dvorak, 2005a). In the latter case, the secretory mechanism involves intragranular compartments organized as tubular vesicles or tubular networks, which bud from donor granules and relocate specific granule products in response to stimulation (Melo et al. 2008). As a result, PMD would accomplish discharge of secretory constituents from storage granules without granule-to-granule and granule-to-plasma membrane fusion events and without direct granule opening to the cell exterior. It is of note that ultrastructural clues diagnostic of PMD have been identified with transmission electron microscopy (TEM) in chromaffin cells from different species including man, mouse, rat, chicken embryo, newt and fish (Crivellato et al. 2003, 2004, 2005, 2006, 2009a,b). In chromaffin cells, this suggestive degranulation pathway may represent a versatile model of cell secretion. On the one hand, it may be part of a generalized secretory reaction to acute adverse conditions, as during exposure to severe osmotic stress in fish (Crivellato et al. 2006). On the other hand, it would exhibit all of the properties of a subtle mechanism for releasing small, and possibly selective, amounts of granule constituents for autocrine and/or paracrine functions. This may be the case in Anuran amphibians, where the adrenal homologue is composed of closely interconnected aminergic chromaffin and inter-renal steroidogenic cells (Crivellato et al. 2009a).

To better define the 3D features of putative PMD-related substructures in chromaffin cells, we have undertaken a study of rat adrenal samples processed according to a modified osmium maceration method for high-resolution scanning electron microscopy (HRSEM) (Riva et al. 1999, 2007). This procedure, which was previously utilized for studying the intracellular structure of secretory and ductal epithelia of the major salivary glands and the substructural organization of the mitochondrial cristae (Riva et al. 1995, 2003, 2005, 2006), provides both panoramic and highly magnified views of the same tissue specimen. In particular, owing to its unique ability to generate stereoscopic images of intracellular organelles and membranaceous surfaces, the osmium maceration method for HRSEM appears to be the instrument of choice for complementing information obtained by TEM on possible PMD specificities in chromaffin cells. In particular, it allows a better definition of some key ultrastructural features that TEM examination brought to light, such as the 3D morphology of the vesicular structures budding from the granule membrane and the spatial relationship between chromaffin granules and detached, shuttling vesicles. In addition, as cytoplasmic organelles may be partially removed during osmium maceration, leaving the cytoplasmic side of the plasmalemma available to inspection, the HRSEM procedure provides an effective strategy for precise investigation of the structural relationship between vesicles located in the subplasmalemmal region and the plasma membrane.

The present study describes the results that we have obtained in rat chromaffin cells examined by the osmium maceration method for HRSEM. In order to gain additional information on the dynamics of granular secretion in these cells, we have compared adrenal samples from control rats and rats stimulated with the chromaffin cell secretagogue angiotensin II.

Matherials and methods

Animals

Ten adult male Sprague-Dawley rats (260 ± 30 g body weight) from a local conventional breeding colony were used in this study. The rats were housed in single cages at 20 °C on a 12-h light/dark cycle and had free access to food and water. Five animals received an intraperitoneal injection of angiotensin II (Sigma Chemical Co., St Louis, MO, USA) at a dose of 1 mg kg−1 body weight. Five animals were treated with a peritoneal injection of saline solution and served as controls. All animals were killed at 24 h after the injection. They were anaesthetized intraperitoneally with pentothal sodium (50 mg kg−1 body weight). The animals were then killed by a lethal dose of pentothal sodium. Compliance was ensured with both the Guide for the Care and Use of Laboratory Animals (NIH) and the European Communities Council Directive governing the maintenance of laboratory animals and their use in scientific experiments (86/609/EEC).

Osmium maceration procedure

Adrenal glands were promptly removed and strips of 1 × 1 × 3 mm were fixed with a mixture of 0.5% glutaraldehyde and 0.5% paraformaldehyde in 0.1 m cacodylate buffer (pH 7.2) for 15 min at room temperature (RT, 21 °C). After fixation, samples were rinsed three times in phosphate-buffered saline (PBS) for 10 min at RT and postfixed with a 1% OsO4/1.25% K4Fe(CN)6 solution in distilled water for 2 h in the dark at 4 °C. After rinsing three times in PBS for 10 min at RT, specimens were embedded in 1% agarose in distilled water and cut into 150-μm-thick sections by a tissue sectioner (TC2, Sorvall, New Town, CT, USA) at RT. Samples were rinsed again and then underwent a second postfixation step with 1% OsO4/1.25% K4Fe(CN)6 solution in distilled water for 1 h in the dark at 4 °C. The maceration step followed. This was protracted for 44–48 h in a 0.1% OsO4 solution in PBS at 25 °C. After rinsing three times in PBS for 10 min at RT, samples were dehydrated through a graded acetone series, followed by critical-point drying with CO2 and coating with platinum (2 nm) by a turbo sputtering apparatus (Emitech 575, Ashford, UK). Specimens were observed by means of a field emission HRSEM (Hitachi S4000, Tokyo, Japan) operated at 15–20 kV.

Results

Viewed at low magnification, chromaffin cells appeared as single cell elements contacting each other side by side (Fig. 1A). As a rule, chromaffin cells contained a round, prominent nucleus and presented with an abundant cytoplasm containing plentiful secretory granules. At intermediate magnification, some subcellular structures could be identified. The nuclear envelope wrapped the chromatin material and exhibited its distinctive complement of nuclear pores (Fig. 1B). In the cytoplasm, many mitochondria of different sizes, along with a number of tubules and cisternae of the endoplasmic reticulum, were recognizable (Fig. 1B). Many tubules and cisternae were covered by ribosomes. Usually, chromaffin cells exhibited a well-developed Golgi complex, whose cisternae revealed characteristic fenestrations (Fig. 1C).

Fig. 1.

Fig. 1

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Rat adrenal medulla treated with the osmium maceration method and viewed by high-resolution scanning electron microscopy. (A) Overview of a portion of a chromaffin cell. pm, plasma membrane; n, nucleus; m, mitochondria. (B) At higher magnification, numerous granules (g) and tubular structures of the endoplasmic reticulum (er) are observable in the cytoplasm. The nuclear envelope (ne) exhibits its complement of nuclear pores (arrowhead). (C) En-face view of the Golgi apparatus (G) of a chromaffin cell, which presents numerous fenestrations. g, chromaffin granules. Bar = 1 μm (A); 0.5 μm (B); 0.25 μm (C).

The most remarkable organelles in chromaffin cells were the chromaffin secretory granules. These appeared as membrane-bound structures filling the cell cytoplasm and exhibiting different shapes, having a size ranging from 137 to 354 nm (Fig. 2A). On TEM examination, two types of chromaffin granules can be easily differentiated, principally on the basis of the electron density of the granule content: the adrenaline-containing granule and the noradrenaline-storing granule. These two types of secretory organelle could not be so clearly distinguished by means of HRSEM because the main differentiating factor, i.e. the core electron density, was not available for inspection. In some instances, granule dimension and shape could help to orient the observer toward one granule type or the other. In general terms, however, there was a certain polymorphism in granule dimension. Viewed by HRSEM, most granules were visible as entire organelles, thus enabling the observer to evaluate their 3D morphology. In some specimens, chromaffin granules appeared as transected structures and their inner organization was exposed. These transected granules mostly appeared as empty organelles. In some instances, however, an inner core was visible that appeared surrounded by a membrane envelope (Fig. 2A).

Fig. 2.

Fig. 2

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Rat adrenal medulla treated with the osmium maceration method and viewed by high-resolution scanning electron microscopy. (A) Two neighboring chromaffin cells exhibit plentiful secretory granules, which appear as intact or transected organelles. When transected, most appear as empty structures (arrows) but, in some instances, the inner core is visible (arrowheads). pm, plasma membrane. (B) Smooth, 30–50-nm-diameter vesicles (arrowheads) are visible near to or attached to granules in the cytoplasm of a control chromaffin cell. (C) The amount of cytoplasmic vesicles (v) is greatly increased after stimulation with angiotensin II. Bar = 0.3 μm (A); 0.1 μm (B); 0.3 μm (C).

At higher magnification, a variable proportion of smooth, 30–60-nm-diameter vesicles could be identified in the intergranular cytosol (Fig. 2B). Some of these vesicles were observed free in the cytoplasm but the majority appeared attached to granules. Remarkably, in the chromaffin cells from angiotensin II-treated animals, the proportion of cytoplasmic vesicles was greatly increased (Fig. 2C). In this case, vesicles were for the most part identified free in the intergranular cytosol, whereas vesicular numbers in the Golgi region remained unaffected. It is of note that some granules presented bulging, pear-like or tail-like protrusions that we interpreted as vesicular structures budding from the granule envelope (Fig. 3A,B). In chromaffin cells from angiotensin II-treated animals, little chains of detached vesicles were recognizable close to single granules, which presumably represented the source of the observed vesicles (Fig. 3C). Interestingly, in some instances the transection plane intersected granules with vesicles emerging from the surface (Fig. 3D). In these cases, the protrusion of the budding vesicle could be observed from the internal side. The osmium maceration method appeared especially useful in visualizing the structural relationship between subplasmalemmal vesicles and the plasma membrane (Fig. 4A–C). These contacts could be examined both in perpendicularly transected membranes and in membranes viewed ‘en face’. Vesicles of the same structure and dimension as those recognized near the granules were observed close to or attached to the cytoplasmic face of the plasma membrane, especially in chromaffin cells from angiotensin II-treated animals (Fig. 4A). In specimens that had previously been shaken with a rotating agitator during maceration, cytoplasmic organelles could be partially removed and the fine structure of the vesicles in contact with the inner side of the plasma membrane emerged most clearly (Fig. 4C).

Fig. 3.

Fig. 3

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Rat adrenal medulla treated with the osmium maceration method and viewed by high-resolution scanning electron microscopy. (A and B) Chromaffin granules from control cells exhibit pear-like or tail-like protrusions (arrowheads) that emerge from the granule surface. In A, one of the pair of budding structures protrudes from a transected granule. (C) A little chain of putative detached vesicles (arrowhead) is recognizable close to a single granule after angiotensin II stimulation. (D) In this transected granule of an angiotensin II-treated chromaffin cell, the internal side of the protruding vesicle (arrowhead) can be inspected. Bar = 0.1 μm (A and B); 0.2 μm (C); 0.15 μm (D).

Fig. 4.

Fig. 4

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Rat adrenal medulla treated with the osmium maceration method and viewed by high-resolution scanning electron microscopy. (A) A remarkable amount of vesicles (arrowheads) is situated close to the plasma membrane (pm) after stimulation with angiotensin II. (B) Some vesicles (arrowheads) in the subplasmalemmal region are also visible in unstimulated chromaffin cells. (C) Cytoplasmic side of the plasma membrane following removal of cytoplasmic organelles. Many vesicles (arrowheads) and vesicotubular structures (arrow) attached to the plasmalemma are visible in this chromaffin cell after stimulation with angiotensin II. Bar = 0.15 μm (A–C).

Discussion

This study provides high-resolution scanning images of rat adrenal chromaffin cells. Some of these 3D ultrastructures were previously investigated with conventional TEM and regarded as highly suggestive of PMD in chromaffin cells (Crivellato et al. 2003, 2004, 2005, 2006, 2009a,b).

First described in the early 1970s, PMD is a slow, particulate and possibly selective process of cell secretion that is especially suitable for the outward transport of small quanta of highly active granule components to avoid local or systemic toxic effects (Dvorak, 2005a,b;). PMD has been thoroughly investigated by TEM (Dvorak, 2005a,b;). Ultrastructural criteria for detecting PMD by means of electron microscopy include specific granular and cytoplasmic changes. Both kinds are recognizable in chromaffin cells (Crivellato et al. 2003, 2004, 2005, 2006, 2009a,b). Granular changes comprise: (i) reduction in granular content, leading to eroded or dissolved matrices; (ii) some tendency to dilation of granular chambers, generating a certain amount of granular polymorphism; and (iii) distortion of granular profiles, with outpouches and projections of their limiting membranes, as the result of vesicular formation and detachment. Cytoplasmic changes consist of a rich supply of membrane-bound, 30–70-nm-diameter, smooth vesicles that are either attached to granules or free in the intergranular cytosol or close to the plasma membrane.

Our HRSEM investigation agrees with previous ultrastructural data that we obtained by conventional TEM, which lent support to the assumption that chromaffin cells may express vesicle-mediated degranulation. One crucial point documented in our HRSEM study is the 3D identification of cytoplasmic vesicles (30–60 nm diameter) localized close to either chromaffin granules or the plasma membrane. Remarkably, the number of these vesicles increases in chromaffin cells from angiotensin II-treated animals and many of these vesicles are identifiable next to or attached to the plasma membrane, a location that suggests vesicle implication in secretory cargo delivery to the extracellular space. In addition, the osmium maceration procedure followed by HRSEM analysis provides 3D images evocative of a process of vesicular generation from the surface of chromaffin granules.

It is interesting that angiotensin II-treated animals show higher quantities of vesicles with a perigranular and juxtaplasmalemmal pattern of intracellular localization. Angiotensin II is a hormone peptide that takes part in the regulation of the stress reaction at all levels of the hypothalamic-pituitary-adrenal axis (Antoni, 1993). Stress increases the production of circulating angiotensin II and the release of catecholamines from the adrenal medulla (Livett & Marley, 1993; Phillips et al. 1993; Leong et al. 2002). Both AT1 and AT2 angiotensin II receptors are expressed in the rat adrenal medulla (Israel et al. 1995). AT1- and AT2-mediated catecholamine secretion is affected by Ca2+ mobilization (Wong et al. 1990; Israel et al. 1995; Belloni et al. 1998; Takekoshi et al. 2001). Remarkably, the stimulation of primary cultures of human adrenal chromaffin cells by angiotensin II determines a 3-, 2- and 12-fold increase in noradrenalin, adrenaline and neuropeptide Y release, respectively (Cavadas et al. 2003). Thus, angiotensin II is a potent synthesizer and releaser of catecholamines and peptides and, as depicted in our study, may affect chromaffin cell secretion by up-regulating the vesicle-mediated mechanism for extracellular transport of granule stored material.

Electron microscopy is not the ideal tool for imaging a continuous, dynamic process of vesicular shuttling from the granular compartment to the plasma membrane and vice versa. This investigative procedure, indeed, provides only static, instantaneous snapshots. In particular, both HRSEM and TEM analyses do not supply information on either the origin or direction of vesicular traffic. There are alternative hypotheses to the one proposed here that could explain the wealth of vesicles found in the cytoplasm of chromaffin cells. We are inclined to exclude the possibility that secretory vesicles might be directly derived from the trans-Golgi network in the context of a constitutive pathway of cell secretion. Although some of the numerous vesicles identified in chromaffin cells both by HRSEM and TEM analysis may actually originate from the Golgi apparatus, it seems unlikely that the great bulk of vesicles would form in this way. Indeed, most vesicles are either attached to granules or interspersed in the intergranular cytosol. This pattern of distribution actually favors the concept of a vesicular origin from donor granules. We would also reject the possibility that cytoplasmic vesicles in chromaffin cells might simply represent transfer conduits involved in transporting secretory material from the trans-Golgi compartment to granular deposits. Although experiments with specific markers for labeling either pre- or postgranular compartments have not yet been reported, much of the previous discussion argues against this hypothesis and favors a vesicular derivation from granules. In our HRSEM study, stimulation of chromaffin cells with the secretagogue angiotensin II does not cause any vesicular increment in the Golgi domain even though the number of vesicles appears to have increased considerably in the perigranular and subplasmalemmal compartments. It is important to bear in mind that catecholamine synthesis is affected by cytosolic and granular enzymes, not by the Golgi molecular machinery, a condition that implies derivation of catecholamine-laden vesicles from the granular compartment and not from the Golgi apparatus (Winkler & Westhead, 1980).

In addition to the classical, large, electron-dense chromaffin granule, chromaffin cells have been shown to possess another type of secretory vesicle that undergoes Ca2+-dependent exocytosis, i.e. the small, clear, synaptic-like microvesicle (SLMV) (Thomas-Reetz & De Camilli, 1994; Aunis, 1998). The SLMV has a diameter of ∼ 50 nm and is similar to a synaptic vesicle because it exhibits an electron-lucent content. SLMVs have been identified in other endocrine cells, such as pancreatic β cells and pinealocytes, as distinct acidic organelles (Moriyama et al. 2000). These secretory organelles contain diverse non-peptide neurotransmitter-like substances, express distinct structural proteins, present specific release kinetics and are supposed to have independent biogenesis (Kasai, 1999). Thus, a proportion of vesicles found in the present HRSEM and previous TEM studies are likely to correspond to SLMVs. Former TEM investigations in various vertebrate species, however, have clearly identified numerous vesicles in chromaffin cells, which are filled by the same electron-dense cargo as chromaffin granules (Crivellato et al. 2003, 2004, 2005, 2006, 2009a,b). These dense vesicles cannot be SLMVs. Thus, the scenario that emerges from a combined TEM/HRSEM examination of chromaffin cells may plausibly fit into a vesicle-mediated mode of cell secretion related to the general schema of PMD.

In conclusion, this study provides HRSEM images that provide evidence for a vesicle-mediated degranulation pattern in adrenal chromaffin cells. The observed vesicular increment in chromaffin cells stimulated by the chromaffin cell secretagogue angiotensin II represents a new finding and suggests that PMD may be a tuneable secretory process that is susceptible to fine regulation.

Acknowledgments

This study has been supported by MIUR local funds to the Department of Medical and Morphological Research, Section of Anatomy, University of Udine, and by Fondazione Banco di Sardegna to the Department of Cytomorphology, University of Cagliari.

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