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Cell Biology International (2007) 31, 413–419 (Printed in Great Britain)
Hsp70 in the atrial neuroendocrine units of the snail, Achatina fulica
M.G. Martynova*, O.A. Bystrova, S.V. Shabelnikov, B.A. Margulis and D.S. Prokofjeva
Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Avenue, 194064 St. Petersburg, Russia


Abstract

Heat shock proteins (Hsps) are evolutionary conserved peptides well known as molecular chaperones and stress proteins. Elevated levels of extracellular Hsps in blood plasma have been observed during the stress responses and some diseases. Information on the cellular sources of extracellular Hsps and mechanisms regulating their release is still scanty. Here we showed the presence and localization of Hsp70 in the neuroendocrine system in the atrium of the snail, Achatina fulica. The occurrence of the peptide in snail atrium lysate was detected by Western blot analysis. Immunoperoxidase and immunogold staining demonstrated that Hsp70-immunoreactivity is mainly confined to the peculiar atrial neuroendocrine units which are formed by nerve fibers tightly contacted with large granular cells. Immunolabelling intensity differed in morphologically distinct types of secretory granules in the granular cells. The pictures of exocytosis of Hsp70-immunolabeled granules from the granular cells were observed. In nerve bundles, axon profiles with Hsp70-immunoreactive and those with non-immunoreactive neurosecretory granules were found. In addition, Hsp70-like material was also revealed in the granules of glia-interstitial cells that accompanied nerve fibers. Our findings provide an immuno-morphological basis for a role of Hsp70 in the functioning of the neuroendocrine system in the snail heart, and show that the atrial granular cells are a probable source of extracellular Hsp70 in the snail hemolymph.


Keywords: Heat shock protein, Granular cell, Nerve fiber, Glia, Western blot, Immunolocalization, Mollusc.

*Corresponding author.


1 Introduction

Heat shock protein 70 (Hsp70) belongs to the family of molecular chaperones which regulate various processes of protein biogenesis and protect cellular homeostasis. Apart from its peptide chaperone capacity, Hsp70 is widely believed to participate in immunity and adaptation of organism to stress. There is evidence of its active role in cardioprotection by myocardial infarction and myocardial ishemia (Snoeckx et al., 2001; Tytell and Hooper, 2001; Yu et al., 2006).

In response to stress, some cells can probably not only enhance the production of Hsp70 but also actively release it into the extracellular environment. Increase of Hsp70 level in the human serum was established in patients with myocardium infarction (Dybdahl et al., 2005), postoperative (Kimura et al., 2004) or acute (Njemini et al., 2003) infection, and by elevated body temperature induced by exercise (Radons and Multhoff, 2005). Circulating Hsps interact with antigen presenting cells through specialized cell surface receptors (Asea, 2003; Binder et al., 2004; Theriault et al., 2005). The cellular sources and mechanisms which regulate the secretion of extracellular Hsp70 remain poorly understood. In vitro, glial (Guzhova et al., 2001), plasmacytoma (Altieri et al., 2004), peripheral blood mononuclear cells (Hunter-Lavin et al., 2004), and A431 human carcinoma cells (Evdonin et al., 2006) were shown to release Hsps.

Hsps are highly conserved throughout evolution and are also reported in some invertebrates, including molluscs. Hsp70 genes were isolated and identified from the mussel, Mytilis galloprovincialis (Kourtidis et al., 2006). It has been shown that various stressors, such as heat shock or exposure to heavy metals, cause Hsp70 gene activation in this species (Franzellitti and Fabbri, 2005). A number of observations suggest that molluscan neuroendocrine system may be involved in regulation of expression of Hsps. Seasonal variations in total amounts of Hsp70 were revealed in the oyster, Crassostrea virginaca (Encomio and Chu, 2005) and the mussels, Mytilus trossulus and M. galloprovincialis (Hofmann and Somero, 1995; Minier et al., 2000). The induction of the Hsp70 gene promoter in hemocytes of the oyster, Crassostrea gigac, and abalone, Haliotis ruberculata, by catecholamines has been shown (Lacoste et al., 2001).

Very specific neuroendocrine complexes exist in the snail atrium. They consist of large secretory granular cells (GCs) integrated in cardiac muscle tissue and tightly contacted with nerve endings. It is proposed that these GCs-nerve units have a role in neurosecretory regulation of heart activity (Erdélyi and Halász, 1972; Volkmer-Ribeiro, 1970; Zs.-Nagy and S.-Rózsa, 1970).

Taken together, the data point to a possible involvement of Hsp70 in the functioning of the neuroendocrine system in the snail heart. The aim of this study was to examine the occurrence and localization of this peptide in the Achatina atrium. The results demonstrate the presence of Hsp70-like substance in the granules of all elements forming the atrial neuroendocrine units and suggest that the atrial granular cells can be the source of extracellular Hsp70.

2 Materials and methods

2.1 Animals

African giant snail, Achatina fulica Férussac, was taken from the colonies reared in this laboratory. The snails were housed in aquarium where they have ad libitum vegetable diet enriched with dry milk and water under standard conditions (25°C, 12-h light/dark cycle).

2.2 Antibodies

The following antibodies were used: monoclonal anti-Hsp70 antibody 3B5 (Lasunskaia et al., 1997), and polyclonal anti Hsp70 antibody R23 (Novoselov et al., 2004).

2.3 Electrophoresis and Western blotting

The atrium was dissected from the specimen and homogenized in cold lysis buffer containing 20mM Tris–HCL (pH 7.4), 20mM NaCl, 1% Tx-100, 2mM EDTA, 2mM PMSF, 1mM DTT. The homogenate was frozen, thawed twice and centrifuged at 13,000rpm for 10min. Sample were then subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in 10% gel. Purified bovine Hsp70 was used as standard. Electrophoresed proteins were then electroblotted with a semi-dry apparatus to nitrocellulose filter. The filter were blocked in PBS buffered saline containing 0.5% Tween 20 and 5% non-fat dry milk, and incubated with polyclonal Hsp70 antibodies diluted 1:10,000. After washing, the blot was incubated in a 1:1000 dilution of the goat anti-rabbit IgG peroxidase linked antibodies, followed by enhanced chemiluminescence reaction.

2.4 Electron microscopy

Tissue pieces were fixed in cacodylate-buffered 2.5% glutaraldehyde solution (pH 7.4) at 4°C overnight, postfixed with 1% osmium tetroxide for 1h, dehydrated and embedded in Epon and Araldite epoxy resin. Ultrathin sections were cut with a diamond knife and contrasted with 70% alcoholic uranyl acetate and Reynold's lead citrate. Material was examined at 80kV in Jem 7A transmission electron microscope. Semi-thin sections were cut with a glass knife, stained with toluidine blue and photographed on Leica DM IRB microscope by Leica DFC Camera using Leica IM50 computer program.

2.5 Light microscopic immunocytochemistry

At a light microscopical level Hsp70 immunoreactivity was visualized by an immunoperoxidase technique using an avidin-biotin peroxidase complex (Hsu et al., 1981). Tissue samples were fixed at room temperature for 24h in a solution of 0.5% Zn acetate and 0.5% ZnCl in Tris-Ca acetate buffer, dehydrated through 70%, 95% ethanol and 100% isopropanol, and embedded in paraffin according to Beckstead (1994). The sections (4μm) were deparaffinized and stained with using DAKO LSAB2 System-HRP Kit peroxidase. All staining procedures were carried out at the room temperature in a moist chamber. The sections were washed in 0.1M Tris buffered saline (TBS), pH 7.4, with 5% DMSO for 10min. After a brief rinse in TBS, endogenous peroxidases were quenched by treatment with 3% H2O2 in water for 5min. After a rinse in TBS, sections were incubated with the primary antibody, monoclonal Hsp70 antibody, diluted 1:100 in 0.1M TBS with 1% bovine serum albumin (BSA) (to block the non-specific binding), for 30min. Following a rinse in TBS the sections were incubated for a further 10min with a biotinylated link antibody containing goat anti-mouse immunoglobulins for 20min and, after a rinse in TBS, with peroxidase-labelled streptavidin for 20min. Staining was completed after an incubation with 3-3′ diaminobenzidine (DAD) Substrate-Chromogen for 10min. The sections were rinsed in distilled water, dehydrated, cleared and mounted under coverslips. Positive cells were labelled brown. Controls of immunocytochemical reactions were performed by substituting primary antibodies with non-immune sera.

2.6 EM immunocytochemistry

Ultrathin sections mounted on nickel grids were first treated with hydrogen peroxide for 20min to loosen the resin. Three washes in PBS for 2min each were followed by incubation with monoclonal Hsp70 antibody diluted 1:2000 in 0.05M Tris/HCl buffer, pH 7.4, containing 1% BSA and 0.1% coldwater fish gelatin overnight, at 4°C in a moist chamber. Gold-conjugated (10nm) goat anti-mouse IgG (Sigma) diluted 1:10 was used as the secondary antibody, and sections were incubated for 1h at room temperature. Finally, the sections were counterstained as described. For the control, the primary antibody was omitted or replaced by irrelevant antibodies.

3 Results

3.1 Structure of the snail heart

The snail Achatina fulica has a two-chambered heart enclosed in a pericardium. The atrial and ventricular walls consist of an outer single-cell layer of coelomic epithelium (epicardium), a myocardium, and an inner discontinuous layer of endothelial cells facing the heart lumen. Numerous granular cells (GCs) were observed scattered throughout the atrium. They are arranged on the luminal surface of the muscular trabeculae under the endothelial cells (Fig. 1A). The atrium is richly innervated. Cardiac nerves enter the atrium and ramifies throughout the organ. Large nerve cords consist of tightly packed axons more or less filled with neurosecretory granules (Fig. 1B). These differ a little in size and electron opacity. A moderate number of additional axon profiles, devoid of neurosecretory granules of any kind, are available. The nerve cords are discontinuously covered by the glio-interstitial cells, long processes of which contain a large granules 300–400nm in diameter called gliosomes (Gilloteaux, 1975). The fine nerve fibers terminate on the myocytes and GCs. No GCs and nerves were seen in the bulk of the ventricle.


Fig. 1

Morphology of the Achatina atrium. (A) Light micrograph of the semithin cross-section through the center of the Achatina atrium. Granular cells (double arrowheads) adjoin muscle cells (M) and are covered by the endothelial cells (arrowheads) on the side of heart lumen (L). Nerve fibers (arrows) are often seen near granular cells. Stained with toluidine blue, original magnification ×40. (B) Transmission electron micrograph of a nerve bundle involving a number of axon profiles and surrounded by glio-interstitial cell (g) which contains gliosomes (arrows). e, endothelial cell. (C) Electron micrograph showing nerve endings (arrows) in close contact with a granular cell. The granular cell completely embraces the nerve processes. Arrowheads indicate a basement lamella surrounding the granular cell. Note very active condition of GC plasmalemma forming numerous vesicles nearby (asterisk). n, nucleus of the granular cell. Bars: 1μm.


The atrial GCs (&007E;50μm in diameter) display a round-to-oval eccentrically located nucleus with clumped chromatin and one nucleolus. The cytoplasm of the mature GC is stuffed with large (0.5–2μm in diameter) endocrine-like, membrane-bound, usually round granules of three morphologically distinct types: electron-dark and electron-light granules of a homogeneous structure, and light granules of a fine fibrous structure. In many GCs, a number of empty granule chambers were also seen (Fig. 1C). The plasma membrane of the GCs appeared hyperactive with numerous surface-associated vesicles obviously originating from it. Thin basement lamella surrounds the cell. Besides mature GCs a relatively less differentiated GCs also occur in the atrium. Their cytoplasm is not so rich in secretory granules and contains well-developed Golgi complexes and abundant cisternae of rough endoplasmic reticulum.

EM showed close apposition of the axon terminals to GCs. The nerve endings devoid of glial elements penetrate beneath the basement lamella of GC and neurilemma forms close junctions with the GC plasmalemma (Fig. 1C). There are no membrane specializations on the pre- or post-synaptic sides.

3.2 Western blot analysis

To determine the presence of Hsp70 in the snail atrium, extracts from the organ were run on gels and probed with anti-Hsp70 antibodies. Identification of Hsp70-immunoreactive proteins in atrium tissue lysate showed the presence of protein recognized by antibody to Hsp70 (Fig. 2). This protein had a molecular mass of &007E;70kDa. Overall these results suggest that the 70kDa protein in the Achatina atrium is Hsp70.


Fig. 2

Western blot analysis of total protein extracts, 50μg/well, from Achatina atrium (line A) and bovine Hsp70, 10ng/well (line B) probed with anti-Hsp70 polyclonal antibodies. Anti-Hsp70 recognized a protein of apparent MW of 70kDa.


3.3 Immunocytochemical localization of Hsp70

Immunoperoxidase staining confirmed the presence of Hsp70-immunoreactive material in the Achatina atrium. Essentially all of the GCs had strong reaction with anti-Hsp70 (Fig. 3). At a higher magnification, intense immunoreactivity of granules in GC was evidenced (Fig. 3, Inset). There was no immunoperoxidase staining with anti-Hsp70 in the snail ventricle. While the GCs because of their size and characteristic morphology are readily identifiable, nerve fibers could hardly be visualized in histological paraffin preparations. More precisely the localization of Hsp70-like immunoreactive material in the Achatina atrial cells was defined by immunoelectronmicroscopy. The results presented in Fig. 4A show that anti-Hsp70 stains the granules of the atrial GCs. The degree of Hsp70 immunolabeling varied among granules of morphologically different types: prominent reactivity was observed in light homogeneous granules, moderate gold labelling was revealed in dark homogeneous granules and only rare gold particles were found over light granules of fibrous structure. Occasional figures of Hsp70-immunolabeled granules releasing from a GC were observed (Fig. 4B). Hsp70-immunoreactive material was also present in the neurosecretory granules of the nerve fibers. The density of Hsp70 immunolabeling in the nerve cords varied among individual axon profiles. Whereas neurosecretory granules in some axons showed strong immunoreactivity, the reactivity of the granules in the neighbouring axons was moderate or absent (Fig. 4C). Moreover, anti-Hsp70 stained specifically the gliosomes in the glio-interstitial cells surrounding the nerve cords (Fig. 4C). A few individual gold particles were also scattered over the cytoplasm and nuclei of different cells.


Fig. 3

Immunoperoxidase localization of Hsp70 within the snail atrium demonstrating increased intensity of staining in the granular cells (arrowheads) adhering the muscular trabeculae (M). Original magnification ×20. Insert: prominent Hsp70-immunostaining of the granules in a GC; original magnification ×40.


Fig. 4

Ultrastructural localization of anti-Hsp70 antibodies in a transverse thin section through the Achatina atrium. (A) Immunogold (10nm) labeling of the granules in a granular cell. Intensivity of Hsp70-immunostaining of morphologically different granules shows significant variation. The greatest labelling was observed over light homogeneous granules (arrowheads), less intensivity of labeling was found over dark homogeneous granules (double arrowhead), and least numerous gold particles were observed over light fibrous granules (triple arrowhead). Note very active cell surface (asterisks). Arrows indicate a basement lamella surrounding the granular cell. (B) Putative exocytosis of an anti-Hsp70 labeled granule from a granular cell. Designation are the same as in (A). (C) Localization of Hsp70-immunoreactive material in the nerve bundle. Axon profiles with intensively labelled neurosecretory granules (arrowheads) neighbour axon profiles with unlabelled neurosecretory granules (double arrowheads). The gliosomes of a glio-interstitial cell also demonstrated Hsp70-immunoreactivity (arrows). Bars: 1μm.



4 Discussion

The GCs in the atrium of A. fulica are integrated in the heart tissue. The snail hemocytes are agranular cells and GCs do not circulate in the hemolymph. Secretory GCs in the snail atrium are in close morphological contact with the nerve endings. Apparently, the nerves and GCs form a permanent structures – the neuroendocrine units. The functional significance of these units is unclear. On the one hand, neuromediators from the nerve fibers may modulate the metabolic and secretory activities of the GCs, and on the other hand, peptides from GCs may influence the central nerve system through the cardiac nerves.

Results of Western blot analysis have demonstrated that Hsp70 is present in the Achatina atrium. Immunocytochemical staining indicated that all elements of the Achatina atrial neuroendocrine system – axons, glio-interstitial cells and GCs – contain Hsp70-like substance in their granules. A number of possible functions of Hsp70 in the neuroendocrine units of the snail atrium can be proposed. (1) Hsp70 might operate at the cellular level inside the granules as a constitutive molecular chaperone influencing the conformation of the granular proteins. (2) Hsp70 might be externalized from the GCs under both normal and stressful conditions through degranulation or be released in the hemolymph due to destruction of the GCs following strong insults. The intensity of these processes may be under direct neural control. Increase of extracellular Hsp70 in the snail hemolymph could perhaps provide stress tolerance at the organism level. (3) Hsp70 might participate in intercourse between nerve fibers and GCs. All these functions might be performed by Hsp70 compatible.

We have observed differences in intensity of Hsp70-like immunoreactivity between individual nerve fibers. While some axon profiles in the nerve bundles exhibited strong anti-Hsp70 staining, the others demonstrated faint or no Hsp70 immunoreactivity. Myogenic molluscan heart is under complex neural control involving inhibitory, excitatory and relaxing motoneurones as well as sensory neurons. The molluscan cardiac nerves are multifunctional and one nerve bundle combines the nerve axons performing different functions and harboring different nerve mediators. Cholinergic and serotonergic innervation was revealed in the heart of the bivalve mollusk, Mercenaria mercenaria (Kuwasawa and Hill, 1997). Neurons innervating the Achatina heart have been shown to elaborate FMRFamide and ACEP-1 (Achatina cardio-excitatory peptide-1) (Fujiwara-Sakata and Kobayashi, 1994). It can be assumed that Hsp70 in nerve fibers co-localize with some definite neuromediators. However, observed differences in Hsp70 presence in nerosecretory granules might also be associated with functional state of the nerve fiber. Further research is necessary to elucidate this point. Neuromediators influence Hsps expression both in vertebrates and invertebrates. In rat, activation of α1-adrenoceptors results in an increase of Hsp72 in the circulation (Johnson et al., 2005). As regards molluscs, noradrenaline has been shown induces the Hsp70 gene promoter in hemocytes of oyster, Crassostrea gigas, and abalone, Haliotis tuberculata via α-adrenargic signaling pathway (Lacoste et al., 2001), and serotonin affects the Hsp70 synthesis in the ocular system of the marine snail, Aplysia (Koumenis et al., 1995).

Hsp70-immunoreactive material was also found in the gliosomes of the glio-interstitial cells. It is sufficiently evidenced that glial cells surrounding the nerve bundles are not merely protection against any mechanical damage. The receptor-mediated signaling between glial and nerve cells ensures glio-interstitial cells an essential role in regulation of nerve functioning (Chiu and Kriegler, 1994). It has been shown that Hsp70 can be released by glial cells and enhance motoneuron survival (Guzhova et al., 2001; Tytell, 2005).

The density of Hsp70 immunolabeling in the GCs varied among morphologically different granules. These granule types, probably, represent advanced stages of granular maturation. The observed pictures of granule exocytosis suggest that the atrial GCs could be a serious source of Hsp70 in the snail hemolymph.

On the basis of metachromasia and alcian blue-safranin staining, the snail heart GCs has been compared with mast cells of vertebrates (Zs.-Nagy and S.-Rózsa, 1970). Indeed, the GCs in snail atrium correspond in their morphological characteristics to mast cells. Besides, mast cells are also closely apposed to nerves in a variety of vertebrate tissues in vivo (Newson et al., 1983; Suzuki et al., 2004; Wiesner-Menzel et al., 1981), and form tight membrane-to-membrane contacts with neurons in vitro (Blennerhassett et al., 1991). Hsp70 was revealed in the granules of the mouse bone marrow derived mast cells (Skokos et al., 2003) and rat basophilic leukemia cells (RBL, homologues of mucosal mast cells) (Bachelet et al., 2002). During degranulation following stress Hsp70 is released from these cells. It is proposed that bidirectional communication occurs between nerve fibers and mast cells. Granular cell-nerve interaction in the snail heart may have many in common with mast cell-nerve co-operation in vertebrates, and therefore, the neuroendocrine units in the molluscan atrium may provide a valuable adjunct to existing models for studying the neuro-endocrine regulatory mechanisms.

Although the precise mechanisms of Hsp70 functioning in the snail heart remain to be elucidated, it is conceivable that this bioactive peptide contributes to the activities of the atrial neuroendocrine units.

Acknowledgments

This work is partly supported by the Russian Foundation for Basic Research (grant 05-04-49393) and Joint research center “Material science and characterization in high technology”.

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doi:10.1016/j.cellbi.2007.01.027


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)