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Immunolocalization of E-cadherin and αE-catenin in rat parotid acinar cell under chronic stimulation of isoproterenol
archives of oral biology 52 (2007) 161–167
available at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/arob
Immunolocalization of E-cadherin and aE-catenin in rat parotid acinar cell under chronic stimulation of isoproterenol Fabio D’Amico a,*, Evangelia Skarmoutsou b a b
Department of Biomedical Sciences, University of Catania, Catania, Italy Department of Animal Biology, University of Catania, Catania, Italy
article info
abstract
Article history:
E-cadherin and aE-catenin were localized in normal and chronically isoproterenol-treated
Accepted 5 September 2006
acinar cells of rat parotid gland by means of immunogold labelling of Lowicryl embedded sections. Immunostaining of both experimental groups with polyclonal antibodies to E-
Keywords:
cadherin and aE-catenin was mainly restricted to the areas of adherens junctions. Surpris-
E-cadherin
ingly, in isoproterenol-treated cell aE-catenin was also found on the secretory granules
aE-catenin
periphery and appeared to encircle a secretory vesicle.
Isoproterenol Rat parotid acinar cell
In isoproterenol-induced cell hyperproliferation, the maintened presence of adherens junctions components, such as E-cadherin and aE-catenin molecules, should be an essential prerequisite for tissue integrity. Our data suggest the presence of a correlation between the organization of actin and the localization of aE-catenin in the chronically isoproterenol-treated acinar cell of rat parotid gland. # 2006 Elsevier Ltd. All rights reserved.
1.
Introduction
Cell-to-cell adhesion is essential for tissue organization, positional information during the development, and for maintenance of tissue structure and integrity in adult organism.1–6 Among the cell junctional complexes, the adherens junctions mediate cell adhesion by classical cadherins,7 which associate with the cortical actin cytoskeleton. Such connections are mediated by the cytoplasmic proteins catenins.8 The transmembrane E-cadherin is a member of classic cadherins and it is expressed by epithelial tissue.9 It contains a single transmembrane domain, five ectodomain repeats and an intracellular actin microfilament connecting domain.10,11 Catenins have been classified into a-, b-, and g-catenin.12,13 They serve to link the cadherin to the actin cytoskeleton.
Catenins are also involved in cellular signalling.14–16 Two acatenins were identified: the neuronal aN-catenin and the epithelial aE-catenin.17–19 In general, the intracellular domain of E-cadherin binds to b-catenin, which is in turn linked to the actin cytoskeleton through a-catenin.20 The treatment with isoproterenol, a b-adrenergic agonist, of rodent salivary glands is widely used to study their biochemistry and physiology. Isoproterenol acts as a stimulant for a large number of intracellular processes,21 and, in particular, it induces the secretory machinery.22 Through the activation of adenylate cyclase and the resulting accumulation of cyclic AMP,23 the chronic administration of isoproterenol induces salivary gland hypertrophy and hyperplasia.24 The agonist also stimulates DNA synthesis,25 increased synthesis of secretory proteins,26,27 and secretory discharge.28
* Corresponding author. Tel.: +39 095 312017; fax: +39 095 312017. E-mail address: [email protected] (F. D’Amico). 0003–9969/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2006.09.003
162
archives of oral biology 52 (2007) 161–167
In order to increase the physiological activity of the parotid gland, we made use of the b-adrenergic agonist isoproterenol. The aim of this study was to investigate the influence of chronic treatment with adrenergic agonist isoproterenol on the immunolocalization of E-cadherin and aE-catenin in rat parotid acinar cell. Using a post-embedding protein G immunogold labelling technique, we showed that E-cadherin and aE-catenin were mainly localized at adherens junctions areas and, in isoproterenol-treated cells, aE-catenin was also found on the secretory granules periphery.
2.
Material and methods
2.1.
Reagents
D-L-Isoproterenol,
bovine serum albumin (BSA), Tween 20, and protein G-colloidal gold (5 nm) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Glycine was obtained from Merck (Darmstadt, Germany) and Lowicryl K4M resin from Polyscience (Warrington, PA, USA). Polyclonal antibodies against E-cadherin (affinity purified and raised against an amino terminus sequence of human Ecadherin) and aE-catenin (affinity purified and raised against a carboxy terminus sequence of human aE-catenin) were purchased by Santa Cruz Biotechnology (Santa Cruz, CA, USA).
2.2.
Animals
Adult male Wistar rats, weighing 150–200 g were used. The animals, divided in two groups (six animals per group), received twice daily intraperitoneal injections for 10 days, respectively with: (a) saline and (b) 25 mg/kg body weight isoproterenol. Twenty-four hours following the last isoproterenol injection and after the same period of fasting, parotid glands were removed under anaesthesia with pentobarbital (approximately 1 ml of 50 mg/ml of anaesthetic). Connective tissue and lymph nodes were carefully removed from glands and fresh parotid mass was obtained with an analytical balance. The average gland wet weights were then determined and expressed by the ratio of mean (S.D.) parotid gland wet weight/100 g body weight.
2.3.
Tissue processing
Small pieces of parotid glands were fixed by immersion in a mixture of 1% glutaraldheyde and 4% para-formaldehyde in 0.1M phosphate-buffered saline (PBS), pH 7.4, for two h at 4 8C. After rinsing with PBS and incubation in 0.15 M glycine in PBS for 1 h at room temperature to block free aldehyde groups, samples were embedded in Lowicryl K4M resin with progressive lowering of temperature (PLT) technique. The resin was polymerised under ultraviolet light for 24 h at 35 8C and continued for 48 h at room temperature. Semithin sections were cut with glass knives, mounted on polylysine coated glass slides, and finally stained according the protocol of Richardson et al.29 Ultrathin sections were cut with diamond knife and mounted on nickel grids coated with formvar.
2.4.
Immunocytochemistry
Each step was performed in a moist chamber, and by floating the grids on drops of the different reagents. After a 10 min treatment with Tris-buffered saline (TBS), sections were incubated overnight at 4 8C with the goat primary polyclonal antibodies against E-cadherin and aE-catenin. Antibodies were diluted at a range of dilutions (1:50, 1:100, 1:150, 1:200, 1:500) in TBS plus 0.5% BSA and 0.05% Tween 20. After washing in TBS, sections were incubated for 1 h at room temperature with protein G-colloidal gold (5 nm), diluted 1:30 in PBS. After washing with PBS and distilled water, grids were counterstained with 2% aqueous uranyl acetate (5 min) and Reynolds lead citrate (5 min). Finally, sections were observed using a Hitachi H-600 transmission electron microscope at 75 kV. As a negative control, some grids were incubated with a solution without primary antibodies or with a pre-immune serum.
2.5.
Quantitative estimations and morphometric analysis
Volume density of secretory granules of normal and isoproterenol-treated gland was stereologically estimated with the point lattice test.30,31 The following simple formula was used: V(sg)/ V(pg), where sg and gp are the number of interceptions of the secretory granules and parotid gland, respectively, with the test lattice. A set of 12 electron micrographs printed at a linear magnification of 34,000 were used. Micrographs were random selected and a point lattice test was random placed over nonrandom section planes. Finally, obtained values were statistically compared with the t-test. Labelling density, expressed as number of gold particles per adherens junction, was determined on a total number of 30 micrographs at a primary magnification of 17,000. Mean and standard error values were determined.
3.
Results
3.1. Isoproterenol treatment causes morphological variations in parotid acinar cells At the end of b-adrenergic treatment, the wet weight of the parotid glands increased approximately three–four-fold. Control glands showed a wet weight of 198 18 mg, whereas treated ones showed a 821 105 mg wet weight (Fig. 1).
Fig. 1 – Parotid gland wet weight of normal and isoproterenol-treated rats. Values are expressed by the ratio of mean W S.D. parotid gland wet weight/100 g body weight. Significance at P < 0.01.
archives of oral biology 52 (2007) 161–167
163
Fig. 2 – Morphological differences between secretory granule of normal and isoproterenol-treated acinar cells of rat parotid gland. The secretory granule of normal acinar cell shows a polymorphic matrix configuration (a), whereas the granule of treated cell (b) shows a homogeneous and lighter configuration. L, acinar lumen. Scale bar: (a) 0.20 mm; (b) 0.25 mm.
At cytological level, in isoproterenol-treated gland, acinar cell showed a more enlarged and distended shape. Rough endoplasmic reticulum seemed to be reduced and Golgi apparatus was more voluminous in comparison to the normal cells. Electron density of secretory granules was decreased and the typical polymorphic configurations of their matrix, owing to the different disposition of light and dark components (for a detailed morphological description refer to D’Amico et al.)32 were disappeared (Fig. 2). The mean diameter of these granules was increased and their volume density was almost duplicated in comparison to the normal cell granules (Fig. 3). Estimations of volume densities of secretory granules of normal (Estnor) and treated cell (Esttrt) were: Estnor = 0.36, Esttrt = 0.65.
3.2. b-Adrenergic stimulation does not change the immunocytochemical pattern in the adherens junctions sites As regards immunolocalization for E-cadherin, it was very similar in the control and treated acinar cells. E-cadherin was
mainly localized at the adherens junctions (Fig. 4a and b), as well as, over the digitiform extroflessions of acinar cells. The immunolabelling for aE-catenin showed a similar pattern to that for E-cadherin in control and treated acinar cells (Fig. 4c and d). The labelling densities for the two molecules under study were similar and their statistical differences were not significant (Fig. 5).
3.3. Treated acinar cells show a new scenario for aEcatenin In isoproterenol-treated glands, several examples of immuolocalization for aE-catenin in the secretory granules have been found. Such an immunolabelling seemed to be localized exclusively on those mature granules nearest the plasma membrane. Moreover, this immunolocalization was observed at the outermost part of the secretory granule. Colloidal gold particles, arranged in groups of two, lined a circular zone, as a
Fig. 3 – Photomicrographs of semithin sections of normal (a) and isoproterenol-treated (b) rat parotid gland stained with Richardson’s technique. The most characteristic features are the number and size increases of secretory granules of agonist treated acinar cells. Scale bar: 50 mm.
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archives of oral biology 52 (2007) 161–167
Fig. 4 – (a) Immunolabelling for E-cadherin is observed over digitiform extroflessions (arrow) and at adherens junctions of normal acinar cell. Scale bar = 0.2 mm; (inset) 0.08 mm. (b) In treated cell, E-cadherin is shown on adherens junctions. Scale bar = 0.08 mm. (c) aE-catenin is localized at adherens junctions of normal acinar cell. Scale bar = 0.15 mm; (inset) 0.1 mm. (d) The immunolabelling for aE-catenin in treated acinar cell shows a similar pattern to that for control ones. Scale bar = 0.2 mm; (inset) 0.15 mm.
vesicle at the granule periphery (Fig. 6). Perhaps a budding or encapsulating process of such vesicles occurred. Unfortunately, there was no observation of any labelled free vesicles in cytoplasm, probably owing to the limitation of the technique used. The negative control immunocytochemical reactions were essentially free of gold particles (Fig. 7).
4.
Fig. 5 – Labelling densities for E-cadherin and aE-catenin in normal and isoproterenol-treated cells. There are no statistically significant differences between the experimental groups.
Discussion
We showed that parotid gland mass was markedly increased by chronic isoproterenol treatment, as also reported by others.24,28,33 The mechanisms of such an isoproterenol-induced hypertrophy are largely unknown. However, the mass increase may reflect, at least partially, the increased size and number of secretory granules of
archives of oral biology 52 (2007) 161–167
Fig. 6 – Colloidal gold particles reveal also the presence of aE-catenin at the periphery of mature secretory granules in isoproterenol-treated acinar cell. Note the couples of colloidal gold labelling the outermost matrix zone of secretory granule. Scale bar = 0.15 mm; (inset) 0.1 mm.
acinar cells. In fact, it is well known that chronic isoproterenol treatment causes mainly an altered expression of secretory proteins,34,35 which could result in the enlargement of acinar cells. Secretory granules matrix of parotid acinar cell in normal rat shows a polymorphic substructure. Such a polymorphism, caused by the differential disposition of dark and light material within the granule matrix has been already described in a previous paper.32 In general, under chronic stimulation with isoproterenol, acinar cell shows an enlarged shape. Rough endoplasmic reticulum seems to be quantitatively reduced, and Golgi stacks seems to be hypertrophic in comparison to the normal cells. Secretory granules electrondensity is remarkably decreased, showing a homogeneous aspect. Our stereological estimations suggest that the mean diameter of secretory granules of treated cells is almost duplicated with respect to normal ones. In this study, the immunolocalization of E-cadherin and aE-catenin in normal and treated cells was found at adherens junctions and are in accordance to the most authors.36–38 In isoproterenol-induced cell hyperproliferation, the unchanged presence of adherens junctions components, such
165
as E-cadherin and aE-catenin molecules, should be an essential prerequisite for tissue integrity. In treated acinar cells, aE-catenin, besides its localization at adherens junctions, is also found over mature secretory granules. This aE-catenin localization was exclusively found at the granule periphery with a circular disposition. Such an immunocytochemical pattern aspect resembles the minor regulated pathway of secretion of parotid gland,39–41 where a vesiculation of membrane around the secretory granules which can occur inward or outward. The minor regulated pathway is very sensitive to secretagogue and it is mobilized by b-adrenergic stimulation.40 Unfortunately, owing to the limitation of used techniques, we can not state if such vesiculation processes follow a centrifugal or centripetal direction, but growing evidence suggests a frequent occurrence of a reversible cytoplasmic a-catenin internalization during actin depolymerization.42–44 Results described here provide evidence that the occurrence of aE-catenin in the secretory granules, may be induced by the agonist chronic treatment, as suggested also by the study of Schnittler et al.,45 who showed that the depolymerization of actin filaments may increase the solubility of the a-catenin. The immunolocalization pattern for aE-catenin on the secretory granules shows these molecules arranged in groups of two. Koslov et al.46 showed that a-catenin exists in a dynamic monomeric–dimeric equilibrium, and that the dimeric state is favoured when the molecule is in pure solution. It is well known that the cytoskeleton plays an important role in the secretion process mechanisms.47,48 Beneath plasma membrane of salivary glands, as in other cell types, an actin network exists.49–51 According to the most studies, under resting condition, the actin cytoskeleton acts as a physical barrier to prevent secretory granules to reach their exocytosis sites.52,53 On the contrary, secretory stimulation, like daily isoproterenol treatment, produces reversible destabilization and disorganization of actin network, allowing secretory granules to reach the exocytosis sites.50,54–58 In conclusion, these data supports the idea of the presence of a correlation between the organization of actin and the localization of aE-catenin. aE-catenin may function as a sensor of the actin status, as previously suggested by Fischer and Quinlan59 who showed that the inability in interacting with cortical actin microfilaments would destabilize aEcatenin. The effects of isoproterenol chronic stimulation on salivary gland physiology and biochemistry are extremely complex. In addition to the well known alterations, such as hypertrophy, altered DNA synthesis and increased and novel synthesis of secretory proteins, additional alteration seem to occur in the physiological machinery of the cadherin catenin complex, as suggested in the present paper. Further studies are necessary in order to study the mechanism by which the isoproterenol treatment induces this novel appearance of aE-catenin in the secretory granules. However, because isoproterenol exerts its action via the cAMP pathway and because adhesion junctions are highly regulated by phosphorylation/dephosphorylation processes of their molecular components60 (Lilien and Balsamo), a real
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archives of oral biology 52 (2007) 161–167
Fig. 7 – Immunocytochemical negative controls in normal (a, for E-cadherin; b, for aE-catenin) and isoproterenol-treated acinar cells (c, for E-cadherin; d, for aE-catenin). Scale bars = 0.2 mm.
possibility is that cAMP-mediated phosphorylation may be involved.
Acknowledgement This work is dedicated in memoriam to Prof. S. Sanfilippo, CMEM—University of Catania, Italy.
references
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21. Cook DI, Van Lennep EW, Roberts ML, Young JA, Secretion by the major salivary glands.Johnson LR, editor. Physiology of the Gastrointestinal Tract, vol. 2. New York: Raven, Press; 1994 . p. 1061–117. 22. Butcher FR, Putney Jr JW. Regulation of parotid gland function by cyclic nucleotide and calcium. Adv Cyclic Nucl Res 1980;13:215–49. 23. Putney Jr JW. Identification of cellular activation mechanisms associated with salivary secretion. Annu Rev Physiol 1986;48:75–88. 24. Selye H, Veilleux R, Cantin M. Excessive stimulation of salivary gland growth by isoproterenol. Science 1961;133: 44–5. 25. Schneyer CA, Humphreys-Beher M. Inhibition of isoproterenol-induced DNA and RNA synthesis in rat parotid and pancreas following removal of submandibular– sublingual glands. Cell Tissue Res 1989;256:361–3. 26. Bogart BI. Secretory dynamics of the rat submandibular gland. An ultrastructural and cytochemical study of the isoproterenol induced secretory cycle. J Ultrastruct Res 1975;52:139–55. 27. Ten Hagen KG, Balys MM, Tabakm LA, Melvin JE. Analysis of isoproterenol-induced changes in parotid gland gene expression. Physiol Genomics 2002;8:107–14. 28. Barka T. Stimulation of DNA synthesis by isoproterenol in the salivary glands. Exp Cell Res 1965;39:355–64. 29. Richardson KC, Jarett L, Finke EH. Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technol 1960;35:313–23. 30. Mayhew TM. A review of recent advances in stereology for quantifying neural structure. J Neurocytol 1992;21:313–28. 31. Cruz-Orive LM, Weibel ER. Sampling designs for stereology. J Microsc 1981;122:235–57. 32. D’Amico F, Skarmoutsou E, Sanfilippo S, Camakaris J. Menkes protein localization in rat parotid acinar cells. Acta Histochem 2005;107:373–8. 33. Onofre MA, de Souza LB, Campos Jr A, Taga R. Stereological study of acinar growth in the rat parotid gland induced by isoproterenol. Arch Oral Biol 1997;42:333–8. 34. Muenzer J, Bildstein C, Gleason M, Carlson DM. Purification of proline-rich proteins from parotid glands of isoproterenol-treated rats. J Biol Chem 1979;254:5623–8. 35. Robinovitch MR, Keller PJ, Johnson DA, Iversen JM, Kauffman DL. Changes in rat parotid salivary proteins induced by chronic isoproterenol administration. J Dent Res 1977;56:290–303. 36. McNeill H, Ozawa M, Kemler R, Nelson WJ. Novel function of the cell adhesion molecule uvomorulin as an inducer of cell surface polarity. Cell 1990;62:309–16. 37. Hirano S, Kimoto N, Shimoyama Y, Hirohashi S, Takeichi M. Identification of a neural a-catenin as a key regulator of cadherin function and multicellular organization. Cell 1992;70:293–301. 38. Groos S, Reale E, Luciano L. Re-evaluation of epoxy resin sections for light and electron microscopic immunostaining. J Histochem Cytochem 2001;49:397–406. 39. Kurosumi K. Ultrastructural immunocytochemistry of the adenohypophysis in the rat: a review. J Electron Microsc Tech 1991;19:42–56. 40. Huang AY, Castle AM, Hinton BT, Castle JD. Resting (basal) secretion of proteins is provided by the minor regulated and constitutive-like pathways and not granule exocytosis in parotid acinar cells. J Biol Chem 2001;276:22296–306. 41. Castle AM, Huang AY, Castle JD. The minor regulated pathway, a rapid component of salivary secretion, may provide docking/fusion sites for granule exocytosis at the apical surface of acinar cells. J Cell Sci 2002;115:2963–73. 42. Hirschberg K, Miller CM, Ellenberg J, Presley JF, Siggia ED, Phair RD, et al. Kinetic analysis of secretory protein traffic
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available at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/arob
Immunolocalization of E-cadherin and aE-catenin in rat parotid acinar cell under chronic stimulation of isoproterenol Fabio D’Amico a,*, Evangelia Skarmoutsou b a b
Department of Biomedical Sciences, University of Catania, Catania, Italy Department of Animal Biology, University of Catania, Catania, Italy
article info
abstract
Article history:
E-cadherin and aE-catenin were localized in normal and chronically isoproterenol-treated
Accepted 5 September 2006
acinar cells of rat parotid gland by means of immunogold labelling of Lowicryl embedded sections. Immunostaining of both experimental groups with polyclonal antibodies to E-
Keywords:
cadherin and aE-catenin was mainly restricted to the areas of adherens junctions. Surpris-
E-cadherin
ingly, in isoproterenol-treated cell aE-catenin was also found on the secretory granules
aE-catenin
periphery and appeared to encircle a secretory vesicle.
Isoproterenol Rat parotid acinar cell
In isoproterenol-induced cell hyperproliferation, the maintened presence of adherens junctions components, such as E-cadherin and aE-catenin molecules, should be an essential prerequisite for tissue integrity. Our data suggest the presence of a correlation between the organization of actin and the localization of aE-catenin in the chronically isoproterenol-treated acinar cell of rat parotid gland. # 2006 Elsevier Ltd. All rights reserved.
1.
Introduction
Cell-to-cell adhesion is essential for tissue organization, positional information during the development, and for maintenance of tissue structure and integrity in adult organism.1–6 Among the cell junctional complexes, the adherens junctions mediate cell adhesion by classical cadherins,7 which associate with the cortical actin cytoskeleton. Such connections are mediated by the cytoplasmic proteins catenins.8 The transmembrane E-cadherin is a member of classic cadherins and it is expressed by epithelial tissue.9 It contains a single transmembrane domain, five ectodomain repeats and an intracellular actin microfilament connecting domain.10,11 Catenins have been classified into a-, b-, and g-catenin.12,13 They serve to link the cadherin to the actin cytoskeleton.
Catenins are also involved in cellular signalling.14–16 Two acatenins were identified: the neuronal aN-catenin and the epithelial aE-catenin.17–19 In general, the intracellular domain of E-cadherin binds to b-catenin, which is in turn linked to the actin cytoskeleton through a-catenin.20 The treatment with isoproterenol, a b-adrenergic agonist, of rodent salivary glands is widely used to study their biochemistry and physiology. Isoproterenol acts as a stimulant for a large number of intracellular processes,21 and, in particular, it induces the secretory machinery.22 Through the activation of adenylate cyclase and the resulting accumulation of cyclic AMP,23 the chronic administration of isoproterenol induces salivary gland hypertrophy and hyperplasia.24 The agonist also stimulates DNA synthesis,25 increased synthesis of secretory proteins,26,27 and secretory discharge.28
* Corresponding author. Tel.: +39 095 312017; fax: +39 095 312017. E-mail address: [email protected] (F. D’Amico). 0003–9969/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2006.09.003
162
archives of oral biology 52 (2007) 161–167
In order to increase the physiological activity of the parotid gland, we made use of the b-adrenergic agonist isoproterenol. The aim of this study was to investigate the influence of chronic treatment with adrenergic agonist isoproterenol on the immunolocalization of E-cadherin and aE-catenin in rat parotid acinar cell. Using a post-embedding protein G immunogold labelling technique, we showed that E-cadherin and aE-catenin were mainly localized at adherens junctions areas and, in isoproterenol-treated cells, aE-catenin was also found on the secretory granules periphery.
2.
Material and methods
2.1.
Reagents
D-L-Isoproterenol,
bovine serum albumin (BSA), Tween 20, and protein G-colloidal gold (5 nm) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Glycine was obtained from Merck (Darmstadt, Germany) and Lowicryl K4M resin from Polyscience (Warrington, PA, USA). Polyclonal antibodies against E-cadherin (affinity purified and raised against an amino terminus sequence of human Ecadherin) and aE-catenin (affinity purified and raised against a carboxy terminus sequence of human aE-catenin) were purchased by Santa Cruz Biotechnology (Santa Cruz, CA, USA).
2.2.
Animals
Adult male Wistar rats, weighing 150–200 g were used. The animals, divided in two groups (six animals per group), received twice daily intraperitoneal injections for 10 days, respectively with: (a) saline and (b) 25 mg/kg body weight isoproterenol. Twenty-four hours following the last isoproterenol injection and after the same period of fasting, parotid glands were removed under anaesthesia with pentobarbital (approximately 1 ml of 50 mg/ml of anaesthetic). Connective tissue and lymph nodes were carefully removed from glands and fresh parotid mass was obtained with an analytical balance. The average gland wet weights were then determined and expressed by the ratio of mean (S.D.) parotid gland wet weight/100 g body weight.
2.3.
Tissue processing
Small pieces of parotid glands were fixed by immersion in a mixture of 1% glutaraldheyde and 4% para-formaldehyde in 0.1M phosphate-buffered saline (PBS), pH 7.4, for two h at 4 8C. After rinsing with PBS and incubation in 0.15 M glycine in PBS for 1 h at room temperature to block free aldehyde groups, samples were embedded in Lowicryl K4M resin with progressive lowering of temperature (PLT) technique. The resin was polymerised under ultraviolet light for 24 h at 35 8C and continued for 48 h at room temperature. Semithin sections were cut with glass knives, mounted on polylysine coated glass slides, and finally stained according the protocol of Richardson et al.29 Ultrathin sections were cut with diamond knife and mounted on nickel grids coated with formvar.
2.4.
Immunocytochemistry
Each step was performed in a moist chamber, and by floating the grids on drops of the different reagents. After a 10 min treatment with Tris-buffered saline (TBS), sections were incubated overnight at 4 8C with the goat primary polyclonal antibodies against E-cadherin and aE-catenin. Antibodies were diluted at a range of dilutions (1:50, 1:100, 1:150, 1:200, 1:500) in TBS plus 0.5% BSA and 0.05% Tween 20. After washing in TBS, sections were incubated for 1 h at room temperature with protein G-colloidal gold (5 nm), diluted 1:30 in PBS. After washing with PBS and distilled water, grids were counterstained with 2% aqueous uranyl acetate (5 min) and Reynolds lead citrate (5 min). Finally, sections were observed using a Hitachi H-600 transmission electron microscope at 75 kV. As a negative control, some grids were incubated with a solution without primary antibodies or with a pre-immune serum.
2.5.
Quantitative estimations and morphometric analysis
Volume density of secretory granules of normal and isoproterenol-treated gland was stereologically estimated with the point lattice test.30,31 The following simple formula was used: V(sg)/ V(pg), where sg and gp are the number of interceptions of the secretory granules and parotid gland, respectively, with the test lattice. A set of 12 electron micrographs printed at a linear magnification of 34,000 were used. Micrographs were random selected and a point lattice test was random placed over nonrandom section planes. Finally, obtained values were statistically compared with the t-test. Labelling density, expressed as number of gold particles per adherens junction, was determined on a total number of 30 micrographs at a primary magnification of 17,000. Mean and standard error values were determined.
3.
Results
3.1. Isoproterenol treatment causes morphological variations in parotid acinar cells At the end of b-adrenergic treatment, the wet weight of the parotid glands increased approximately three–four-fold. Control glands showed a wet weight of 198 18 mg, whereas treated ones showed a 821 105 mg wet weight (Fig. 1).
Fig. 1 – Parotid gland wet weight of normal and isoproterenol-treated rats. Values are expressed by the ratio of mean W S.D. parotid gland wet weight/100 g body weight. Significance at P < 0.01.
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Fig. 2 – Morphological differences between secretory granule of normal and isoproterenol-treated acinar cells of rat parotid gland. The secretory granule of normal acinar cell shows a polymorphic matrix configuration (a), whereas the granule of treated cell (b) shows a homogeneous and lighter configuration. L, acinar lumen. Scale bar: (a) 0.20 mm; (b) 0.25 mm.
At cytological level, in isoproterenol-treated gland, acinar cell showed a more enlarged and distended shape. Rough endoplasmic reticulum seemed to be reduced and Golgi apparatus was more voluminous in comparison to the normal cells. Electron density of secretory granules was decreased and the typical polymorphic configurations of their matrix, owing to the different disposition of light and dark components (for a detailed morphological description refer to D’Amico et al.)32 were disappeared (Fig. 2). The mean diameter of these granules was increased and their volume density was almost duplicated in comparison to the normal cell granules (Fig. 3). Estimations of volume densities of secretory granules of normal (Estnor) and treated cell (Esttrt) were: Estnor = 0.36, Esttrt = 0.65.
3.2. b-Adrenergic stimulation does not change the immunocytochemical pattern in the adherens junctions sites As regards immunolocalization for E-cadherin, it was very similar in the control and treated acinar cells. E-cadherin was
mainly localized at the adherens junctions (Fig. 4a and b), as well as, over the digitiform extroflessions of acinar cells. The immunolabelling for aE-catenin showed a similar pattern to that for E-cadherin in control and treated acinar cells (Fig. 4c and d). The labelling densities for the two molecules under study were similar and their statistical differences were not significant (Fig. 5).
3.3. Treated acinar cells show a new scenario for aEcatenin In isoproterenol-treated glands, several examples of immuolocalization for aE-catenin in the secretory granules have been found. Such an immunolabelling seemed to be localized exclusively on those mature granules nearest the plasma membrane. Moreover, this immunolocalization was observed at the outermost part of the secretory granule. Colloidal gold particles, arranged in groups of two, lined a circular zone, as a
Fig. 3 – Photomicrographs of semithin sections of normal (a) and isoproterenol-treated (b) rat parotid gland stained with Richardson’s technique. The most characteristic features are the number and size increases of secretory granules of agonist treated acinar cells. Scale bar: 50 mm.
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Fig. 4 – (a) Immunolabelling for E-cadherin is observed over digitiform extroflessions (arrow) and at adherens junctions of normal acinar cell. Scale bar = 0.2 mm; (inset) 0.08 mm. (b) In treated cell, E-cadherin is shown on adherens junctions. Scale bar = 0.08 mm. (c) aE-catenin is localized at adherens junctions of normal acinar cell. Scale bar = 0.15 mm; (inset) 0.1 mm. (d) The immunolabelling for aE-catenin in treated acinar cell shows a similar pattern to that for control ones. Scale bar = 0.2 mm; (inset) 0.15 mm.
vesicle at the granule periphery (Fig. 6). Perhaps a budding or encapsulating process of such vesicles occurred. Unfortunately, there was no observation of any labelled free vesicles in cytoplasm, probably owing to the limitation of the technique used. The negative control immunocytochemical reactions were essentially free of gold particles (Fig. 7).
4.
Fig. 5 – Labelling densities for E-cadherin and aE-catenin in normal and isoproterenol-treated cells. There are no statistically significant differences between the experimental groups.
Discussion
We showed that parotid gland mass was markedly increased by chronic isoproterenol treatment, as also reported by others.24,28,33 The mechanisms of such an isoproterenol-induced hypertrophy are largely unknown. However, the mass increase may reflect, at least partially, the increased size and number of secretory granules of
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Fig. 6 – Colloidal gold particles reveal also the presence of aE-catenin at the periphery of mature secretory granules in isoproterenol-treated acinar cell. Note the couples of colloidal gold labelling the outermost matrix zone of secretory granule. Scale bar = 0.15 mm; (inset) 0.1 mm.
acinar cells. In fact, it is well known that chronic isoproterenol treatment causes mainly an altered expression of secretory proteins,34,35 which could result in the enlargement of acinar cells. Secretory granules matrix of parotid acinar cell in normal rat shows a polymorphic substructure. Such a polymorphism, caused by the differential disposition of dark and light material within the granule matrix has been already described in a previous paper.32 In general, under chronic stimulation with isoproterenol, acinar cell shows an enlarged shape. Rough endoplasmic reticulum seems to be quantitatively reduced, and Golgi stacks seems to be hypertrophic in comparison to the normal cells. Secretory granules electrondensity is remarkably decreased, showing a homogeneous aspect. Our stereological estimations suggest that the mean diameter of secretory granules of treated cells is almost duplicated with respect to normal ones. In this study, the immunolocalization of E-cadherin and aE-catenin in normal and treated cells was found at adherens junctions and are in accordance to the most authors.36–38 In isoproterenol-induced cell hyperproliferation, the unchanged presence of adherens junctions components, such
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as E-cadherin and aE-catenin molecules, should be an essential prerequisite for tissue integrity. In treated acinar cells, aE-catenin, besides its localization at adherens junctions, is also found over mature secretory granules. This aE-catenin localization was exclusively found at the granule periphery with a circular disposition. Such an immunocytochemical pattern aspect resembles the minor regulated pathway of secretion of parotid gland,39–41 where a vesiculation of membrane around the secretory granules which can occur inward or outward. The minor regulated pathway is very sensitive to secretagogue and it is mobilized by b-adrenergic stimulation.40 Unfortunately, owing to the limitation of used techniques, we can not state if such vesiculation processes follow a centrifugal or centripetal direction, but growing evidence suggests a frequent occurrence of a reversible cytoplasmic a-catenin internalization during actin depolymerization.42–44 Results described here provide evidence that the occurrence of aE-catenin in the secretory granules, may be induced by the agonist chronic treatment, as suggested also by the study of Schnittler et al.,45 who showed that the depolymerization of actin filaments may increase the solubility of the a-catenin. The immunolocalization pattern for aE-catenin on the secretory granules shows these molecules arranged in groups of two. Koslov et al.46 showed that a-catenin exists in a dynamic monomeric–dimeric equilibrium, and that the dimeric state is favoured when the molecule is in pure solution. It is well known that the cytoskeleton plays an important role in the secretion process mechanisms.47,48 Beneath plasma membrane of salivary glands, as in other cell types, an actin network exists.49–51 According to the most studies, under resting condition, the actin cytoskeleton acts as a physical barrier to prevent secretory granules to reach their exocytosis sites.52,53 On the contrary, secretory stimulation, like daily isoproterenol treatment, produces reversible destabilization and disorganization of actin network, allowing secretory granules to reach the exocytosis sites.50,54–58 In conclusion, these data supports the idea of the presence of a correlation between the organization of actin and the localization of aE-catenin. aE-catenin may function as a sensor of the actin status, as previously suggested by Fischer and Quinlan59 who showed that the inability in interacting with cortical actin microfilaments would destabilize aEcatenin. The effects of isoproterenol chronic stimulation on salivary gland physiology and biochemistry are extremely complex. In addition to the well known alterations, such as hypertrophy, altered DNA synthesis and increased and novel synthesis of secretory proteins, additional alteration seem to occur in the physiological machinery of the cadherin catenin complex, as suggested in the present paper. Further studies are necessary in order to study the mechanism by which the isoproterenol treatment induces this novel appearance of aE-catenin in the secretory granules. However, because isoproterenol exerts its action via the cAMP pathway and because adhesion junctions are highly regulated by phosphorylation/dephosphorylation processes of their molecular components60 (Lilien and Balsamo), a real
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Fig. 7 – Immunocytochemical negative controls in normal (a, for E-cadherin; b, for aE-catenin) and isoproterenol-treated acinar cells (c, for E-cadherin; d, for aE-catenin). Scale bars = 0.2 mm.
possibility is that cAMP-mediated phosphorylation may be involved.
Acknowledgement This work is dedicated in memoriam to Prof. S. Sanfilippo, CMEM—University of Catania, Italy.
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