THE ANATOMICAL RECORD 228:lll-122 (1990)

Redistribution of Membranes and Cytoskeletal Proteins in Chicken Oxyntic Cells During the HCI Secretory Cycle: Ultrastructural and lmmunofluorescence Study CECILIA S. KOENIG AND MONICA DABIKE Departamento de Biologia Celular, Pontificia Universidad Catolica de Chile, Alameda Santiago, Chile

ABSTRACT Changes in ultrastructure and cytoskeletal organization by avian oxyntic cells, a t the onset of HC1 secretion, were analysed. Cells in resting state, induced by fasting and cimetidine, were compared with histamine stimulated secreting cells. Ultrastructural studies were done by transmission electron microscopy; the distribution of prekeratin, myosin, and filamin-like protein, by immunofluorescence; and that of F-actin using FITC-phalloidin. Resting cells show short pericellular clefts. These are increasingly deepened in secreting cells by a reorganization of the lateral cell borders involving displacement of the junctional complexes toward the cell base and incorporation of the tubular system to the luminal plasma membrane. In secreting cells, the processes of the secretory surface are concentrated in a pericellular groove. Histamine stimulation induces a drastic redistribution of cytoskeletal proteins. In chicken oxyntic cells, in addition to the F-actin cytoskeleton associated with the membranes of the secretory surface, there is a cytoskeletal ring containing F-actin, myosin, and a filamin-like protein, located at the level of the junctional complexes. In resting cells, filaments and masses of cytoskeletal matrix are associated with the zonula adherens. In secreting cells, the junctional complexes maintain their association with the filamentous ring, while the amorphous matrix is replaced by microfilaments that support the processes of the luminal surface. Intermediate filaments form a peripheral ring probably associated with the zonula adherens, and project from the ring toward the cell cytoplasm. Thus, with the onset of HC1 secretion, the apical cytoskeletal ring of resting cells displaces toward the cell base. A role for this cytoskeletal ring in the changes in shape parallel to HCl secretion is discussed. Translocation of organelles and changes in cell shape depend on the integrated action of cellular membranes and the cytoskeletal network, but the localization of cytoskeletal proteins and the way they interact in vivo are not well known (Jacobson, 1983; Niggli and Burger, 1987). Gastric oxyntic cells, which undergo remarkable changes in shape during the HC1 secretory cycle, are a n excellent model for studying the role of the cytoskeleton in complex and transient processes occurring in differentiated epithelial cells (Vial and Garrido, 1979; Forte et al., 1981). A distinctive feature of oxyntic cells is their system of endocellular membranes- currently designated the tubulovesicular, or tubular system (Vial, 1982)-which is incorporated into the luminal plasma membrane under the action of HC1 physiological secretagogues (Vial and Orrego, 1960; Sedar and Friedman, 1961; Toner, 1963; Ito and Schoffield, 1974; Vial and Garrido, 1979; Forte et al., 1981). After the onset of HC1 secretion, these membranous systems of the secretory pole change their relation with the actin cytoskeleton (Vial and Garrido, 1976; Vial et al., 1979; Jiron et al., 1984; Vial e t al., 1985; Koenig et al., 1987). In resting cells, (c)

1990 WILEY-LISS, INC.

the plasma membrane of the smooth luminal surface is separated from the tubular system by a cortical actin meshwork. Stimulation induces a massive expansion of the secretory surface (Helander and Hirschowitz, 1972) which becomes filled with actin containing cytoplasmic processes. These changes may reflect the regulation of the output of acid through the translocation of the membranes containing the proton pump, from the endocellular compartment to the luminal cell surface (Smolka et al., 1983; Koenig, 1984; Fujimoto et al., 1986). In fact, stimulated cells develop a million-fold proton gradient across the secretory membrane (Diamond and Machen, 1983). Purified oxyntic cell extracts contain actin, accessory proteins capable of gelating actin, and myosin, which

Received February 13, 1989; accepted February 6 , 1990. Address reprint requests to Cecilia S. Koenig, Unidad de Ultraestructura y Funcion Celular y Tisular, Departamento de Biologia Celular, Facultad de Ciencias Biologicas, Pontificia Universidad Catolica de Chile, Casilla 114-D, Santiago, Chile.

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induces contraction of the gelled extracts (Vial et al., 1979; Koenig et al., 1981; Wolosin e t al., 1983). In these gels, some polypeptides show molecular weights in the range of high molecular weight-ABPs, such as filamin, the HMW-macrophage ABP (Stossel, 1983; Langanger e t al., 1984). Myosin and a filamin-like protein have been located by immunofluorescence at the secretory pole of amphibian oxyntic cells, and the behaviour of this contractile system in vitro has been related to the mechanism responsible for the redistribution of the secretory membranes (Dabike et al., 1986). On the other hand, the mobilization of actin and spectrin associated with the secretory membranes redistribution has been established in rat oxyntic cells (Mercier et al., 1989). Moreover, in amphibian and mammalian oxyntic cells, intermediate filaments (IF) have been localized in relation to the secretory pole (Dabike e t al., 1981; Dabike and Koenig, 1983). A function has been proposed for I F in the generation and maintenance of cell shape, and in the structural organization of the cytoplasm, including cell organelle position (Foisner et al., 1988). The role of cytoskeletal elements in the morphofunctional changes related to the onset of HC1 secretion could be further studied by comparing structural reorganization and immunocytochemical distribution of cytoskeletal proteins in vertebrate oxyntic cells with different morphological arrangements in their secretory pole, but with the same reorganization of the membranous systems, induced by the HC1 secretory process. In bird oxyntic cells, in contrast with what has been shown in mammals and amphibians, the luminal surface rearrangements take place in deep intercellular clefts (Toner, 1963; Vial et al., 1979), and the structural reorganization induced by the onset of HC1 secretion is particularly evident a t the light microscopy level, after cytochemical staining of the secretory membrane marker enzymes (Koenig, 1984; Koenig et al., 1987). Thus, these cells seem to be a n adequate model for studying the relations between the reorganization of the secretory pole membranes and the distribution of intermediate filaments, actin, and actin-associated cytoskeletal proteins. This paper presents a comparison of chicken oxyntic cells in two defined states: a resting secretory state induced by fasting and cimetidine administration, and a histamine-induced HC1 secretory state. The ultrastructural changes undergone by the cells were studied by electron microscopy; distribution of prekeratin, myosin, and filamin, by indirect immunofluorescence; and distribution of F-actin, with FITC-labelled phalloidin as specific probe. MATERIALS AND METHODS

Healthy, adult white Leghorn chickens (Gallus domesticus) weighing 1.5-1.8 kg were used. They were fasted for 24 h before the experiment, with water ad libitum. To obtain resting glands, two doses of cimetidine were injected intraperitoneally (2 mglkg body weight) 15 h and 1 h before cervical decapitation of quiet animals. To obtain HC1 secreting glands, 0.7 mg of histamine-base per kg of body weight was injected subcutaneously, 40 min before decapitation (Koenig, 1984; Koenig et al., 1987). Glandular stomachs were quickly removed. To determine the secreting state of

gastric glands, the pH of gastric contents and lobular duct lumens were measured with pHydrion paper. The pH of the glandular effluent in resting glands was always over 6.0. Electron Microscopy

Small pieces of mucosa were fixed for 6 h a t room temperature, in a solution containing 3% glutaraldehyde, 100 mM KCl, 2 mM MgCl,, 0.25 M sucrose, and 0.1 M buffer PIPES pH 6.9. Glands were washed overnight in 100 mM KC1, 2 mM MgCl,, 0.25 M sucrose, and 0.1 M buffer PIPES pH 6.9, a t 2°C. The tissue was postfixed for 1h in 1%OsO, in 0.1 M cacodylate buffer pH 7.2, block stained in aqueous 1% uranyl acetate, dehydrated in acetone, and embedded in Epon. Thin sections were obtained in a MT-2 Porter-Blum ultramicrotome, counterstained with 2% uranyl acetate and lead citrate, and examined in a Siemens Elmiskop 102 electron microscope. lrnrnunofluorescence Microscopy

Pieces of gastric mucosa were quickly immersed in M-L embedding matrix (Lipshaw) and frozen in liquid nitrogen. Sections of 4 km were stained as described elsewhere (Dabike e t al., 1986). To stain actin filaments, fixed sections were treated for 60 minutes a t room temperature with fluorescein isothiocyanate (F1TC)-labelled phalloidin (Sigma Chem. Co) diluted to a final concentration of 4 pgiml in PBS (Slepecky, 1989). The preparation and characterization of rabbit antisera against bovine hoof prekeratin, human platelet myosin heavy chain, and chicken gizzard filamin have been described elsewhere (Dabike et al., 1981, 1986). The specific antisera were diluted 150. FITClabelled goat antibodies against rabbit IgG (Sigma) were used as second antibodies. Controls consisted of the substitution of the immune sera by preimmune sera, the absorption of immune sera with the purified antigen, and the staining of the sections with FITClabelled antibody only. RESULTS Oxyntic Cell Structure

Resting oxyntic cells display a columnar shape. Their apices, though rather irregular, always show a smooth surface. The junctional complexes lie near the glandular lumen and delimit extremely short intercellular clefts surrounding the luminal cell pole (Fig. 1). These apical junctional complexes are formed by a n occluding tight junction, a prominent belt-like zonula adherens extending approximately 0.5 -0.8 pm along the membrane, and small spot-like desmosomes. The tubular system, formed by tightly packed endocellular membranous tubules, is located at the cell luminal border (Fig. 1).The basolateral cell surfaces are rather straight, with loosely interdigitated short cytoplasmic processes (Figs. 1,5a). The nucleus is a t the base of the cell. The cytoplasm is filled with mitochondria, closely associated with ergastoplasmic cisternae, mostly located a t the supranuclear cytoplasm. Zymogen granules concentrate in this region (Fig. 1). After histamine stimulation, oxyntic cells are still columnar, but their shape has been markedly modified (Fig. 2). The nucleus is displaced toward the prominent secretory pole, over the level of the junctional com-

STRUCTURAL REORGANIZATION OF CHICKEN OXYNTIC CELLS

Fig. 1. Ultrastructure of resting chicken oxyntic cells, in a crosssectioned glandular tubule. The short intercellular clefts (asterisks) are delimited by junctional complexes (arrowheads) near the glandu-

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lar lumen (L). The apical cytoplasm contains the tubular system. Masses of cytoplasmic matrix (arrows) are next to the junctional complexes. X 7,500.

plexes. Mitochondria, associated with ergastoplasm tact with the tubule membranes (Fig. 3a, arrow) and cisternae, are evenly distributed throughout the cyto- connected to the luminal plasma membrane by lateral plasm, while zymogen granules are concentrated in the bridges (Fig. 3, inset). The cell cortex, close to the juncinfranuclear region. Junctional complexes are now tional complexes, is continuous with thick masses of found near the base of the cell delimiting the deep in- cytoplasmic matrix, which are rich in granular and tercellular clefts that encircle the peculiar secretory filamentous elements (Figs. 1, 3b). These masses, conpole of avian oxyntic cells. Over the junctional com- nected to tight junctions and zonulae adherens, seem to plexes, the oxyntic cell cytoplasm shows a n invagina- form a continuous structure in the periphery of the tion that forms a pericellular groove which is continu- cytoplasm. Bundles of microfilaments and I F are seen ous with the intercellular cleft (Fig. 2a,b). The luminal connected to the cytoplasmic dense plaques of the cell surface shows the elaborate arrangement charac- zonula adherens, and centrioles and microtubules are teristic of active HCl secretion. Nevertheless, in frequently found (not shown). In stimulated cells, the masses of amorphous matrix chicken oxyntic cells, these cytoplasmic processes correspond mostly to branched and anastomosed lamellar associated with the junctional complex are not present. cytoplasmic projections which fill the intercellular The components of the secretory pole cytoplasmic maclefts. These are particularly abundant toward the cleft trix are now associated to the luminal processes, to the bottom (Fig. 2a) and in the pericellular groove (Fig. adjacent cytoplasmic border, and to the junctional com2b). Some tubular bundles appear in the apical cyto- plexes. The cytoplasm of the luminal processes conplasm (Fig. 2b). tains sparse 10-20 nm granules, and fine 6-8 nm filIn resting cells, the tubular system is interposed be- aments (Fig. 4). These microfilaments, which run tween the supranuclear mitochondria and the apical parallel to the plasma membrane of the processes, are cell cortex, which appears as a n amorphous matrix ad- occasionally seen connected to the membrane (Figs. jacent to the luminal plasma membrane (Fig. 3). The 4a,b). They penetrate the underlying cytoplasm, and tubules near the cellular surface are embedded in this intertwine with microfilaments running parallel to the matrix (Fig. 3a). Microfilaments are found both in con- cell surface and associated with the zonula adherens

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Fig. 2. Ultrastructure of secreting chicken oxyntic cells. a: Crosssection of a glandular tubule. L; glandular lumen. Two cells are longitudinally sectioned across the central region, and one cell is cut along its periphery. The deep intercellular clefts (asterisks) are delimited by junctional complexes (arrowheads).Cytoplasmic processes

are concentrated a t the bottom of the cleft. x 6,600. b: Longitudinal section of the cortical cytoplasm showing just a cap of nucleus. A pericellular groove (asterisks)separates the apical and basal borders of the intercellular cleft. An enteroendocrine cell is seen ( e ) . x 8,800.

Fig. 3. Secretory pole of resting oxyntic cells. a: At the luminal cell surface. L: glandular lumen. the tubular system is separated from the cell membrane by a cytoskeletal cortex. Microfilaments are associated with the tubule membrane (arrow) and to the luminal plasma membrane (arrow, inset). x 30,000. b Next to the intercellular cleft (asterisk) masses of cytoplasmic matrix associated with the junctional complexes are between the tubular system and the plasma membrane. x 20,000.

Fig. 4. Intercellular cleft and pericellular groove in stimulated ox-

yntic cells. a: Longitudinal section through a pericellular groove basal zone delimited by two junctional complexes. tj, tight junction; za, zonula adherens. Microvillar and lamellar processes fill the lumen. Microfilaments run parallel to membrane processes (arrows);10 nm filaments and microtubules (arrowheads)are found in the subjacent cytoplasm. x 20,000. b: Tangential section through the junctional complex zone (za) beneath the pericellular groove. Microfilaments of the luminal processes intertwine with microfilaments connected to the zonula adherens (arrows); 10 nm filaments lie underneath the microfilaments (arrowhead). x 32,000.

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bution of the staining is observed. The fluorescent line bordering the luminal region of resting cells displaces toward the basal third in stimulated cells (Fig. 7a). Considering the peculiar shape of secreting cells, immunoreactivity in sectioned cells is seen a s fluorescent masses or as semicircular structures in the cytoplasm adjoining the bottom of the clefts (Fig. 7a). At higher magnification, fine fibrillar bundles are seen projecting from the fluorescent ring toward the apical and basal regions of the cell (Fig. 7b). In cross-sectioned cells, the staining appears framing peripheral rings in the basal zone (Fig. 7a, arrow, 7c). These images show that I F are mostly located in the apical peripheral cytoplasm adjacent to the intercellular clefts and in the cortical basal cytoplasm. Distribution of actin

In resting glands stained with FITC-phalloidin, oxyntic cells sectioned along their peripheral cytoplasm show a strong fluorescent continuous line bordering their apices [Fig. 8a,b (arrowhead), c]. In sections through the center of the cells, the immunoreactivity concentrates in masses apparently located a t the level of the intercellular junctions (Fig. 8a,b), while the luminal border appears delimited by a faint, thin fluorescent line (Fig. Sb, arrow). The basolateral cell surfaces are also stained, displaying a punctate pattern (Fig. Sb). In cross-sectioned cells, the peripheral concentration of actin is clearly evidenced; at the luminal border, the polygonal lattice is delimited by a strong fluorescent line (Fig. Sd), while near the cell base Fig. 5. Basolateral borders of chicken oxyntic cells. a: Resting cells. Arrowhead, junctional complex. x 15,000. b: Stimulated cells. Arrow- staining is rather discontinuous (Fig. Sd, arrow). head, junctional complex; asterisk, intercellular cleft. X 15,000. After histamine stimulation, a clear change is observed in the distribution pattern of actin. Staining is conspicuous along the lateral cell borders, while in the luminal cell apices, i t is seen as a faint line (Fig. 9a,b). (Fig. 4b). Filaments of 10 nm also run parallel to the In longitudinal sections through the intercellular cleft, cell surface, and are connected to the cytoplasmic dense the basal zone appears strongly decorated, while a plaques of the zonula adherens (Figs. 4a,b). Centrioles weaker staining is observed toward the apical border. and microtubules are present in the cytoplasm adjacent The basal zone of the cells is deeply stained (Fig. 9b). In to the junctional complexes (Fig. 4a). Microtubules are longitudinal sections through the cell periphery, a also scattered in the cell cytoplasm and in the vicinity strong fluorescent line delimits the basal third of the of the nucleus (not shown). cells, in the region that would correspond to the periA comparison between the structure of the lateral cellular groove (Fig. 9b, arrow). Around the intercellusurface in resting (Fig. 5a) and stimulated cells (Fig. lar cleft, phalloidin-stained actin shows a meshwork5b) shows that, after histamine stimulation, the baso- like distribution (Fig. 9c). Strongly stained polygonal lateral plasma membranes appear deeply folded, pre- arrays are observed in cross-sectioned cells (Fig. 9d). In senting lamellar evaginations interdigitated with contrast with resting cells, the staining of the secreting those of neighbouring cells. cells borders appears fuzzy (Fig. 9b,d). lmmunofluorescence Localization of Cytoskeletal Proteins Distribution of cytokeratin

In resting glands, stained with antiprekeratin antibodies, the luminal border of the oxyntic cells lining the tubules shows a strong fluorescence. In sectioned cells, the staining appears as a bright line, a s fluorescent discontinuous masses (Fig. 6a), or as semicircular structures surrounding the apical borders (Fig. 6b). In tangential sections through the cell apex, staining concentrates in a peripheral ring (Fig. 6c). At the same time, decorated fine fibrillar bundles project from the apical ring to the basal cytoplasm (Fig. 6a,d). The columnar cells of the mucous epithelium are strongly stained (data not shown). In secreting glands, a striking change in the distri-

Distribution of myosin and filamin

In resting glands, the use of antimyosin and antifilamin antibodies shows that myosin and filamin co-localize a t the glands’ luminal border. The staining is seen as a fine discontinuous line or as discrete fluorescent aggregations, bordering the apical pole of the sectioned oxyntic cells (Fig. 10a, 12a).Tangential sections through the cell luminal pole show that these proteins form an apical peripheral ring (Figs. lob, 12b). In addition, antifilamin antibodies decorate the basal margin of the cells (Fig. 12c). Strongly stained elements, probably muscle cells, are seen in the interglandular connective tissue. The mucous cells of the lining epithelium do not stain (data not shown). In secreting glands, a clear redistribution of the im-

STRUCTURAL REORGANIZATION OF CHICKEN OXYNTIC C E L L S

Figs. 6, 7. Immunofluorescence staining with antiprekeratin antibodies. L, lumen; arrowheads, stroma. Flg. 6. Resting glands. In longitudinal sections (a),the staining concentrates in the luminal border of the cells. In tangential sections (b),fine fluorescent lines are seen embracing the apical cell border. In transversal sections through the luminal pole (c), the organization of immunoreactivity in a polygonal lattice is clearly visualized. Note that a fine fibrillar meshwork projects from the apical fluorescent line toward the basal cytoplasm (a,d).a-c, x 880; d, x 1,400.

munoreactivity is observed. Staining concentrates in the basal third of sectioned cells, framing either a fine fluorescent line, or discontinuous masses (Figs. 1la, 13a). In sections where displacement of the intercellular junctions is evident, myosin and filamin concentrate a t the bottom of the intercellular clefts, in the adjoining cytoplasm (Figs. l l b , 13b,c), framing a polygonal lattice (Figs. l l c , 13d). These changes in the localization of antimyosin and antifilamin antibodies are similar to those observed in the peripheral cytoskeletal ring stained with antiprekeratin antibodies. In both secreting states, myosin and filamin are sparsely distributed in the rest of the cytoplasm. DISCUSSION

Our results on the ultrastructure of resting and stimulated cells (Fig. 14) show that, in chicken oxyntic cells, the changes in shape related to the secretory state are more complex than those previously described by Toner (1963), Vial and Garrido (1979), and Vial et al. (1979). In contrast with what has been reported for resting

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Flg. 7. Secreting glands. Small arrowheads, luminal cell border. In sectioned cells the staining is seen as a continuous dense line, discrete fluorescent masses (a,b),or semicircular structures delimiting the basal third of the cells (c). Higher magnifications show that the immunoreactive material is located in the cytoplasm adjoining the bottom of the clefts (arrows in b). Fine fibrillar bundles are observed radiating from the fluorescent line toward the apical and basal regions of the cells (a,b).In cross sections through the basal cytoplasm the stain is seen as a polygonal array (c, arrow in a). a,c, x 880; b, x 1,400.

cells, the tubular system is concentrated a t the luminal border, and the intercellular clefts are very short. Moreover, the junctional complexes are associated both with the masses of cytoskeletal matrix continuous with the luminal cell cortex, and with the peripheral filamentous cytoskeletal ring. The absence of the deep intercellular cleft which we observed in non-secreting cells may be attributed to the use of more adequate conditions for obtaining the resting state. In chickens fasted for 24 h we observed some variability among cell structures, but most of them had a deep and smooth intercellular cleft. In these conditions, however, there was a basal acid production amounting to about 2 5 4 of the maximal secretion obtained after histamine stimulation (Long, 1967; Gonzalez, personal communication). When the resting state was induced by fasting, blocking of histamine H2-receptors, and avoiding chicken stress (Burhol, 1982), the gastric gland effluents always had a pH higher than 6.0. After this treatment, nearly 1 0 0 8 of the oxyntic cells examined showed a short intercellular cleft (Koenig, 1984; Koenig et al., 1987).On the other hand, when the rest-

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Figs. 8,9.Actin staining with FITC-phalloidin. L, lumen; arrowheads, stroma. Fig.8.In sections ofresting glands, fluorescence concentrates in the luminal border of oxyntic cells as a continuous sheet (a,c)or as discontinuous masses; a thin line delimits the luminal cell border (arrow in b and d);a punctate staining is present in the basolateral region cell border (a,b,c).Cross-sectioned cells clearly show the peripheral concentration of actin (d). a,d, x 880; b, x 1,400; c, x 1,200.

ing state was induced by anoxia or o-DNP (Vial and Garrido, 1979; Vial et al., 19791, the cells could reorganize the tubular system in the absence of metabolic energy, but no modification was observed in their intercellular clefts. Stimulated oxyntic cells display a prominent secretory pole characterized by a remarkable increase in secretory cell surface and displacement of the junctional complexes toward the basal zone. Our results in chicken cells show that the cytoplasmic processes of the secretory surface, formed by insertion of the tubular system into the luminal membrane (Koenig, 1984),are concentrated in the pericellular groove. The microfilaments of these processes are associated with the microfilaments adjacent to the zonula adherens. At the same time, a change occurs in the supramolecular organization of actin, apparently similar to that described in pigeon (Vial et al., 19791, and in other vertebrate oxyntic cells, using heavy meromyosin (HMM)decoration (Vial and Garrido, 1976). The outcome of this structuraI rearrangement is to form a well-defined expanded pericellular zona that contains the H ' pump. In this compartment, circumscribed by the secretory

Fig. 9.Cross-sections of stimulated glands show F-actin concentration in the lateral border of oxyntic cells (a,b).In longitudinal sections through the cell periphery a strong fluorescent line is seen located in the basal third of the cells ( c , arrows). In cross-sectioned cells, the staining shows a fuzzy aspect (d). Small arrowhead, luminal cell border. a,d, x 880; b,c, x 1,200.

membrane, the composition of the luminal fluid would be different from the secretion in the glandular tubule. The importance of a restricted canalicular lumen for the ionic exchanges involved in HC1 secretion is quite clear in mammalian oxyntic cells (Diamond and Machen, 1983). This organization of the secretory pole might be significant, since chickens secrete more HC1 per gram of glandular stomach than any other vertebrate (Long, 1967). The remarkable difference in the immunocytochemical localization of cytoskeletal proteins in resting and stimulated chicken cells shows that histamine induces a massive mobilization of prekeratin, F-actin, myosin, and filamin. This is concomitant with the incorporation of the intracellularly stored secretory membranes to specific zones of the cell surface. In resting cells, F-actin is clearly concentrated a t the luminal cell pole. The distribution of the FITC-phalloidin staining agrees well with that of the amorphous matrix located a t the sub-luminal plasma membrane cortex and next to the junctional complexes. These cytoskeletal matrices would contain actin microfilaments, since phalloidin stains small actin polymers

STRUCTURAL REORGANIZATION OF CHICKEN OXYNTIC CELLS

Figs. 10, 11. Immunofluorescence staining with antimyosin antibodies. L, lumen; arrowheads, stroma.

Fig. 10. Resting glands. In longitudinally sectioned glands (a),the fluorescence concentrates in the luminal border of the cells. In tangential sections through the apical cytoplasm (h), the decoration forms a polygonal lattice. Strongly stained elements, probably smooth muscle cells, are between the glandular tubules. x 880.

forming a meshwork of randomly oriented short filaments, which are not well preserved during processing for electron microscopy (Slepecky, 1989). The presence of HMM decorated microfilaments in the subplasmalemma1 zone of the luminal pole of resting avian oxyntic cells has been previously reported (Vial et al., 1979). The localization in chicken resting oxyntic cells of a filamin-like protein, concentrated in a peripheral ring next to the junctional complexes, suggests that this HMW-ABP (Weeds, 1982; Craig and Pollard, 1982; Stossel, 1983) is associated with the luminal actin network (Dabik6 et al., 1986). In stimulated cells, F-actin maintains its association with the cell secretory pole. In FITC-phalloidin stained cells a thin fluorescent line is seen in the upper cell border, while a marked concentration is observed toward the deeper zone of the intercellular cleft, where the luminal cell processes are more abundant. Neither filamin nor myosin are present in this region of the intercellular clefts. Therefore, the actin cytoskeleton present in the processes of the secretory surface of stimulated cells would exclude filamin and myosin as associated proteins. In resting and stimulated cells, filamin and myosin co-localize with cytokeratin and actin in a narrow peripheral cytoskeletal ring, apparently located a t the level of the junctional complexes, since the cytoplas-

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Fig. 11. Stimulated glands. Small arrowhead, luminal cell border. In longitudinally sectioned cells (a), the fluorescence concentrates in a band or in discrete masses toward the basal region of the cells. A fine fluorescent line is seen in the cytoplasm adjoined to the bottom of the clefts (h). In sections across the basal third of the cells, the fluorescence concentrates a t the cell periphery framing a polygonal lattice (c).The rest of the cytoplasm stains diffusely. x 880.

matic plaques of the zonula adherens are associated with filamentous bundles containing microfilaments and 10 nm filaments. The deep intercellular clefts seem to be formed by a displacement of the junctional complexes toward the cell base through a complicated cellular movement that has not been described in other vertebrate oxyntic cells. This involves the protrusion of the apical cytoplasm and the folding of the basolateral cell surfaces, with a relocalization of tubular system membranes and other organelles. Through these changes, the junctional complexes seem to keep their position in the plasma membrane. A circumferential contractile actomyosin ring has been described in at least two types of highly polarized non-glandular epithelial cells (Owaribe et al., 1981; Owaribe and Matsuda, 1982; Klotz et al., 1988). Chicken retinal pigmented cells display thick circumferential microfilaments bundles associated with the basally displaced zonula adherens (Sandig and Kalnis, 1988).At the same time, in intestinal epithelial cells, a circumferential contractile band of microfilaments enriched with myosin, filamin, and other actin-associated proteins, is attached to the adherens junction (Bretscher and Weber, 1978; Hull and Staehelin, 1979; Hirokawa e t al., 1982, 1983; Mooseker, 1983, 1985). The significance of the actomyosin ring and its contractility are not known, but the fact

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Figs. 12, 13. Immunofluorescence staining with antifilamin antibodies. The distribution of immunoreactivity in resting and stimulated cells is similar to that observed using antimyosin antibodies. L, lumen; arrowheads, stroma.

Fig. 13. Stimulated glands. Small arrowhead, luminal cell border. a: Cross-sectioned glands. b,c: Sectioned cells showing the fluorescent line a t the bottom of the intercellular cleft. d Section across the basal third of the cells. x 880.

Fig. 12. Resting glands. a: Cross-sectioned glands. b: Tangential section through the luminal zone of the cells. c: Longitudinal sections showing the basal cell margin. a,c, x 880; b, x 1,400.

Fig. 14. Diagram summarizing the main structural differences between resting and stimulated chicken oxyntic cells. In resting cells, the junctional complexes near the glandular lumen and the intercellular cleft are short; the tubular system occupies the apical cell border and the basolateral cell surface is rather smooth. In stimulated cells, the junctional complexes located toward the cell base and the intercellular cleft are deep; the luminal secretory processes are concentrated in a pericellular groove a t the bottom of the cleft, and the basolateral cell surface is markedly folded (for details, see text).

that it is present in epithelial cells performing quite different functions suggests a structural role in preserving and stabilizing the cellular pattern of all

simple epithelia (Volk and Geiger, 1986). In chicken oxyntic cells, the cytoskeletal ring containing myosin and filamin could stabilize the structure of resting cells. Stimulation could induce the participation of the cytoskeletal ring in framing the pericellular groove, either by mediating its contraction, or by acting as a n organizing center for the F-actin polymerization associated with the luminal processes membrane (Vial and Garrido, 1976; Vial e t al., 1979; Volk and Geiger, 1986). On the other hand, membranes of the secretory pole could be mobilized by the interaction of actin microfilaments associated with the secretory membranes with the myosin present in the actomyosin ring. In amphibian oxyntic cells, in which the reorganization of the secretory pole membranes occurs in the luminal border of the cells, the apical myosin and filamin containing cytoskeletal ring maintains its position in resting and secreting cells (Dabike et al., 1986; Herman e t al., 1981). The translocation of the cytoskeletal ring to the cell basal zone could be mediated by its interactions with other cytoskeletal components, such a s the F-actin associated with the basolateral borders, and the IF present in the cortical cytoplasm. It has been proposed that microtubules participate in HC1 secretion (Kas-

STRUCTURAL REORGANIZATION OF CHICKEN OXYNTIC CELLS

bekar and Gordon, 1979; Tsunoda and Mizuno, 1985). The vicinity of centrioles and microtubules with the junctional complexes suggests that they could also play a role in the framing of the length of the deep intercellular cleft. In this context, i t has been shown that contraction of teleost retinal cones requires the concomitant depolymerization of microtubules (O’Connor and Burnside, 1982). The distribution of cytokeratin in chicken oxyntic cells suggests t h a t this system contributes toward stabilization of cell components not engaged in the membranous rearrangements, since they form a filamentous network projecting from the peripheral ring toward the base of resting cells, and toward the apical and basal zone of secreting cells. This is consistent with findings in mammalian oxyntic cells showing that IF concentrate beneath the intracellular canaliculus of stimulated cells, enclosing the secretory membranes (Dabike and Koenig, 1983); and in amphibian oxyntic cells, in which I F form a basket-like structure that surrounds the luminal secretory membranes (Dabike et al., 1981). Black et al. (1982) reported that cytochalasin B produces a n inhibition of HCl secretion accompanied by ultrastructural modifications, including collapse of the canalicular and glandular lumina and gross disorganization of the microfilamentous system. These results agree with the idea that microfilaments are involved in the translocation of the tubular system to the apical surface and t h a t their disorganization may impair the changes in shape of oxyntic cells (Vial and Garrido, 1976; Vial e t al., 1979). However, cytochalasin B might interfere with HC1 secretion by acting on the two microfilament systems present in the luminal pole. This could be achieved, first, by disruption of microfilaments associated with the cytoplasmic processes in secreting cells, directly involved in the translocation of the tubular system; and second, by disorganization of the cytoskeletal ring that could constitute a structural framework for the cell luminal pole. Undoubtedly, the integrity of the epithelial sheet is essential for the ultrastructural changes and the maintenance of HC1 secretion. At the same time, until the exact role of the cytoskeletal peripheral ring is established, we cannot discard a direct participation of this structure in the translocation of membranes, so that any alteration of its integrity would affect the structural modifications induced by secretagogues. The displacement of the cytoskeletal ring in relation to the chicken oxyntic cells’ secretory cycle points to these cells as a valuable tool for studying the relation of this ring with the other cytoskeletal components, i.e., actin associated with the secretory pole and actin present in the basolateral cell surface, the IF cytoskeleton, and microtubules. It would be interesting to find out how alterations in the cytoskeletal ring interfere with the morphofunctional changes undergone by oxyntic cells during their secretory cycle. ACKNOWLEDGMENTS

The excellent technical assistance of Alejandro Munizaga is gratefully acknowledged. This work was supported by the Fondo de Investigaciones de la Pontificia Universidad Catolica de Chile.

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