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an immunogold electron-microscope study. Jacqueline S. Weinman, Jacqueline M. Feinberg, Dominique P. Rainteau, Bruno Della Gaspera, Serge J. Weinman.
Cell Tissue Res (1994) 278:389-397

Cell&Tissue Research 9 Springer-Verlag 1994

Annexins in rat enterocyte and hepatocyte: an immunogold electron-microscope study Jacqueline S. Weinman, Jacqueline M. Feinberg, Dominique P. Rainteau, Bruno Della Gaspera, Serge J. Weinman D~partement de Biochimie,UFR Biom~dicaledes Saints-P6res, Universit~ Ren~ Descartes, 45 Rue des Saints-P~res, F-75270 Paris Cedex 06, France Received: 2 September 1993/Accepted: 30 March 1994

Abstract. In the present study, immunogold labeling of ultrathin sections of rat small intestine and liver has been used to obtain insights into the ultrastructural localization and possible functions of annexins. In enterocytes, annexins II, IV, and VI are found at the periphery of the core of each microvillus and of the rootlets, but are absent from the interrootlet space. Annexins II, IV, and VI are also observed close to the interdigitated plasma membrane. In hepatocytes, only annexin VI is found to be concentrated within the microvilli in the bile canaliculi, on the inner face of the sinusoidal cell surface, particularly in the space of Disse, and all along the plasma membrane. Annexin VI is also detected in mitochondria of enterocytes and hepatocytes. These localizations are in agreement with the concept of a close calcium-dependent association of annexins with membranes and cytoskeletal proteins, particularly with actin. Moreover, they support the hypothesis of an involvement of annexins in exocytotic and endocytotic processes, which take place in epithelial cells. Key words: Annexins - Actin - Enterocyte - Hepatocyte - Immunogold electron microscopy - Rat (Sprague Dawley)

Introduction Within the past decade, the search for intracellular calcium sensors has led to the identification of a new family of calcium-binding proteins, viz., the annexin family. Annexins are closely related water-soluble proteins that associate reversibly with membrane phospholipids in a Ca2§ manner (for a review, see Moss 1992). Members of this family are widely distributed in a variCorrespondence to: Dr. S. Weinman

ety of tissues. Six annexins have been found in the intestinal mucosa. Annexins II and IV have been isolated from membrane vesicles derived from porcine intestinal brush border (Gerke and Weber 1984; Shadle et al. 1985). Subsequently, annexins I1 to VI have been purified from bovine intestinal mucosa (Pepinsky et al. 1988). In addition, immunolocalization of annexins II and IV has been performed in enterocytes at the lightmicroscopic level. Annexin II has thus been detected in the terminal web (Gerke and Weber 1984; Gould et al. 1984), and annexin IV has been found to be associated with the basolateral membrane (Massey et al. ] 991). Recently, a novel intestine-specific N-myristoylated annexin has been characterized and shown to be associated with the plasma membrane of undifferentiated proliferating crypt epithelial cells, and with differentiated villus enterocytes. In polarized enterocytes, the highest concentrations of this intestine-specific annexin are found at the apical membrane (Wice and Gordon 1992). Annexins II, IV, and VI have been characterized in porcine liver by Shadle et al. (1985). Small amounts of annexins I and II have been detected in rat hepatocytes (Karasik et al. 1988). Annexins III and IV are also found at low concentrations in rat liver whereas annexin V is present at higher levels (Kaetzel et al. ] 989). In contrast, annexin VI is strongly expressed (Mathew et al. 1986; Smith and Dedman 1986). Thus, annexin VI appears to predominate in rat liver. The role of annexins is not fully understood (Moss 1992). Knowledge of their location might clarify their functions with respect to their interaction with the cytoskeleton and the cell membrane. We have therefore determined the subcellular localization of annexins in two different kinds of polarized epithelial cells, viz., enterocytes and hepatocytes, and compared it with the localization of actin, a major cytoskeletal protein. Ultrastructural immunogold studies have been performed using glutaraldehyde-fixed rat small intestine and liver embedded in Lowicryl K4M.

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Materials and methods

Animals and specimens Young adult female rats (Sprague Dawley; 250 g) were anesthetized with ketamine (Parke-Davis, France; 15 mg/kg, intraperitoneal). For Western-blot analysis, samples of the small intestine and liver were removed and processed immediately. Alternatively, for immunocytochemistry, samples were obtained from rats flushed by an in vivo perfusion with 2.0% formaldehyde, 0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 37~ C.

Chemicals and reagents All chemicals were of the highest purity available and purchased from: Merck (Darmstadt, Germany), Fluka (Buchs, Switzerland), Sigma (St. Louis, Mo.), Bio-Rad (Richmond, Calif.) for polyacrylamide gel electrophoresis reagents and nitrocellulose membranes; Ladd (Burlington, Vt.) for glutaraldehyde; and Chemische Werke Lowi (Waldkraiburg, Germany) for Lowicryl K4M. Goat anti-rabbit, goat anti-mouse, and rabbit anti-sheep peroxidase-conjugated IgG antibodies were from Pasteur Diagnostics (Marne la Coquette, France), Dakopatts (Glostrup, Denmark), and Nordic (Tilburg, Netherlands), respectively. Colloidal-gold(5 or 10 nm)-labeled secondary antibodies were purchased from Janssen (Beerse, Belgium).

Antibody production and purification Annexins I and II, and affinity-purified antibodies to annexins I and II were prepared according to Glenney et al. (1987). Annexins III, IV, and V were purified from rat liver according to Kaetzel et al. (1989), and annexin VI according to Mathew et al. (1986). Antibodies to annexins III-VI were raised in sheep, affinity-purified according to Dedman et al. (1978), and controlled by cross-adsorption according to the method described by Smith and Dedman (1986). Two monoclonal mouse antibodies to actin, viz., C4 and HUC 1-1, were isolated and characterized as described by Lessard (1988) and Sawtell et al. (1988). The C4 monoclonal antibody was raised against chicken gizzard actin. It reacts with all known vertebrate actins, plant actin, and slime mold actin. It has no known spurious reactivities. The HUC 1-1 monoclonal antibody was raised against human vascular smooth muscle actin (umbilical artery). It reacts with all mammalian muscle isoactins, and has little or no reactivity toward the cytoplasmic actins. Actin from chicken gizzard (Sigma) was used for control experiments in Western blot analysis and immunoelectron microscopy.

liver were homogenized in 2,5 vol 4 mM EDTA, 100 mM 2-mercaptoethanol, 20 mM phosphate buffer, pH 6.8 (buffer A), containing 0.2 mM phenylmethylsulfonylfluoride, 2 gg/ml antipain, 1 gg/ ml pepstatin, 1 lxg/ml leupeptin, and 10 gg/ml aprotinin as antiproteolytic agents. The suspension was centrifuged at 30000 g for 30 rain. The supernatant was referred to as the "soluble fraction" as proteins contained in this extract were directly soluble in buffer A. Subsequently, the pellet was extracted in 2.5 vol buffer A containing 10% SDS, and was sedimented. The supernatant was referred to as the "particulate fraction" as proteins present in the second extract were soluble in buffer A only in the presence of SDS. Soluble and particulate proteins (100 gg) were electrophoresed on a 13 % polyacrylamide gel (Laemmli 1970) and transferred to a nitrocellulose membrane (Towbin et al. 1979).

Immunoelectron microscopy Samples of small intestine and liver of rat perfused in vivo were removed and fixed at 4~ C for 1 h in 0.5% glutaraldehyde, 5 mM CaCI> 0.1 M cacodylate buffer, pH 7.2, embedded in Lowicryl K4M, and processed for immunogold labeling, as described by Weinman et al. (1986), using the primary antibodies at an IgG concentration of 25-50 gg/ml and the appropriate gold (5 or 10 nm)-labeled secondary antibodies at a dilution of 1:50. The specificity of immunolabeling was assessed by comparison with labeling patterns obtained from antibodies preabsorbed with a 10 M excess of the complementary antigen. Gold grids were covered with a carbon film, and sections were viewed in a Philips EM 300 electron microscope operated at 80 kV. All immunocytochemical localizations shown in this paper were performed at least four times using independent preparations from different animals. Similar patterns were observed, and the trends and ratios were the same in each case.

Results

Specificity of antibodies As Shown in Fig. 1, the affinity-purified a n t i b o d i e s to a n n e x i n s I - V I a n d the m o n o c l o n a l a n t i b o d i e s to actin reacted only with the c o r r e s p o n d i n g antigen. N o other cross-reactive p o l y p e p t i d e b a n d s were detected b y immunoblotting.

Identification of annexins and actin in rat small intestine epithelium and liver

Specificity of antibodies The specificity of the purified antibodies to annexins and monoclonal antibodies to actin was examined by Western-blot analysis of a mixture of purified annexins and actin (15 ng each). The proteins were resolved by SDS polyacrylamide gel electrophoresis, (Laemmli 1970), transferred to a nitrocellulose membrane (Towbin et al. 1979), and reacted individually with an antibody to annexins I, II, III, IV, V, or VI, or to actin. All antibodies were detected with an appropriate second antibody by standard methods.

Western-blot analysis of biological samples The small intestine was excised and washed with an ice-cold buffer that contained 137 mM NaC1, 1.5 mM KC1, 1.5 mM KH2PO4, 8.1 mM NaaHPO~, pH 7.2. The epithelium was detached from the smooth muscle by gentle scraping. Intestinal epithelial cells and

W e s t e r n - b l o t analysis o f the soluble a n d p a r t i c u l a t e extracts o f the e p i t h e l i u m o f rat small intestine (Fig. 2) a n d liver (Fig. 3) was p e r f o r m e d p r i o r to the i m m u n o c y t o c h e m i c a l analysis described below. I n i n t e s t i n a l epithelium, there was n o reactivity with a n t i - a n n e x i n I. T h e a n t i b o d i e s to a n n e x i n s recognized o n l y a single p r o t e i n b a n d at 3 6 - 3 2 k D a for a n n e x i n s I I - V a n d at 67 k D a for a n n e x i n VI in b o t h soluble a n d p a r t i c u l a t e fractions. The a m o u n t s o f a n n e x i n s III a n d V were low; these p r o t e i n s were barely detectable. A n n e x i n IV was f o u n d m a i n l y in the p a r t i c u l a t e fraction. T h e C4 a n d H U C 1-1 m o n o c l o n a l a n t i b o d i e s to actin b o u n d o n l y to a polypeptide b a n d o f 46 k D a in b o t h soluble a n d p a r t i c u l a t e fractions. I n liver, the a n t i b o d i e s to a n n e x i n VI a n d the m o n o c l o n a l a n t i b o d y C4 to actin each reacted with o n l y

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Figs. 1-3. 1 Western-blot analysis of the specificity of the antibodies against annexins I-VI, and of the C4 and HUC 1-1 monoclonal antibodies against actin. A mixture of annexins I VI and actin was resolved by SDS gel electrophoresis, and reacted with an antibody to annexins I, II, III, IV, V, or VI, or to actin. Each antibody reacted only with the corresponding antigen. 2 Demonstration of the presence of annexins II-VI and actin in the epithelium of the rat small intestine. Purified annexins I-VI or actin (lanes 1) were co-electrophoresed with soluble (lanes 2) and particulate (lanes 3) extracts (100 gg protein) from the epithelium of the rat small intestine, and reacted with the antibodies against annexins I-VI (anti-AI

one band, at 67 k D a and 46 k D a , respectively, in b o t h soluble and particulate fractions. N o other cross-reactive polypeptide b a n d s were detected by i m m u n o b l o t t i n g with the antibodies utilized in this study.

SubcelIular distribution of annexins and actin in rat small intestine enterocytes In the apical area o f the enterocytes, the a n t i b o d y to annexin II labeled the microvilli and the rootlets (Fig. 4a). In cross-sections o f the brush border, gold particles were f o u n d essentially at the periphery o f the core

to anti-AVI) and the C4 and HUC 1-1 monoclonal antibodies against actin (anti-actin C4, HUC 1-1). Antibodies bound specifically to the corresponding antigens present in both the soluble and the particulate extracts from small intestine epithelium. Mr Relative molecular mass. 3 Western-blot analysis of the specificity of the antibodies against annexin VI (anti-AVI) and actin (antiactin C4) in rat liver. Annexin VI or actin (lanes 1) were co-electrophoresed with soluble (lanes 2) and particulate (lanes 3) extracts (100 gg protein) from rat liver. The antibodies bound specifically to the corresponding antigens present in both the soluble and the particulate extracts. Mr Relative molecular mass

o f each microvillus (Fig. 4b). Their density was higher in rootlets t h a n in microvilli (Table 1). The label observed in the terminal web space between the rootlets was negligible; m o s t o f the gold particles were associated with the rootlets, whereas only a few o f t h e m were encountered in the inter-rootlet space (Fig. 4b). Spot desm o s o m e s were also reactive (Fig. 4a, c). Slight i m m u n o labeling was o b t a i n e d with the a n t i b o d y to annexin IV (Fig. 4 d, e). It was present along the p l a s m a m e m b r a n e o f microvilli, where gold particles were r a n d o m l y distributed. The a n t i b o d y to annexin VI b o u n d to the periphery o f the core o f the microvilli and rootlets (Fig. 4f, g). Label was m o r e p r o m i n e n t a r o u n d the rootlet core (Ta-

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Fig. 4a-n. Immunogold electron micrographs of adult rat enterocytes using antibodies against annexin II (a-e), annexin IV (d, e), annexin VI (f-h), C4 monoclonal antibody against actin (i, k, 1), and HUC 1-1 monoclonal antibody against actin (m, n). a, d, f, i, m Longitudinal sections of both the microvilli (MV) and the

rootlets inside the terminal web (TW). b, e, g, 1, n Cross-sections of both the microvilli and the rootlets, e, h, k Spot desmosomes. D Spot desmosomes; z o zonula occludens; z a zonula adherens. x 14700. B a r : 0.5 pm

393 Table 1. Morphometric quantitation of an-

nexin II, annexin IV, annexin VI, and actin localization in enterocyte and hepatocyte

Cell type

Enterocytes Microvilli longitudinal section cross-section Rootlets longitudinal section cross-section Lateral membrane Mitochondria Cytoplasm apicalportion basalportion Nucleus Hepatocyte Bile canaliculi Lateral membrane Sinus Mitochondria Cytoplasm Nucleus

Staining intensity (gold particles"/gm a -+SEM) Ann II

Ann IV

Ann VI

Actin C4

44+ 4 46-+ 4

8_+2 5+1

106-+ 8 46-+ 4

262-+20 201 -+ 17

76_+6 36_+4

118__+ 9 148-+11 66-+ 5 15 -+ 2

11-+2 18-+2 27 -+3 9 -+1

6 3 2 - + 4 6 434+_37 332___27 376-+29 154-+ 11 78 -+ 8 134 -+20 22 +_ 3

116_+9 80_+6

10-+ 1 10-+ 2 15-+ 2

8+_1 2_1 15-+2

29+ 3 62-+ 5 38+ 4

8-+ 2 4-+ I 5-+ 1

302-1-27 250-+ 22 334-+ 29 97-+ 8 66-+ 7 28 -+ 4

130-+11 87 + 9 105 -+ 11 17-+3 6-+ 2 4-+ 2

Actin HUC 1-1

a Gold particle diameter: 10 nm for annexin I1, annexin IV, actin C4, and actin HUC 1-1 ; 5 nm for annexin VI

ble 1). Spot desmosomes were unreactive. In the apical portion of the enterocytes, the C4 antibody to actin (Fig. 4i, k, 1) displayed immunogold labeling within microvilli and rootlets. The gold particle density was higher in the rootlets than in the microvilli (Table 1). Labeling was also found in the cytoplasm adjacent to apical cell junctions, including the zonula occludens and zonula adherens. No label was detected in association with desmosomes. The H U C 1-1 antibody to actin (Fig. 4m, n) showed labeling of the core, from the tip of the microvilli to the base of the rootlets. It was more prominent within the rootlets than the microvilli (Table 1). The inter-rootlet space was reactive neither to C4 nor to H U C 1-1 antibodies. The interdigitated part of the lateral plasma membrane was the site of significant immunolabeling by the antibodies to annexins II, IV, and VI, and by the C4 antibody to actin (Fig. 5 a, b, c, d, respectively, Table 1), whereas the cytoplasm was unreactive. The type II plaques (Drenckhahn and Franz 1986) were heavily labeled by the antibody to annexin VI (Fig. 5 c). In the basolateral area of the enterocytes, sparse gold labeling was obtained with the antibody to annexin II (Fig. 5 e). In contrast, the plasma membrane, the cortical cytoplasm (Fig. 5 f), and the crests of the mitochondria (Fig. 5 g) showed intense labeling (Table 1) with the antibody to annexin VI. No significant labeling was detected over the nucleus with either of these antibodies. The C4 antibody to actin labeled the entire basal membrane and the adjacent cytoplasm (Fig. 5 h).

Subcellular distribution of annexins and actin in rat hepatocytes Immunostaining with the antibody to annexin V was diffuse in the cytoplasm in hepatocytes. No specific labeling could be detected (data not shown). As shown in Fig. 6 and Table 1, immunoreactivity with the antibody to annexin V! was intense in the bile canalicular microvilli, along the lateral plasma membrane (Fig. 6 a), and on the inner face of the basal sinusoidal cell surface, particularly in the processes in the space of Disse (Fig. 6b). Mitochondria were also labeled. The immunolabel of hepatocytes obtained with the C4 monoclonal antibody to actin (Fig. 6c, d) was prominent within the bile canalicular microvilli and all along the lateral plasma membrane, the basal sinusoidal cell surface, and the perisinusoidal space of Disse.

Discussion

In this study, Western-blot analysis has demonstrated the presence of annexins II, III, IV, V, and VI in rat enterocytes; annexin VI is the major annexin in rat hepatocytes. These findings are reinforced by unpublished results from our laboratory showing the presence of the corresponding m R N A s in these cells. Moreover, immunogold labeling with specific antibodies has allowed a comparison of the ultrastructural localization of annexins and actin associated with the subcellular organelles of enterocytes and hepatocytes. As annexins are thought to be involved in the promotion of membrane contacts (Creutz 1992), the formation or modulation of ion channels (Pollard et al. 1992; Diaz-Munoz

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Fig. 5 a-h. Immunogold electron micrographs of adult rat enterocytes using antibodies against annexin II (a, e), annexin IV (b), annexin VI (e, f, g), and C4 monoclonal antibody against actin

(d, h). a-d Interdigitated lateral plasma membrane (m). e, f, h Basolateral area of the enterocyte, g Mitochondria. M Mitochondria; Nnucleus; cyt cytoplasm, x 15000. Bar: 0.5 gm

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Fig. 6a-d. Immunogold electron micrographs of adult rat hepatocytes using antibodies against annexin VI (a, b) and C4 monoclonal antibody against actin (e, d). BC Bile canaliculus; m plasma mem-

brane; S sinus; PS perisinusoidai space of Disse; M mitochondria; N nucleus; end endothelial cell. • 12600. Bar: 0.5 gm

et al. 1990), and the organization or membrane attachment of cytoskeletal elements (Glenney et al. 1987), we now wish to consider the possible physiological relevance of the distribution of annexin and actin in enterocytes and hepatocytes.

The core is bound at its periphery to the brush border myosin-I-calmodulin (Cheney et al. 1993; Weinman et al. 1994), forming a system capable of mechanochemical activity (Zot et al. 1992; Wolenski et al. 1993). This complex might be tethered to the membrane by annexins II, IV, and VI, whose role would be to orient actin filaments, myosin-I, and membrane phospholipids so as to support motility (Zot et al. 1992). The core of the rootlets is composed of the same two isoactins. As in the microvilli, these actins may well be organized by annexins II and VI. Such a scheme is consistent with the actin localizations presented by Hagen and Trier (1988), and Drenckhahn and Dermietzel (1988). It is also in full agreement with the results of Sawtell et al. (1988), who have demonstrated that the enterocyte brush border contains two unique isoactins that share features of nonmuscle and muscle isoactins. It is also in accord with the proposed role of annexins in linking cytoskeletal elements to membranes (Creutz 1992). Moreover, as villin crosslinks the actin filaments to form bundles at low Ca a+ concentrations and severs them into short fragments at high Ca 2 + concentrations, the preferential localization of annexins II and VI, and their Ca 2 +-buffering capacity may help to explain the known resistance of the basal portions of the core to Ca 2 § solutions, even at high Ca 2 + concentrations (Mooseker 1985).

Enterocytes Microvilli and rootlets. For the first time, the presence

and co-localization o f two isoactins with annexins II and VI have been demonstrated within microvilli and rootlets by immuno-electron microscopy. The four proteins extend over the entire length of these organelles, from the tips of the microvilli to the bases of the rootlets. Actin is localized within the core, in microvilli and in rootlets. Its density, as revealed by the C4 and H U C 1-1 monoclonal antibodies, is higher in the rootlets than in the microvilli. Annexins II and VI are found at the periphery of the actin filament bundles. They are more concentrated in the rootlets than in the microvilli. These findings allow a new scheme for the structure of the microvilli and the rootlets to be proposed. The microvillous core consists of a bundle of filaments composed of two isoactins, a/~- and a 7-species, that both contain the C4 and the H U C 1-1 epitopes (Sawtell et al. 1988).

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Plasma membrane. A striking feature in the enterocytes is the distribution of annexins II, IV, and VI close to the plasma membrane, where they are co-localized with actin, as demonstrated in this paper, and fodrin, as shown by Gould et al. (1984), Gerke and Weber (1984), Drenckhahn and Bennett (1987), and K o o b et al. (1987). This finding affords an additional illustration of the calcium-dependent binding of annexins to cytoskeletal proteins (Glenney 1986; Cheney and Willard 1989). Hepatocytes Hepatocytes are unique a m o n g the simple epithelia studied so far in that only annexin VI appears at a significant level, and in a non-polarized fashion at apical (canalicular), lateral and basal (sinusoidal) cell surfaces, in contrast to other epithelial cells. Thus, in enterocytes, other annexins, such as annexins II and IV (this paper), and intestine-specific annexin (Wice and G o r d o n 1992), are associated with annexin VI to form a complex network with the cytoskeletal proteins, essentially at the apical p a r t of the cell. In secretory ameloblasts, annexin VI is restricted to the apical Tome's processes (Goldberg et al. 1991). Nevertheless, in hepatocytes, as in both enterocytes and ameloblasts, a c o m m o n feature is the close co-localization of annexin VI with actin. Finally, the abundance of annexin VI extending over the entire plasm a m e m b r a n e of hepatocytes is in full accordance with the proposed role of this protein in exocytosis and endocytosis.

Mitochondria The only calcium-binding protein present at a detectable level in mitochondria o f enterocytes and hepatocytes is annexin VI, which appears to be closely associated with the inner membrane. This finding agrees with our previous localization of annexin VI within the mitochondria of rat secretory ameloblasts and odontoblasts (Goldberg et al. 1991), and ram germ cells (Feinberg et al. 1991). The function of annexin VI in mitochondria is not yet understood. It m a y play a role in regulating Ca a § fluxes between mitochondria and the surrounding cytoplasm, or between mitochondrial compartments. In this respect, the function of annexin VI would be similar to that of annexins V and VII (synexin) whose interaction with phospholipid bilayers induces the f o r m a t i o n of calciumspecific voltage-gated channels (Pollard et al. 1992). Furthermore, this hypothesis is reinforced by the finding that annexin VI binding to phospholipid monolayers results in an increase in surface pressures, indicating a rearrangement of m e m b r a n e structure (Newman et al. 1989). In the present work, we have examined the presence and the localizations of annexins in two different cells of epithelial origin, viz., rat enterocytes and hepatocytes, by an immunochemical and an immunocytochemical approach. Our results provide support for the involvement of the different annexins in regulating physiological pro-

cesses occurring in the epithelial cells. In this respect, it is noteworthy that both the apical and basal portions of these highly polarized cells contain at least one annexin, annexin VI, which occurs in close association with plasma m e m b r a n e and cytoskeletal proteins, such as actin. Another prominent feature is the close association of annexin VI with mitochondrial membranes. These findings reinforce our previous observations in secretory ameloblasts and odontoblasts of rat incisor (Goldberg et al. 1991). Nevertheless, other annexins, such as annexins II and IV and the intestine-specific annexin described by Wice and G o r d o n (1992), are involved in processes taking place in enterocytes. Thus, the data presented here show the unique distribution and possible specific functions of annexins in enterocytes and hepatocytes.

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