Cytochemical properties of mitochondria in the gastric parietal cell.

The matrix of some mitochondria in gastric parietal cells of rat and guinea pig evidenced affinity for the high iron diamine method which localizes sulfated complex carbohydrates selectively by light and electron microscopy. Such staining has not been observed elsewhere in the stomach. The high iron diamine reactive mitochondria about equaled in number those which were unreactive, and the two groups were indistinguishable morphologically. The distinction was not apparent either when mitochondria were stained by other cytochemical procedures including dialyzed iron for acidic complex carbohydrates, 3-3' diaminobenzidine-H2O2 at pH 6.0 for cytochrome oxidase, and Kominick's pyroantimonate osmium tetroxide for antimonate precipitable cations. The dialyzed iron method stained acid glycoconjugates in the outer intermembrane space in parietal cell mitochondria. These mitochondria stained more strongly with dialyzed iron than have any others examined heretofore with this method and comprised the only reactive mitochondria in the stomach. Parietal cell mitochondria also stained strongly for cytochrome oxidase but those of other gastric cells failed to evidence this reactivity.     

Ultrastructural carbohydrate cytochemistry of gastric epithelium

Synopsis On examination with ultrastructural methods for visualizing thevicinal glycols and acid groups of complex carbohydrates, the most superficial surface epithelium of the rat gastric corpus displayed biphasic mucous droplets consisting of a cortex of hexose-rich (i.e. periodate-reactive) neutral mucosubstance and an uncharacterized denser core plus monophasic droplets with the neutral mucosubstance. In many surface epithelial cells of the foveolae, the biphasic and monophasic droplets with the neutral mucosubstance intermingled in varying proportions with monophasic droplets showing uniform periodate reactivity, a variable degree of dialyzed ironbinding—demonstrative of acidic glycoconjugate, and high iron—diamine affinity—demonstrative of sulphomucin. Deep foveolar epithelium displayed only monophasic droplets, most of which contained acidic periodate-reactive complex carbohydrate. Underiying cells, designated isthmus cells, exhibited monophasic or occasional biphasic granules containing sulphated, hexose-rich mucosubstance. Nascent droplets or granules near the Golgi zone differed from the mature organelles in the distribution of the glycoconjugate. Mucous neck cells occupied a deeper stratum and displayed a uniform population of monophasic mucous droplets with a loose meshwork of neutral mucosubstance.Techniques for demonstrating hexoses ultrastructurally stained all Golgi cisternae in the mucigenic epithelium, showing increasing reactivity toward the maturing face. Distinctive cistemae with moderate reactivity in the Golgi complex of isthmus cells were interpreted as GERL. Acidic mucosubstances were visualized only in the inner, mature cisternae of the Golgi complex of cells storing acidic glycoconjugates, and not in cisternae interpretable as GERL.The apical plasmalemma of isthmus cells uniquely exhibited abundant sulphated glycoconjugate and that of parietal cells revealed a less prominent, periodic neutral mucosubstance. Lateral and basal plasmalemmae varied from unstained to slightly reactive; basement membranes showed moderate reactivity with methods for visualizing complex carbohydrates. Abundance of glycogen further characterized surface epithelial cells of the corpus and of some parietal cells     

The surface characteristics of the plasma membrane of the exocrine pancreas.

Regional differentiation of the plasma membrane and related structures of the exocrine pancreas has been studied ultrastructurally and cytochemically. Fixation with an osmium tetroxide-silver acetate solution produced abundant fine precipitates on the luminal and basal surface of the centroacinar but not the acinar cells. Staining with dialyzed iron (DI) revealed the heaviest concentration of anionic sites on the luminal plasma membrane of the acinar cells, including the surface of both the intercellular canaliculi and the main lumen. The reactive sites on the apical acinar plasmalemma appeared to consist of discrete globules. DI-reactivity of the lateral basal membranes was most prominent in the centroacinar cells and essentially absent in the acinar cells but was weak relative to that of the acinar-cell apical plasmalemma. The lamina lucida of the basement membrane of the duct stained with DI, but that of basement membrane under acinar cells did not. Sialidase digestion prior to DI staining abolished the staining of plasma membranes. These results indicate that duct epithelial cells, including most prominently the centroacinar cells, are chiefly responsible for electrolyte and fluid transport.     

Gastric K+-stimulated p-nitrophenylphosphatase cytochemistry

A cytochemical study of gastric K+-stimulated p-nitrophenylphosphatase (K-NPPase) activity, corresponding to a K+-stimulated phosphoprotein phosphatase of H-K-ATPase system, has been made by a new cytochemical method. Sections of fixed guinea pig gastric mucosa in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde, were incubated with the incubation medium (1.0 M glycine-0.1 M KOH buffer, pH 9.0, 2.5 ml; 1.1 M KCl, 0.5 ml; 10 mM lead citrate dissolved in 50 mM KOH, 4 ml; levamisole, 6.0 mg; dimethyl sulfoxide, 2.0 ml; 0.1 M p-nitrophenylphosphate (Mg-salt), 1.0 ml; ouabain, 73.0 mg) for 30 min at room temperature. Under a light microscope the specific gastric K-NPPase reaction was distributed only in the parietal cells of the fundic glands. The electron microscopic cytochemistry showed that the gastric K-NPPase activity was localized on the membrane lining the apical surfaces, secretory canaliculi and tubulovesicles. On the other hand, ouabain-sensitive K-NPPase activity (Na-K-ATPase) was demonstrated to localize only in the basolateral membrane of parietal cells with Mayahara's method. These findings support the interrelationships between the apical surface membrane, secretory canalicular membrane and tubulovesicles, and the functional differentiation of the membrane between the secretory membrane and basolateral membrane.     

Ultracytochemical study of ATP-ase activity in mucosal parietal cells and in the cells of human adenocarcinoma of the stomach]

Ultracytochemical investigation of ATP-ase activity was carried out in parietal cells of the mucosa and in cancer cells of human stomach carcinoma possessing a similar ultrastructure. In parietal cells the reaction product of ATP-ase was observed on the membranes of microvilli of intracellular canaliculi, on the membranes delineating the lateral intercellular space, on the basal plasmolemma and in the nucleoli. The reaction product was absent on the membranes of tubuvesicles and on the apical surface of the plasmalemma. In cancer cells the reaction product was found on the membranes of the microvilli of the intracellular canaliculi, basal plasmolemma and in the nucleoli. Comparative examination of ATP-ase activity in these cells implies that at least the part of the mechanism of hydrochloric acid secretion which is involved in the transfer of H+ and Cl- is retained in cancer cells. A steady decrease in hydrochlorid acid secretion observed in the stomach mucosa in cancer as well as in the tumour itself seems to be associated with other mechanisms.     

Ionic components of secretory cell surfaces in relation to secretory function

Synopsis Rat pancreas was examined by ultrastructural cytochemical methods for localizing cations and anions, as well as polyanions and, more specifically, sulphated mucosubstance. Exceptionally abundant antimonate-precipitable cation was demonstrated between pancreatic acinar cells and at the base of the centro-acinar and other duct epithelial cells. Precipitates of nuclear heterochromatin appeared lighter whereas those of mitochondria and cytoplasm were coarser and more conspicuous in acinar that duct cells. Stimulation with synthetic secretin at a low level diminished antimonate reactivity of nuclei as well as the precipitation at the basement membrane of centro-acinar cells. At a higher dose, secretin selectively eliminated precipitation between and below centro-acinar and other duct cells while inducing increased antimonate-reactive cation in centro-acinar cells and the acinar lumens. Pancreozymin stimulation elimated antimonate-precipitable cations between acinar cells and, to a much lesser extent, those between duct cells and increased cytoplasmic precipitates on granular reticulum of acinar cells.Silver-precipitable anions were localized on the luminal surface of the apical plasma lemma and the outer surface of the latero-basal plasmalemma of centro-acinar cells but not on acinar cell surfaces. Silver precipitates also occurred on junctional complexes of acinar and duct epithelial cells and at tight junctions of acinar cells and on the inner face of the lateral plasmalemma of acinar cells.Dialysed iron staining demonstrated the most number of sites of acid mucosubstance on the luminal surface of the plasmalemma of acinar cells. Lateral and basal plasmalemmas of centro-acinar and more distal duct cells stained lightly with dialysed iron but those of acinar cells did not. Dialysed iron visualized acid mucosubstance in the lamina lucida of the basement membrane of duct but not of acinar cells. Dialysed iron staining of the plasma membranes succumbed to prior sialidase treatment whereas that of basement membrane resisted digestion. High iron diamine staining demonstrated sulphated mucosubstance in the lamina lucida of the duct basement membrane exclusively. The cytochemical results implicate centro-acinar cells as primarily responsible for contributing fluid and electrolytes to pancreatic secretion.     

Lectin-binding pattern in normal human gastric mucosa. A light and electron microscopic study

Normal human gastric mucosal cells were examined by light and electron microscopy using lectins as a probe. The ABC method was used with biotinylated lectins for light microscopy and HRP-labeled lectins for electron microscopy. The human gastric mucosal cells revealed specific binding patterns for each lectin by light microscopy. Among the lectins tested, in particular, DBA gave a characteristic pattern. It specifically stained the supranuclear region of surface epithelial cells and the perinuclear region of parietal cells. By electron microscopy, the stacked cisternae and the vesicles of the Golgi apparatus of the surface epithelial cells were positive for the DBA staining. These results show that the DBA-positive supranuclear region observed by light microscopy corresponds to the Golgi apparatus. In the parietal cells, DBA, RCA and ConA bound to the intracellular secretory canaliculi which are invaginations of the cell membrane running around the nucleus in the cytoplasm. Therefore, the tubular perinuclear positive region observed by light microscopy corresponds to the membranes of the intracellular secretory canaliculi. In addition, the ConA reagent stained the endoplasmic reticulum, Golgi apparatus, nuclear envelope, and cell membrane of the parietal cell, which explains the diffuse cytoplasmic staining observed at the light microscopic level with this lectin. Lectins have proved to be very useful for the evaluation of in situ cytochemical aspects of the glycoconjugates characteristic to human gastric mucosal cells.     

High-pressure freezing of isolated gastric glands provides new insight into the fine structure and subcellular localization of H+/K+-ATPase in gastric parietal cells.

High-pressure freezing (HPF) is currently the most reliable method to obtain an adequately frozen sample for high-resolution morphological evaluation. Here we applied the HPF technique to isolated rabbit gastric glands to reveal structural evidence that may be correlated with functional activity of gastric parietal cells. This approach provided well-preserved fine structure and excellent antigenicity of several parietal cell proteins. Microtubules were abundant in the cytoplasm and frequently appeared to be associating with tubulovesicles. Interestingly, many electron-dense coated vesicles were apparent around the intracellular canaliculi (IC) of resting parietal cells, consistent with active membrane retrieval from the apical membranes. Immunolabeling of H+/K+-ATPase was evident on the endocytic components (e.g., multivesicular bodies) and tubulovesicles. After histamine stimulation, the parietal cells characteristically showed expanded IC membranes with varied features of their apical microvilli. The labeling density of H+/K+-ATPase was four-fold higher on the IC membrane of stimulated parietal cells than on that of resting parietal cells. Immunolabeling of ezrin was clearly identified on the IC and basolateral membranes of parietal cells, corresponding to their F-actin-rich sites. The present findings provide a new insight into the correlation of cell structure and function in gastric parietal cells.     

Lectin-binding pattern in normal human gastric mucosa

Summary Normal human gastric mucosal cells were examined by light and electron microscopy using lectins as a probe. The ABC method was used with biotinylated lectins for light microscopy and HRP-labeled lectins for electron microscopy. The human gastric mucosal cells revealed specific binding patterns for each lectin by light microscopy. Among the lectins tested, in particular, DBA gave a characteristic pattern. It specifically stained the supranuclear region of surface epithelial cells and the perinuclear region of parietal cells. By electron microscopy, the stacked cisternae and the vesicles of the Golgi apparatus of the surface epithelial cells were positive for the DBA staining. These results show that the DBA-positive supranuclear region observed by light microscopy corresponds to the Golgi apparatus. In the parietal cells, DBA, RCA and ConA bound to the intracellular secretory canaliculi which are invaginations of the cell membrane running around the nucleus in the cytoplasm. Therefore, the tubular perinuclear positive region observed by light microscopy corresponds to the membranes of the intracellular secretory canaliculi. In addition, the ConA reagent stained the endoplasmic reticulum, Golgi apparatus, nuclear envelope, and cell membrane of the parietal cell, which explains the diffuse cytoplasmic staining observed at the light microscopic level with this lectin. Lectins have proved to be very useful for the evaluation of in situ cytochemical aspects of the glycoconjugates characteristic to human gastric mucosal cells.     

CORRELATION OF THE FINE STRUCTURE OF THE GASTRIC PARIETAL CELL (DOG) WITH FUNCTIONAL ACTIVITY OF THE STOMACH

The fine structure of the parietal (oxyntic) cell in the gastric glands (corpus of the stomach) of the dog was examined under conditions of active gastric acid secretion and compared with cellular structure in the non-acid-secretory (basal) state. Animals, in both acute and chronic experiments, were equipped with gastric fistulae so that gastric juice could be collected for analysis of total acidity, free acidity, volume, and pH prior to biopsy of the gastric mucosa. The specimens of mucosa were fixed in buffered OsO₄ and embedded in n-butyl methacrylate and the thin sections were stained with lead hydroxide before examination in the electron microscope. A majority of parietal cells showed an alteration of fine structure during stimulation of gastric acid secretion by a number of different techniques (electrical vagal stimulation, histamine administration, or insulin injection). The changes in fine structure affected mainly the smooth surfaced vesicular elements and the intracellular canaliculi in the cytoplasm of the cell. The mitochondria also appeared to be involved to some extent. During acid secretion a greater concentration of smooth surface profiles is found adjacent to the walls of the intracellular canaliculi; other parietal cells exhibited a marked decrease in number of smooth surfaced elements. Intracellular canaliculi, always present in non-acid-secreting oxyntic cells, develop more extensively in cells of acid-secreting gastric glands. The surface area of these canaliculi is greatly increased by the elaboration of a large number of closely approximated and elongated microvilli. Still other parietal cells apparently in a different stage of the secretory cycle exhibit non-patent canaliculi lacking prominence; such cells have very few smooth surfaced vesicular elements. These morphological findings correlated with the acid-secretory state of the stomach provide evidence that the parietal cell participates in the process of acid secretion.     

Polarized distribution of actin isoforms in gastric parietal cells.

The actin genes encode several structurally similar, but perhaps functionally different, protein isoforms that mediate contractile function in muscle cells and determine the morphology and motility in nonmuscle cells. To reveal the isoform profile in the gastric monomeric actin pool, we purified actin from the cytosol of gastric epithelial cells by DNase I affinity chromatography followed by two-dimensional gel electrophoresis. Actin isoforms were identified by Western blotting with a monoclonal antibody against all actin isoforms and two isoform-specific antibodies against cytoplasmic beta-actin and gamma-actin. Densitometry revealed a ratio for beta-actin/gamma-actin that equaled 0.73 +/- 0.09 in the cytosol. To assess the distribution of actin isoforms in gastric glandular cells in relation to ezrin, a putative membrane-cytoskeleton linker, we carried out double immunofluorescence using actin-isoform-specific antibodies and ezrin antibody. Immunostaining confirmed that ezrin resides mainly in canaliculi and apical plasma membrane of parietal cells. Staining for the beta-actin isoform was intense along the entire gland lumen and within the canaliculi of parietal cells, thus predominantly near the apical membrane of all gastric epithelial cells, although lower levels of beta-actin were also identified near the basolateral membrane. The gamma-actin isoform was distributed heavily near the basolateral membrane of parietal cells, with much less intense staining of parietal cell canaliculi and no staining of apical membranes. Within parietal cells, the cellular localization of beta-actin, but not gamma-actin, isoform superimposed onto that of ezrin. In a search for a possible selective interaction between actin isoforms and ezrin, we carried out immunoprecipitation experiments on gastric membrane extracts in which substantial amounts of actin were co-eluted with ezrin from an anti-ezrin affinity column. The ratio of beta-actin/gamma-actin in the immunoprecipitate (beta/gamma = 2.14 +/- 0.32) was significantly greater than that found in the cytosolic fraction. In summary, we have shown that beta- and gamma-actin isoforms are differentially distributed in gastric parietal cells. Furthermore, our data suggest a preferential, but not exclusive, interaction between beta-actin and ezrin in gastric parietal cells. Finally, our results suggest that the beta- and gamma-actin-based cytoskeleton networks might function separately in response to the stimulation of acid secretion.     

Ultrastructural localization of carbonic anhydrase in gastric parietal cells with the immunoglobulin-enzyme bridge method

Summary Ultrastructural immunostaining of carbonic anhydrase in gastric parietal cells was accomplished with the immunoglobulin-peroxidase bridge procedure applied to cryostat sections of fixed guinea-pig stomach prior to dehydration and embedment. Of a variety of fixatives tested, only freshly prepared paraformaldehyde buffered with calcium acetate provided both immunostaining and adequate preservation of ultrastructural morphology. Delipidization or exposure of specimens to detergent prior to staining enhanced the intensity of the immunostaining and increased the sensitivity of the method. Increased diaminobenzidine concentration in the peroxidase substrate appeared also to intensify the densification at the reactive site. Carbonic anhydrase was localized ultrastructurally with this pre-embedment immunobridge procedure in the hyaloplasm of gastric parietal cells and less consistently in the superficial surface epithelium. The basal portion of the parietal cells stained more intensely than the apical region and immunoreactivity appeared concentrated at the plasmalemma and around mitochondria.     

Gastric K+-stimulated p-nitrophenylphosphatase cytochemistry

Summary A cytochemical study of gastric K⁺-stimulated p-nitrophenylphosphatase (K-NPPase) activity, corresponding to a K⁺-stimulated phosphoprotein phosphatase of H-K-ATPase system, has been made by a new cytochemical method.Sections of fixed guinea pig gastric mucosa in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde, were incubated with the incubation medium (1.0 M glycine-0.1 M KOH buffer, pH 9.0, 2.5 ml; 1.1 M KCl, 0.5 ml; 10 mM lead citrate dissolved in 50 mM KOH, 4 ml; levamisole, 6.0 mg; dimethyl sulfoxide, 2.0 ml; 0.1 M p-nitrophenylphosphate (Mg-salt), 1.0 ml; ouabain, 73.0 mg) for 30 min at room temperature. Under a light microscope the specific gastric K-NPPase reaction was distributed only in the parietal cells of the fundic glands. The electron microscopic cytochemistry showed that the gastric K-NPPase activity was localized on the membrane lining the apical surfaces, secretory canaliculi and tubulovesicles. On the other hand, ouabain-sensitive K-NPPase activity (Na-K-ATPase) was demonstrated to localize only in the basolateral membrane of parietal cells with Mayahara's method.These findings support the interrelationships between the apical surface membrane, secretory canalicular membrane and tubulovesicles, and the functional differentiation of the membrane between the secretory membrane and basolateral membrane.In honour of Prof. P. van DuijnPart of this paper was presented at the 24th Annual Meeting of the Japan Society of Histochemistry and Cytochemistry held in Nagoya, October 27–28, 1983 (Ogawa KS, Fujimoto K, Ogawa K (1983) A new lead citrate method for the cytochemical demonstration of the H⁺–K⁺-ATPase with p-NPP as a substrate. Acta Histochem Cytochem 16:662)This study was supported by Grants-in-Aid for Encouragement of Young Scientists No. 60770019 to K. Fujimoto from the Ministry of Education, Science and Culture, the Japanese Government     

Basolateral localization of anion exchanger 2 (AE2) and actin in acid-secreting (parietal) cells of the human stomach

Basolateral uptake of chloride by the HCl-secreting parietal cells of the gastric (oxyntic) glands is most likely mediated by a HCO^– ₃/Cl^– anion exchange mechanism. Circumstantial evidence indicates that in rodents the anion exchange proceeds through an anion exchanger 2(AE2)-like membrane protein. In the present study, we raised antibodies against a bacterial fusion protein expressing a -26-kDa portion of the human AE2 sequence. These antibodies were used to identify and localize AE2 in the human stomach. Here we report that the mucosa of the human stomach expresses an 160-kDa immunoreactive form of AE2 containing the AE2-specific exoplasmic domain (Z-loop) as identified by polymerase chain reaction. Immunostaining specific for AE2 was restricted to the basolateral membrane domain of parietal cells and was also detected in small epithelial cells localized in the glandular isthmus region. The latter cells most likely represent pre-parietal cells. Parietal cells were identified by simultaneous and sequential labelling with antibodies against the gastric H⁺, K⁺-ATPase and actin, respectively. Both actin and the H⁺, K⁺-ATPase were localized along the apical membrane of parietal cells and the membrane of their secretory intracellular canaliculi. In addition, actin was shown to be colocalized with AE2 along the basolateral cell surface. Discontinuous staining for AE2 coincided with infoldings of the basolateral plasma membrane labelled by the actin antibody. These observations indicate that AE2 might be placed at specialized (folded) microdomains of the basolateral cell surface by linkage to the actin-based cytoskeleton.Large parts of this publication belong to the MD thesis of B. Warrings. B. Warrings and T. Jöns should be considered alphabetically listed first authors who made equally strong contributions to this study     

The unique ultrastructure of secretory membranes in gastric parietal cells depends upon the presence of H+, K+ -ATPase

Ion transporters play a central role in gastric acid secretion. To determine whether some of these transporters are necessary for the normal ultrastructure of secretory membranes in gastric parietal cells, mice lacking transporters for H+, K+, Cl-, and Na+ were examined for alterations in volume density (Vd) of basolateral, apical, tubulovesicular and canalicular membranes, microvillar dimensions, membrane flexibility, and ultrastructure. In mice lacking Na+/H+ exchanger 1 (NHE1) or the Na+-K+-2Cl- cotransporter (NKCC1), the ultrastructure and Vd of secretory membranes and the secretory canalicular to tubulovesicular membrane ratio (SC/TV), a morphological correlate of secretory activity, were similar to those of wild-type mice. In mice lacking Na+/H+ exchanger 2 (NHE2) or gastric H+, K+ -ATPase alpha- or beta-subunits, the SC/TV ratio and Vd of secretory membranes were decreased, though canaliculi were often dilated. In H+, K+ -ATPase-deficient parietal cells, canalicular folds were decreased, normally abundant tubulovesicles were replaced with a few rigid round vesicles, and microvilli were sparse, stiff and short, in contrast to the long and flexible microvilli in wild-type cells. In addition, microvilli of the H+, K+ -ATPase-deficient parietal cells had centrally bundled F-actin filaments, unlike the microvilli of wild-type cells, in which actin filaments were peripherally positioned concentric to the plasmalemma. Data showed that the absence of H+, K+ -ATPase produced fundamental changes in parietal cell membrane ultrastructure, suggesting that the pump provides an essential link between the membranes and F-actin, critical to the gross architecture and suppleness of the secretory membranes.     

SAP 97 is a potential candidate for basolateral fixation of ezrin in parietal cells

 Acid secretion in gastric parietal cells is preceded by a dramatic increase in surface area of the apical membrane compartment, due to fusion of the H⁺/K⁺-ATPase-containing tubulovesicles. The resulting canaliculi must be fixed for a period of minutes by cytoskeletal elements to sustain acid secretion. Using immunofluorescence microscopy, the cytoskeletal linker molecule, ezrin, localizes to the apical canalicular membrane of parietal cells. Antibodies against ezrin precipitate H⁺/K⁺-ATPase and β-actin. In addition to its apical localization, ezrin is found to be colocalized at the basolateral compartment with synapse-associated protein (SAP) 97. Immunoprecipitation confirms a direct binding of SAP 97 and ezrin. We conclude that ezrin is fixed to the basolateral compartment by SAP 97. Upon stimulation of acid secretion, ezrin moves to the apical surface where it might stabilize the canalicular microvilli by connecting to β-actin and H⁺/K⁺-ATPase, thereby sustaining acid secretion. Accepted: 14 January 1999     

Cytochemical demonstration of phosphatases in membrane-recycling structures of endodermal cells

We applied cytochemical procedures to demonstrate the presence of acid and alkaline phosphatase in the visceral yolk-sac endoderm of rats using frozen, aldehyde-fixed tissue with cerium as the capture agent. This procedure allowed more detailed topochemical localization than was possible using unfrozen tissue or with lead as the capture agent. Acid phosphatase was found to be present in lysosomes as well as in a small number of apical canaliculi, which are thought to be recycling structures of the cell membranes in endodermal cells. Reaction products of alkaline phosphatase were observed on the outer surface of apical, lateral, and basal cell membranes. In addition, some apical vacuoles contained alkaline phosphatase, and more apical canaliculi were positive for alkaline phosphatase than for acid phosphatase. However, most of the apical canaliculi were negative for both enzymes. It is suggested that acid and alkaline phosphatase are taken up by different numbers of apical canaliculi during the detachment of apical canaliculi from lysosomes and resorption vacuoles.     

Distribution of sialoglycoconjugates on acinar cells of the mammalian pancreas

Using the sialic acid-specific lectin, limulin (LPA; from Limulus polyphemus hemolymph), the distribution and nature of sialoglycoconjugates on the surface of rat pancreatic cells has been investigated. Binding of rhodaminated LPA (Rh-LPA) or horseradish peroxidase-conjugated LPA (HRP-LPA) to fixed-frozen sections of adult rat pancreas resulted in intense linear staining of the apical surface of acinar cells with fainter staining on the basal but not the lateral cell surfaces. LPA binding was specific in that it could be abolished by 1) pretreatment of tissue sections with neuraminidase or periodic acid; 2) competition with sialic acid; and 3) incubation in Ca2+ -free buffers. Pretreatment of sections with proteases abolished LPA binding to the apical surfaces of acinar cells and also enhanced LPA binding to the lateral cell surface. Lipid extraction of sections following protease treatment markedly reduced LPA binding to the acinar cell periphery. These results suggest that LPA binding sites on the acinar cell apical surface may be primarily sialoglycoproteins, while those on the basolateral surfaces may consist in part of gangliosides. Electron microscopy of collagenase-dispersed acini exposed to HRP-LPA confirmed binding of LPA to the basal plasmalemma and, in addition, revealed staining of basal lamina when present. LPA binding to the acinar cell surface was not affected by digestion of tissue sections with hyaluronidase, heparinase, collagenase, or 6 M guanidine-HCl. Control experiments indicated that rat pancreatic secretory proteins contain undetectable amounts of sialoglycoproteins and thus that the apical localization of LPA is not due to adherent secretory proteins. Islets of Langerhans were always uniformly and heavily stained with LPA conjugates; this staining was protease insensitive. Appearance of LPA binding sites was examined on embryonic pancreatic epithelia. At day 15 of gestation, Rh-LPA stained the entire periphery of the epithelial cells, including the lateral cell surface, although more intense staining was already noted on the apical surface. This pattern persisted through day 17 of gestation, but by day 19 an adult staining pattern was observed with loss of staining of the lateral cell surfaces.     

Mice with a targeted disruption of the AE2 Cl-/HCO3- exchanger are achlorhydric

The AE2 Cl-/HCO3- exchanger is expressed in numerous cell types, including epithelial cells of the kidney, respiratory tract, and alimentary tract. In gastric epithelia, AE2 is particularly abundant in parietal cells, where it may be the predominant mechanism for HCO3- efflux and Cl- influx across the basolateral membrane that is needed for acid secretion. To investigate the hypothesis that AE2 is critical for parietal cell function and to assess its importance in other tissues, homozygous null mutant (AE2(-/-)) mice were prepared by targeted disruption of the AE2 (Slc4a2) gene. AE2(-/-) mice were emaciated, edentulous (toothless), and exhibited severe growth retardation, and most of them died around the time of weaning. AE2(-/-) mice exhibited achlorhydria, and histological studies revealed abnormalities of the gastric epithelium, including moderate dilation of the gastric gland lumens and a reduction in the number of parietal cells. There was little evidence, however, that parietal cell viability was impaired. Ultrastructural analysis of AE2(-/-) gastric mucosa revealed abnormal parietal cell structure, with severely impaired development of secretory canaliculi and few tubulovesicles but normal apical microvilli. These results demonstrate that AE2 is essential for gastric acid secretion and for normal development of secretory canalicular and tubulovesicular membranes in mouse parietal cells.