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Volume 52 (1): 77-86, 2004
Copyright ©The Histochemical Society, Inc.
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
Department of Molecular and Cell Biology (AS,JGF) and Electron Microscopy Lab (KLM), University of California, Berkeley, California
Correspondence to: Prof. John G. Forte, Dept. of Molecular and Cell Biology, University of California, 241 LSA, Berkeley, CA 94720-3200. E-mail: jforte{at}uclink.berkeley.edu
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Summary |
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 Top Summary IntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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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.
(J Histochem Cytochem 52:77–86, 2004)
Key Words: high-pressure freezing • electron microscopy • isolated gastric gland • parietal cell • transformation • immunogold labeling • H+/K+-ATPase • ezrin
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Introduction |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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CRYOFIXATION is generally accepted as the best initial fixation step to provide superior preservation of cell and tissue ultrastructure. High-pressure freezing (HPF) followed by freeze-substitution is currently the most reliable method to obtain a high yield of adequately frozen samples (Moor 1987
; Dahl and Staehelin 1989). The application of high pressure (in the range of 2100 bars) lowers the freezing point and reduces the rate of ice nucleation and ice crystal growth, giving high quality tissue preservation. Recently, the combined use of HPF/freeze-substitution and low-temperature embedding has been applied to histochemical investigations to preserve the antigenicity of samples (Monaghan et al. 1998; McDonald 1999).
Gastric parietal cells undergo morphological transformations in response to stimulation of acid secretion. Correlation of parietal cell structure and function was facilitated by the establishment of isolated gastric glands (Berglindh and Öbrink 1976) and cultured parietal cells (Chew et al. 1989) as experimental models. The HPF technique has been recently applied to morphological studies on gastric parietal cells (Okamoto et al. 2000; Duman et al. 2002; Sawaguchi et al. 2002b), bringing new insights into fine structural features.
The gastric proton pump, also known as the H+/K+-ATPase, is the major cargo protein of tubulovesicles in parietal cells. On the basis of membrane recruitment and recycling hypothesis, it is now generally accepted that the H+/K+-ATPase-rich tubulovesicles are recruited by fusion to the apical membrane on stimulation and then recycled back to the cytoplasm on return to the resting state (Forte et al. 1977; Forte and Yao 1996). Ezrin is an actin-binding protein that may play a key role in the stimulation-associated remodeling of apical microvilli and ultimately in the regulation of acid secretion (Yao et al. 1995; Urushidani et al. 1997). Hanzel et al. (1991) reported on the subcellular localization of ezrin using ultrathin cryosections of parietal cells processed by mild fixation. However, detailed study has been hindered due to the sensitive antigenicity of ezrin and the poor morphological preservation by the mild fixation.
Recently, we introduced a procedure using poly-L-lysine-coated aluminum planchettes for direct attachment and HPF of isolated gastric glands in defined physiological states (Sawaguchi et al. in press). The direct attachment of samples to the planchettes not only improved manipulation but also minimized physical and physiological damage to the samples. Preliminary observations demonstrated excellent ultrastructure and well-preserved antigenicity in the glands. Here we describe the detailed fine structure of parietal cells and stimulation-associated morphological transformations in isolated gastric glands processed by the poly-L-lysine technique. Immunocytochemical studies also show sub- cellular localization of H+/K+-ATPase and ezrin that is improved by the well-preserved antigenicity.
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Materials and Methods |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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High-pressure Freezing of Isolated Gastric Glands
Gastric glands were isolated from gastric mucosa of New Zealand White rabbits as previously described (Ammar et al. 2002
). All procedures involving procurement of animal tissues were reviewed by the Berkeley Animal Care and Use Committee. Isolated gastric glands were held in the resting state with 100 µM cimetidine at 37C for 30 min or stimulated with 100 µM histamine and 30 µM 3-isobutyl-1-methyl-xanthine (IBMX) at 37C for 30 min. Histamine stimulation was quantified in the same preparation using the aminopyrine (AP) uptake assay (Berglindh 1990). Just before cryoimmobilization by HPF, the glands were plated onto poly-L-lysine-coated type B planchettes (Sawaguchi et al. in press) and covered with a 200-µm-deep well of a type A aluminum planchette (Engineering office M. Wohlwend; Sennwald, Switzerland). This assembly was immediately frozen at 2100 bars in an HPF machine (HPM 010; BAL-TEC, Liechtenstein).
Freeze-substitution and Embedding
Freeze-substitution was carried out in 1% osmium tetroxide plus 0.1% uranyl acetate in acetone, in a Leica AFS machine (Leica; Vienna, Austria). After programmed warming from -155C to -90C at 5C/hr, samples were kept at -90C for 3 days and then gradually warmed to 20C at 10C/hr. After three 10-min rinses in pure acetone, samples were infiltrated and embedded in Epon within flat-bottom embedding capsules (Ted Pella; Redding, CA). The aluminum planchette was removed from the Epon bloc by pliers after careful trimming around the planchette. Semithin (1-µm) sections were cut and stained with 1% toluidine blue/1% sodium tetraborate solution for light microscopic observation. Ultrathin sections (60–80 nm thick) were cut and stained with 2% uranyl acetate in 70% methanol and Reynolds' lead citrate, and observed in a TECNAI 12 (FEI; Eindhoven, Netherlands) transmission electron microscope operating at 100 kV.
For immunolabeling of ezrin, freeze-substitution was carried out in pure acetone. After programmed warming from -155C to -90C at 5C/hr, samples were kept at -90C for 3 days and then gradually warmed to -35C at 10C/hr. The substitution medium was replaced with pure ethanol (three changes each of 10-min duration) and infiltrated directly with 100% Lowicryl HM20 for 2 hr at -35C. The polymerization was performed using an ultraviolet lamp of the AFS machine for 24 hr at -35C and for a further 8 hr at 18C.
Immunolabeling of H+/K+-ATPase and Ezrin
Ultrathin sections of Epon-embedded gastric glands were picked up on 200-mesh nickel grids coated with Formvar film and treated with 5% sodium metaperiodate in distilled water for 30 min (Bendayan and Zollinger 1983). After rinsing in distilled water, sections were incubated in 2% bovine serum albumin (BSA) in PBS for 10 min to block nonspecific binding and then incubated with a mouse monoclonal antibody, 2G11, against the ß-subunit of H+/K+-ATPase (Affinity Bioreagents, Golden, CO; diluted with 2% BSA in PBS) at 4C overnight. After rinsing in PBS, the sections were incubated for 30 min with 10-nm colloidal gold-conjugated goat anti-mouse IgG (British Biocell International, Cardiff, UK; diluted 1:80 with 2% BSA in PBS). After rinsing in PBS and drying, sections were post-stained as described above. For controls, 2G11 was omitted from the procedure.
For immunolabeling of ezrin, ultrathin sections of Lowicryl HM20-embedded glands were used. Sections were incubated in 2% BSA in PBS for 10 min and then incubated with a mouse monoclonal antibody, 4A5, against ezrin (Hanzel et al. 1989; diluted 1:4 with 2% BSA in PBS) at 4C overnight. After rinsing in PBS, the sections were incubated for 30 min with 10-nm colloidal gold-conjugated goat anti-mouse IgG and rinsed in PBS before post-staining as described above. For controls, 4A5 was omitted from the procedure.
Quantification of Immunolabeling
The comparison of the labeling density of H+/K+-ATPase was performed between resting and stimulated parietal cells. To normalize the conditions, the immunolabeling was carried out at the same time on resting and stimulated samples. Micrographs at a magnification of x23,000 were randomly taken from nine parietal cells in each sample. The micrographs were scanned through an Epson Color Image Scanner (U.S. Epson; Long Beach, CA) and the membrane length of apical membrane and tubulovesicles was measured in each digitized micrograph with NIH Image software (version 1.62; NIH, Bethesda, MD). Gold particles present on the membrane or within 20 nm of the membrane were counted. The mean densities were expressed as the number of gold particles/µm ± SEM.
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Results |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Resting Parietal Cells in Cimetidine-treated Gastric Glands
Most of the parietal cells were morphologically in the resting state in cimetidine-treated gastric glands, characterized by narrow IC and numerous tubulovesicles (Figures 1A and 1B) . In the cytoplasm, many microtubules were clearly observed, some of which appeared to be associating with tubulovesicles (Figures 1C and 1D). As previously noted in cultured parietal cells (Sawaguchi et al. 2002b
), small vesicular inclusions (designated here as microvesicles) were found within some tubulovesicles (Figure 1D). Characteristically, many electron-dense coated pits and vesicles were present around the IC of resting parietal cells (Figure 1E). In the resting state, multivesicular bodies were frequently observed in the cytoplasm. It should be noted that small dense vesicles were scattered throughout the cytoplasm (Figures 1C–1F) and abundant around the MVB (Figure 1F).
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At the base region of the glands, exocytosed zymogenic contents were observed as electron-dense droplets in the glandular lumen (Figure 2A ; Sawaguchi et al. 2002a
). Interestingly, electron-dense deposits similar to zymogenic contents were occasionally found in the IC of parietal cells (Figure 2B).
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Stimulated Parietal Cells in Histamine-treated Gastric Glands
To evaluate the effectiveness of histamine stimulation, we performed the AP uptake assay in the same preparations of isolated gastric glands as were used for HPF. The AP accumulation ratio is expressed as the ratio of intracellular to extracellular concentration of AP ± SE, and is an estimate of acid accumulation by the glands (Berglindh 1990
). For cimetidine-treated glands the AP ratio was 45.2 ± 1.6, somewhat higher than we normally measure for resting preparations (Ammar et al. 2002). However, treatment with histamine plus IBMX resulted in significant stimulation and an increase in the AP ratio to 126.5 ± 28.3. Therefore, the glands were well stimulated. Histamine-stimulated parietal cells prepared for electron microscopy exhibited an expansion of the IC that was readily visible at the light microscopic level (Figures 3A and 3B) . Some histamine-stimulated glands also exhibited expanded glandular lumens (Figure 3B). At the electron microscopic level, the features of expanded IC varied as follows: (a) moderately expanded IC lined with many long microvilli (Figure 3C); (b) highly expanded IC lined with many microvilli (Figure 3D); (c) extraordinarily expanded IC lined with few microvilli (Figure 3E). Interestingly, stimulated parietal cells with the extraordinarily expanded IC showed a smooth basolateral surface devoid of surface folds.
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Subcellular Localization of H+/K+-ATPase
Well-preserved antigenicity enabled H+/K+-ATPase labeling even in the osmicated Epon-embedded samples. Consequently, the well-defined morphology helped us to clarify a precise localization of the H+/K+-ATPase. In resting parietal cells, H+/K+-ATPase labeling was clearly evident on tubulovesicular membranes (Figure 4B) as well as on the Golgi apparatus and throughout the cis- to trans-Golgi (Figure 4A). It should be noted that the microvesicles apparent within the tubulovesicles were specifically labeled for H+/K+-ATPase (Figure 4B, inset). In addition, the H+/K+-ATPase labeling was seen on diverging tubulovesicles (Figure 4C) and on more complicated structures such as concentric multi-laminae (Figure 4D).
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The presence of H+/K+-ATPase was demonstrated on various endocytic components within parietal cells, such as the electron-dense coated vesicles (Figure 4E) and MVBs (Figure 4F). On the MVBs, most of the H+/K+-ATPase labeling was present on the inner vesicles and was rarely seen on the outer limiting membrane of the organelles. In addition, the small dense vesicles that abound throughout the cytoplasm were specifically labeled for the H+/K+-ATPase. Figure 4G depicts the H+/K+-ATPase labeling on a dense MVB whose inner vesicles were highly condensed.
In cimetidine-treated glands, H+/K+-ATPase labeling was almost always observed on the apical microvillar membranes of IC. However, the labeling density did not appear as intense as on tubulovesicles (Figure 5A). In the resting state, we also occasionally found H+/K+-ATPase labeling on an occluded IC whose microvilli were closely packed as shown in Figure 5B. After stimulation by histamine, the IC plasma membrane was intensely labeled for H+/K+-ATPase (Figure 5C). Furthermore, as expected, the smooth surface of extraordinarily expanded IC was also specifically labeled for H+/K+-ATPase (Figure 5D). Because of the retained antigenicity of the morphologically superior Epon-embedded, osmicated samples, we were able to carry out a quantitative comparison of the labeling densities of membrane compartments in the resting and stimulated states. The data in Table 1 compare the relative labeling of the two primary H+/K+-ATPase-rich membrane compartments in resting and stimulated parietal cells. The labeling density of tubulovesicles of resting cells was approximately the same as for the remnant tubulovesicles of stimulated cells. However, the labeling density of H+/K+-ATPase was fourfold higher on the IC plasma membrane of stimulated parietal cells compared to that of resting cells.
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Subcellular Localization of Ezrin in Parietal Cells
Immunolabeling of ezrin did not survive osmication and Epon embedding. To preserve the sensitive antigenicity of ezrin, freeze-substitution was carried out in pure acetone without fixative, followed by low-temperature Lowicryl HM20 embedding (see Materials and Methods). Although the morphological representation of Lowicryl embedded samples was not as good as Epon it was superior to that obtained with LR White in parietal cells (Okamoto et al. 2000
). Ezrin labeling was clearly evident on microvilli of the IC and the subapical region of resting parietal cells, whereas tubulovesicles and most other organelles seemed to be immunonegative for ezrin (Figure 6A)
. In addition, the basolateral membrane and its surface folds were highly labeled for ezrin (Figure 6B). After histamine stimulation, the intense ezrin labeling persisted on the long microvilli of the IC (Figure 6C). On close examination it appeared that most of the ezrin labeling was found on the periphery of the apical microvilli (Figure 6D and 6E, respectively), consistent with its putative role as a membrane–cytoskeleton linker protein. Ezrin labeling was also seen on the basolateral membrane and surface folds of stimulated parietal cells (not shown).
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Discussion |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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We have described the fine structure and subcellular localization of H+/K+-ATPase and ezrin in rabbit gastric parietal cells processed by HPF/freeze substitution. The use of poly-L-lysine for direct attachment to aluminum planchettes provided a high yield of adequately frozen isolated gastric glands and enabled us to examine parietal cells free from ice crystal damage. The deep vitreous freezing permitted cryoimmobilization not only of the epithelial cells but also of the intraluminal fluid contents in the gastric glands. The present results ensure that HPF of isolated gastric glands will be a useful model to elucidate the structural evidence that can be correlated to functional activity of the glands.
In cimetidine-treated glands, resting parietal cells showed abundant tubulovesicles in the cytoplasm. Pettitt et al. (1995) reported a network of helically coiled tubules in mouse gastric parietal cells processed by rapid freezing/freeze-substitution (but not HPF). Indeed, the helically coiled structures were not seen in our extensive examinations, but the present study did demonstrate other membrane complexes, such as the concentric multi-laminae and microvesicles. Interestingly, both of these membrane complexes were labeled for H+/K+-ATPase, indicating that the character of these membranes is similar to that of tubulovesicles. However, their physiological significance remains unclear.
Ogata and Yamasaki (2000) demonstrated three-dimensional networks of tubules and cisternae in rat gastric parietal cells by using rapid freezing and high-resolution scanning electron microscopy. It is likely that the present divergent and multi-connected structures are sectioned features of those tubulocisternal networks in the TEM study. Duman et al. (2002) recently reconstructed three-dimensional models of tubulovesicles from serial sections and tomograms of resting parietal cells processed by HPF/freeze-substitution. The models demonstrated that the tubulovesicular compartment was chiefly composed of small stacks of cisternae, designated as tubulocisternae. However, the present study demonstrated that tubulovesicles can also have more complicated structural features. Such discrepancy might be explained by the different manipulations of the glands before cryoimmobilization by HPF. In fact, previous methods required a centrifugation step to harvest the glands, in which G-forces might induce an ultrastructural alteration (Okamoto et al. 2000; Duman et al. 2002). On the other hand, the present poly-L-lysine technique avoids the harsh pelleting step and minimizes the manipulation before HPF (Sawaguchi et al. in press). Consequently, we assume that the presence of complicated characteristics is more consistent with the real structure of tubulovesicles.
Several structural features are much more readily apparent in parietal cells prepared by HPF than by conventional fixation methods, especially microtubules and the variety of small vesicular elements that may be associated with membrane recycling. Although microtubules have been identified and even implicated in parietal cell function (Kasbekar and Gordon 1979), we are struck by their abundance and location throughout the compartment of tubulovesicles. Based on the membrane recruitment and recycling hypothesis (Forte et al. 1977; Forte and Yao 1996), the H+/K+-ATPase-rich tubulovesicles are recruited by fusion to the apical membrane on stimulation. However, little is known about the machinery for vesicular transport in the parietal cell. The present results suggest a close morphological association between the microtubules and tubulovesicles, with occasional points of contact. For some systems it is believed that vesicular transport is directed by microtubular motor proteins, such as kinesin, in an ATP-dependent manner (for review see McNiven and Marlowe 1999), but it still remains to be clarified whether and which motor proteins exist and operate on the microtubules in gastric parietal cells.
Stimulation-associated volume expansions have been previously observed in the internalized apical membrane vacuoles of cultured parietal cells (Chew et al. 1989; Mangeat et al. 1990). It has been proposed that the volume expansion was due to the concomitant secretion of hydrochloric acid and water (Mangeat et al. 1990). In the present study, extensive volume expansion was also observed in the glandular lumen of some histamine-stimulated glands, suggesting an accumulation of glandular secretory products, e.g., hydrochloric acid, mucin, zymogen. It could be speculated that an extreme volume expansion might subtend the microvillar extensions, resulting in the smooth surface profile of apical membrane that was seen in these cases. Importantly, to the best of our knowledge, the volume expansion has never been reported in gastric glands and biopsy specimens taken directly from the stomach, most likely because the confinement of the surrounding connective tissue and the gastric peristaltic movement expel the glandular contents into the stomach lumen through channels in the mucous layer (Johansson et al. 2000; Sawaguchi et al. 2002a).
The present study demonstrated that the labeling density of H+/K+-ATPase was fourfold higher on the IC membrane of stimulated parietal cells than that of resting parietal cells. By contrast, the labeling density was approximately the same on tubulovesicles of resting and stimulated cells. These results were consistent with the previous work of Scott et al. (1993) using the covalent binding of [3H]-omeprazole as a probe for H+/K+-ATPase. It can be safely assumed that the increase of the H+/K+-ATPase density on the IC resulted from the recruitment of H+/K+-ATPase-rich tubulovesicles to the apical membrane rather than the increase of the H+/K+-ATPase density on the tubulovesicles on stimulation. In addition, previous morphometric studies reported that the apical secretory surface area increased four- to tenfold in stimulated parietal cells (Helander and Hirschowitz 1972; Black et al. 1981). Therefore, on stimulation, the total amount of H+/K+-ATPase would significantly increase in the apical membrane via direct recruitment of tubulovesicles, supporting the principal secretory function of the parietal cell.
The present study clearly demonstrated H+/K+-ATPase in a variety of endocytic components, such as the electron-dense coated vesicles. To the best of our knowledge, this is the first report demonstrating H+/K+-ATPase in the MVBs. Based on current knowledge of the endocytic pathway, the H+/K+-ATPase might be retrieved from the apical membrane and transported into an early endosome that differentiates into the MVB. In this study, small dense vesicles with the H+/K+-ATPase were frequently seen around the MVB, implying a role for these vesicles as an H+/K+-ATPase carrier to the endosomes. To date, however, little is known about the fate of the internalized proteins in the MVBs as to whether they will be degraded or recycled back (reviewed by Piper and Luzio 2001). The H+/K+-ATPase in the dense MVB (Figure 4G) seems to be in the degradation pathway rather than the recycling pathway because the dense MVB could be classified as a primary lysosome. Further studies will be of great interest to elucidate the sorting system and the fate of H+/K+-ATPase in the endocytic pathway, including the possible recycling pathway.
In cimetidine-treated glands, we occasionally found occluded IC whose microvilli were in close contact with one another. Similar structures have previously been reported in the recovering state (i.e., from secreting to resting) of piglet parietal cells after withdrawal of histamine (Forte et al. 1977). It could be postulated that the occluded IC were formed in recovering parietal cells induced by cimetidine treatment in this study. To date, surprisingly, there have been relatively few studies concerning the morphological changes in the recovering state (Helander and Hirschowitz 1972; Forte et al. 1977; Schofield et al. 1979; Mangeat et al. 1990), even though there is general acceptance of the membrane recruitment and "recycling" hypothesis. It will be of great interest to morphologically examine the recovering state of parietal cells using HPF and to elucidate the physiological significance of the occluded structures as well as other forms of membrane retrieval in relation to the regulation of acid secretion.
The exocytosed zymogenic contents showed a droplet-like appearance in the glandular lumen of isolated rabbit gastric glands, consistent with previous observations of in vivo rat gastric glands (Sawaguchi et al. 2002a). Interestingly, the zymogenic contents were occasionally found in the IC of parietal cells, suggesting a dynamic flow in the glandular lumen. This finding raised the question of whether the zymogenic enzymes such as pepsin, whose optimal pH is about 2.0, are active or inactive in the IC of acid-secreting parietal cells.
The combined use of HPF/acetone freeze-substitution and low-temperature embedding has improved the subcellular localization of ezrin in gastric parietal cells. The use of absolute acetone as freeze-substitution medium preserved satisfactory morphology as well as antigenicity, even without any fixative. As a result, the present study demonstrates the precise localization of ezrin in gastric parietal cells. Ezrin has been implicated as a cytoskeleton–membrane linker protein and is co-localized with ß-actin in the parietal cell (Yao et al. 1995). The present results confirmed by electron microscopy that ezrin was present on the IC and the basolateral membrane, corresponding to the F-actin-rich sites in the parietal cell. Moreover, most of the apical ezrin was present on the periphery of apical microvilli, consistent with their key role as a cytoskeleton-membrane linker at the microvilli.
In conclusion, application of the HPF technique provided the excellent ultrastructure and antigenicity of the parietal cells in isolated rabbit gastric glands. The ultrastructure was also excellent in other cell types, such as the mucous cells and the chief cells. Isolated gastric glands have been an outstanding experimental model for studies of gastric secretion because their physiological and biochemical functions can be conveniently regulated and monitored. Therefore, it is highly anticipated that the HPF of isolated gastric glands will be a powerful tool to correlate cell structure and immunohistochemical localization with functional activity of the glands.
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Acknowledgments |
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Supported by NIH Grants DK10141 and DK38972.
We thank Reena Zalpuri, Serhan Karvar, David A. Ammar, Rihong Zhou, Waylan Wong, and Jennifer Su for assistance with experimental procedures.
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Footnotes |
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Received for publication June 25, 2003; accepted September 3, 2003
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