ARTICLE |
Correspondence to: Tatsuo Suganuma, Dept. of Anatomy, Miyazaki Medical College, 5200 Kihara, Kiyotake-cho, Miyazaki 889-1692, Japan. E-mail: suganumat@post1.miyazaki-med.ac.jp
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Summary |
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The high-pressure freezing/freeze substitution technique followed by Lowicryl K4M embedding provided an excellent ultrastructure and retention of antigenicity of rat gastric glands as well as the intraluminal fluid contents. By taking this advantage, we histochemically investigated the excretory flow of the zymogenic and mucin contents in rat gastric glandular lumen at the ultrastructural level. The combination of KMnO4-UA/Pb staining for zymogenic contents and Griffonia simplicifolia agglutinin-II (GSA-II) labeling for mucous neck cell (MNC) mucin distinguished the exocytosed zymogenic contents from the MNC mucin in the glandular lumen. Interestingly, at the base and neck regions, the zymogenic contents showed a droplet-like appearance, forming a distinct interface with the MNC mucin. At the pit region, the GSA-II labeling demonstrated restricted paths, designated as MNC mucous channels, which flowed into the surface mucous gel layer. It should be noted that the interface between exocytosed zymogenic contents and MNC mucin disappeared, and that the zymogenic contents merged into the MNC mucous channels. At the top pit region, the surface mucous gel layer showed laminated arrays of three types of gastric mucins. On the basis of these ultrastructural findings, we propose a model of the excretory flow in rat gastric gland. (J Histochem Cytochem 50:223234, 2002)
Key Words: high-pressure freezing, gastric gland, fluid dynamics, excretory flow, intraluminal channel, zymogenic contents, mucin contents, immunogold labeling, permanganate oxidation
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Introduction |
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A CONTINUOUS VISCOELASTIC MUCOUS LAYER, secreted by the surface epithelial and the mucous neck cells, covers the gastric mucosa. It is primarily responsible for the protective properties and critical in the stomach. Zymogenic contents and acid secreted into the lumen of the gastric gland have to traverse the mucous layer to contribute to the digestion of food. Thus far, how they reach the gastric lumen is not fully understood. In in vitro studies,
The high-pressure freezing (HPF) technique is capable of freezing up to 0.2-mm-thick biological specimens in a vitreous or microcrystalline state suitable for high-resolution structural analysis (
Recently, we have developed the potassium permanganate oxidationuranyl acetatelead citrate (KMnO4 UA/Pb) staining, which gives a differential staining of the zymogen granules of rat gastric chief cells on Lowicryl K4M ultrathin sections prepared by HPF/freeze substitution. It has been suggested that the KMnO4 oxidation evokes anionic sites on the zymogen granules which contribute to the binding of uranyl acetate (
The mucous-secreting cells have been mainly classified into two types, i.e., the mucous neck cell (MNC) and the pit cell. The MNC mucin is specifically labeled with Griffonia simplicifolia agglutinin-II (GSA-II) (
The aim of this study was to elucidate the in vivo fluid dynamics of the excretory flow in rat gastric gland. From the base to the top pit region, we carried out the combination of KMnO4UA/Pb staining and colloidal gold labeling for various gastric mucins on Lowicryl K4M ultrathin sections prepared by HPF/freeze substitution. On the basis of our observations, we first propose a model of the excretory flow of zymogenic and mucin contents in rat gastric gland.
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Materials and Methods |
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Reagents
The monoclonal antibody RGM11 was prepared by Dr. Ishihara (1 (PLC
1) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Biotinylated GSA-II, goat anti-mouse IgM, and goat anti-biotin antibody were obtained from Vector Laboratories (Burlingame, CA). Goat anti-mouse IgG was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Poly-L-lysine hydrobromide with molecular weight range of >300 kD was purchased from Sigma Chemical (St Louis, MO). N-Acetyl-D-glucosamine was from Nacalai Tesque (Kyoto, Japan).
Preparation of Colloidal Gold (CG), CCG, and ImmunoglobulinGold Complex
Monodisperse CG of 8 nm in diameter was prepared by the method of
High-pressure Freezing
Male Wistar rats, 810 weeks old, were deeply anesthetized with diethylether and sodium pentobarbital and small fragments of the stomach were excised. They were promptly cut into 0.20.3-mm slices and sandwiched in the cavity of a supporting aluminum plate (
Freeze Substitution and Embedding
Freeze substitution was carried out using a Reichert AFS system (Leica; Vienna, Austria). After programmed warming to -90C at 3C/hr, samples were freeze-substituted at -90C for 20 hr in acetone containing 0.5% glutaraldehyde in which a molecular sieve had been added before use. After programmed warming to -30C at 10C/hr, the substitution medium was replaced with pure ethanol (three changes each of 10-min duration) and then gradually raised to 18C and left for 2 hr to remove the remaining hydration shell of a protein (
Chemical Fixation
As a control experiment for cryoimmobilization, we prepared specimens by conventional processing. Rats were deeply anesthetized with diethylether and sodium pentobarbital and then perfused with 2% paraformaldehyde2.5% glutaraldehyde in 0.1 M phosphate buffer (PB). The stomach was removed and further fixed by immersion in the above fixative at 4C overnight. After washing in PBS for 2 hr, specimens (about 1 mm3 in size) were dehydrated in a graded series of ethanol in water at progressively lower temperature: 30% for 30 min at 0C; 50% for 60 min at -20C; 70, 90, and 100% for 60 min at -35C. Infiltration with Lowicryl K4M and polymerization were performed as described above.
KMnO4UA/Pb Staining
The KMnO4UA/Pb staining was performed as described in the previous publication (
Combination of PLC1 Immunolabeling and KMnO4UA/Pb Staining
Ultrathin sections of the cryoimmobilized specimens were treated with 50 mM Tris-HCl buffer, pH 7.2, containing 5% normal goat serum (NGS), 0.2% BSA, 0.3 M NaCl, and 0.1% Tween-20 for 30 min to block nonspecific binding and then incubated with anti-PLC1 antibody (diluted 1:30 with 1% BSA in PBS) at 4C overnight as a specific marker for the zymogen granules of rat gastric chief cells (
1 antibody was replaced by normal mouse serum or omitted from the procedure.
Combination of GSA-II Labeling and KMnO4UA/Pb Staining
Ultrathin sections of the cryofixed specimens were treated with 1% BSA in PBS for 10 min to block nonspecific binding and then incubated with 5 µg/ml biotinylated GSA-II (diluted with 1% BSA in PBS) for 40 min at RT. After washing with PBS, the sections were incubated for 30 min with goat anti-biotin-IgG-CG (8 nm) conjugate (diluted with 1% BSA in PBS). After washing with DW and drying, the KMnO4UA/Pb staining was performed as mentioned above. As a control experiment, GSA-II was pre-incubated with 0.2 M N-acetyl-D-glucosamine for 30 min and then applied to sections.
Combination of CCG Labeling at pH 1.0 and KMnO4UA/Pb Staining
After blocking with 1% BSA in PBS for 10 min, the sections were passed through 50 mM Tris-HCl buffer containing 0.2% BSA (Tris-HCl/BSA) at pH 1.0 and then incubated with CCG (8 nm) at pH 1.0 for 40 min at RT. After brief incubation with Tris-HCl/BSA at pH 1.0, the sections were washed with DW. Subsequently, the KMnO4UA/Pb staining was performed. As a control experiment, sections were pre-incubated in 0.1 mg/ml non-labeled poly-L-lysine in Tris-HCl/BSA at pH 1.0 for 30 min before incubation with CCG at pH 1.0.
Dual Labeling of CCG at pH 1.0 and GSA-II Followed by KMnO4UA/Pb Staining
Ultrathin sections were first labeled with CCG (8 nm) at pH 1.0 and then labeled with the biotinylated GSA-II followed by goat anti-biotinIgGCG (14 nm) conjugate, according to each protocol mentioned above. Subsequently, the KMnO4UA/Pb staining was performed.
Dual Labeling of RGM11 Antibody and GSA-II Followed by KMnO4UA/Pb Staining
Ultrathin sections were first labeled with GSA-II as mentioned above. After washing with PBS, the sections were incubated with hybridoma medium of RGM11 antibody overnight at 4C. After washing with PBS, the sections were incubated with a mixture of goat anti-biotinIgGCG (14 nm) conjugate and goat anti-mouse IgM antibodyCG (8 nm) conjugate for 30 min. Subsequently, the KMnO4UA/Pb staining was performed. For controls, RGM11 antibody was replaced by normal mouse serum or omitted from the procedure.
GSA-II Labeling for Conventionally Fixed Specimens
To compare with the cryoimmobilized specimens, ultrathin sections of the chemically fixed ones were stained with the biotinylated GSA-II followed by goat anti-biotinIgGCG (8 nm) conjugate described above. Then the sections were contrasted with UA/Pb staining.
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Results |
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Base Region
The PLC1 immunolabeling was clearly co-localized on the zymogen granules as well as the exocytosed zymogenic contents, which intensively reacted to KMnO4UA/Pb staining (Fig 1A). The combination of GSA-II labeling for MNC mucin and KMnO4UA/Pb staining distinguished the zymogenic contents from the MNC mucin in the gastric glandular lumen (Fig 1B). The electron-opaque exocytosed zymogenic contents showed a droplet-like appearance forming the distinct seromucous interface. The intense GSA-II labeling was seen exclusively on the electron lucent area of the glandular lumen as well as the bipartite granules of intermediate cell (
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Neck Region
The combination of GSA-II labeling and KMnO4UA/Pb staining revealed that the exocytosed zymogenic contents, ascending to the pit region, still kept the seromucous interface and remained the droplet-like appearance at the neck region (Fig 2A). It should be noted that the GSA-II labeling was seen on the electron-lucent area but was absent on the exocytosed zymogenic contents in the glandular lumen. For the chemically fixed samples, the intense GSA-II labeling was confined to the mucous granules and the apical membrane of MNCs. In contrast, only a few gold particles were found in the glandular lumen (Fig 2B). This indicated poor preservation of the intraluminal fluid contents processed by conventional fixation. It was noteworthy that the PLC1 immunolabeling still remained within the exocytosed zymogenic contents (Fig 2C).
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Isthmus Region
Undifferentiated, granule-free stem cells predominated in this region. The appearance of excretory flow in the glandular lumen was almost the same as that of the neck region (not shown).
Low and Mid Pit Regions
Another type of mucin, sulfated mucin, which was specifically labeled with CCG at pH 1.0, was secreted from the low and mid pit cells. In the glandular lumen, dual labeling of CCG at pH 1.0 and GSA-II differentially stained the sulfated mucin and the MNC mucin, respectively (Fig 3). On a cross-section at the low pit region, the MNC mucin was confined to the central portion of the glandular lumen, surrounded by the sulfated mucin secreted from the low pit cells (Fig 3A). It should be noted that a distinct interface was formed between the two types of mucins. At the mid pit region, the MNC mucin, the sulfated mucin, and the non-sulfated pit cell mucin faced each other, spatially occupying the distinct territories in the glandular lumen (Fig 3B).
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High Pit Region
The combination of GSA-II labeling and KMnO4UA/Pb staining demonstrated restricted paths, designated as channels, in the glandular lumen (Fig 4A and Fig 4B). It should be noted that the appearance of exocytosed zymogenic contents changed into several electron-dense streaks with KMnO4UA/Pb staining (Fig 4A). Close to the top pit region, the streaks of zymogenic contents were vividly labeled with GSA-II. These results imply that the zymogenic contents merge into the MNC mucin, forming several streaks. With dual labeling of RGM11 antibody and GSA-II, the MNC mucous channels were topologically surrounded by RGM11-positive mucin in the glandular lumen (Fig 4C). On the other hand, CCG labeling at pH 1.0 clearly demonstrated the sulfated mucous channels in the glandular lumen (Fig 4D). Furthermore, the dual labeling of CCG at pH 1.0 and GSA-II verified that both types of mucous channels merged with each other, forming topological compartments at the high pit region (Fig 4E).
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Top Pit Region
The combination of GSA-II labeling and KMnO4UA/Pb staining demonstrated the laminated arrays of the MNC mucin in the surface mucous gel layer (Fig 5A). The MNC mucin laminae showed high electron density with KMnO4UA/Pb staining, indicating the presence of the zymogenic content. The CCG labeling at pH 1.0 also demonstrated the sulfated mucin laminae in the surface mucous gel layer (Fig 5B). Furthermore, dual labeling with the RGM11 antibody and GSA-II demonstrated an alternating laminated array consisting of MNC mucin and RGM11-positive mucin (Fig 5C).
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Discussion |
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The HPF/freeze substitution method provided the deep vitreous freezing that permitted the cryo-immobilization of fluid contents in rat gastric glandular lumen. As shown in Fig 2A and Fig 2B, the glandular lumen of the cryofixed gastric glands was filled with GSA-II-reactive mucin content and exocytosed zymogenic content, whereas that of the chemically fixed specimens was almost negative for GSA-II labeling. These results imply that conventional processing is inadequate to examine the intraluminal fluid contents and that HPF processing therefore appears to be superior in terms of preservation of extracellular fluid content close to the living state. By means of the combination of KMnO4UA/Pb staining and colloidal gold labeling for various gastric mucins, we demonstrated the excretory flow in rat gastric fundic gland, from the base to the pit region, as summarized in Fig 6. As previously reported, the KMnO4UA/Pb staining enhanced the overall contrast, without remarkable reduction of immuno-gold labeling, which allowed us to define the antigen localization with high accuracy (
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The combination of PLC1 immunolabeling and KMnO4UA/Pb staining demonstrated that most of the exocytosed PLC
1 molecules were co-localized to the electron-opaque areas, reactive with KMnO4UA/Pb staining, in the glandular lumens. These results reconfirmed that KMnO4UA/Pb sequence produced adequate differential staining not only of zymogen granules but also of exocytosed zymogenic contents in the glandular lumen (
The combination of GSA-II labeling and KMnO4UA/Pb staining spatially distinguished the exocytosed zymogenic contents from the MNC mucin in the glandular lumen. Interestingly, at the base and neck regions, the exocytosed zymogenic contents showed a droplet-like appearance forming the distinct seromucous interface between the MNC mucin and the zymogenic contents. Previously, we applied periodic acidthiocarbohydrazidesilver proteinate (PATCHSP) staining on rat gastric glands processed by HPF/freeze substitution (
On reaching the mid or the high pit region, the droplet-like appearance of the zymogenic contents was altered in the bundles of several streaks and merged with the MNC mucous channels. These remarkable changes in rheological property may be explained by the proteolytic degradation of the MNC mucin (
The present study, to the best of our knowledge, provides the first evidence of the in vivo intraluminal mucous channels in rat gastric glandular lumen. Ever since
The surface mucous gel layer at the top pit region consisted of alternating laminated arrays of MNC mucin, sulfated mucin, and RGM11-positive pit mucin.
These results are likely to reflect some physicochemical disparities among the different mucins. The rheological properties of a mucous gel are characterized by the parameters of viscosity and elasticity (
In conclusion, we have found the in vivo paths of the excretory flow of zymogenic and mucin contents in rat gastric glands. The present histochemical findings are capable of providing some clues to the rheological and biochemical analysis of the fluid dynamics of gastric secretions.
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Acknowledgments |
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Supported by grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (08670024) and from the Japan Society for the Promotion of Science (13670019).
We thank Yoshiteru Goto (Division of Electron Microscopy, Central Research Laboratories, Miyazaki Medical College), Soyuki Ide, and Eiko Matsuura (Department of Anatomy, Miyazaki Medical College) for expert assistance.
Received for publication July 13, 2001; accepted September 26, 2001.
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