ARTICLE |
Correspondence to: Eleni Roussa, Anatomisches Institut, Medi-zinische Fakultät, Universität des Saarlandes, 66421 Homburg/Saar, Germany.
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Summary |
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Using antibodies against the 31-kD and 70-kD subunits of vacuolar type H+-ATPase (V-ATPase) and light microscopic immunocytochemistry, we have demonstrated the presence of this V-ATPase in rat submandibular gland. We have also investigated the adaptive changes of this transporter during acid-base disturbances such as acute and chronic metabolic acidosis or alkalosis. Our results show intracellularly distributed V-ATPase in striated, granular, and main excretory duct cells in controls, but no V-ATPase immunoreaction in acinar cells. Both acute and chronic metabolic acidosis caused a shift in V-ATPase away from diffuse distribution towards apical localization in striated and granular duct cells, suggesting that a V-ATPase could be involved in the regulation of acid-base homeostasis. In contrast, during acidosis the main excretory duct cells showed no changes in the V-ATPase distribution compared to controls. With acute and chronic metabolic alkalosis, no changes in the V-ATPase distribution occurred. (J Histochem Cytochem 46:91-100, 1998)
Key Words: salivary glands, proton pump, immunocytochemistry, ion transport
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Introduction |
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The two-stage hypothesis of salivary secretion, first described by
Studies of secretory mechanisms in the duct system were originally performed in the main excretory duct because of its accessibility for microperfusion studies (
The salivary duct epithelium responds to acid-base changes in the blood by marked changes in secretion of HCO3-. During metabolic acidosis, ductal HCO3- secretion is abolished, whereas during metabolic alkalosis HCO3- secretion is enhanced (
In the kidney the cellular mechanisms involved in the regulation of acid-base homeostasis have been studied in more detail (
A vacuolar proton pump that is physiologically and structurally identical to the kidney V-ATPase has also been found in osteoclasts, which appears to be responsible for creation of the acidic microenvironment fundamental to bone resorption (
In the present study, we have determined the localization of the V-ATPase in the secretory endpieces and segments of the duct system in rat submandibular gland by immunohistochemistry. Furthermore, we have assessed the distribution of this enzyme during acute and chronic metabolic acidosis or alkalosis.
Our results demonstrate the presence of an intracellularly distributed V-ATPase in the entire duct system but its absence in acinar cells of the rat submandibular gland. Metabolic acidosis induces a redistribution of the H+-ATPase to the apical side in striated and granular duct cells but not in main excretory duct cells. In contrast, no adaptive change of the distribution of V-ATPase was found during metabolic alkalosis.
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Materials and Methods |
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Materials
Picric acid, formaldehyde, acetic acid, Triton X-100, and Tween 20 were obtained from Merck (Darmstadt, Germany). Gelatin, 3,3-diaminobenzidine, naphthol AS-MX phosphate, and 4-benzoylamino-2,5-dimethoxy-benzenediazonium chloride hemi [zinc chloride]salt (Fast Blue RR Salt) were from Sigma (Deisenhofen, Germany). Dimethylformamide was obtained from Serva (Heidelberg, Germany). Vogel Histo-Comp was from Vogel (Giessen, Germany). Enhanced chemiluminescence reagents were from Amersham-Buchler (Braunschweig, Germany). Nonfat dry milk and prestained protein standards were obtained from Bio-Rad (Munich, Germany). Polyvinylidene difluoride (PVDF) membranes and X-ray films were from Dupont (Bad Homburg, Germany). Pefabloc SC was obtained from Boehringer (Mannheim, Germany). All other reagents were of analytical grade.
Methods
Thirty-six male Wistar rats (200-300 g), six rats for each experimental group, were used. Acute metabolic acidosis and alkalosis were induced by gastric administration of 1.5 M NH4Cl (2.4 ml/100 g bw) or of 0.976 M NaHCO3 (5 ml/100 g bw), respectively. Control animals were treated with 1.37 M NaCl (2.4 ml/100 g bw).The animals were sacrificed 4-6 hr later. Chronic acidosis and alkalosis were induced according to
The animals were anesthetized with pentobarbital. After collection of arterial blood from the abdominal aorta and urine from the urinary bladder, pH, pCO2, HCO3-, base excess, and %O2 saturation were determined in blood and urine. Subsequently, the animals were sacrificed and the submandibular glands were removed. Glandular tissue was fixed by immersion in Bouin's fixative (water-saturated 71.4% picric acid, 23.8% formaldehyde, and 4.76% acetic acid) for 48 hr at 4C, dehydrated through graded series of isopropanol (80%, 96%, and 100%) and methylbenzoate (100%), and embedded in paraffin. The paraffin blocks were cut in serial 5-µm sections.
Immunocytochemistry
For localization of the vacuolar-type H+-ATPase, indirect enzyme immunocytochemistry was applied. Antibodies against the A- and E-subunits of the bovine H+-ATPase were produced by immunizing rabbits with synthetic peptides coupled to hemocyanin, containing the amino acid sequence of the C-terminus of bovine H+-ATPase A- and E-subunits, DMQNAFRSLED and CGANANRKFLD, respectively, similarly as described by
After deparaffinization the sections were rinsed with water for 20 min and washed three times for 5 min with 0.05 M Tris buffer, pH 7.4. The slides were incubated with the primary antibody (1:200 dilution) at room temperature (RT) overnight in 0.05 M Tris buffer containing 0.04% sodium azide, 0.25% gelatin, and 0.06% Triton X-100 to reduce nonspecific background staining. Slides were washed three times for 5 min in 0.05 M Tris buffer and incubated with the secondary antibody (1:50 dilution) for 2 hr at RT in 0.05 M Tris buffer containing 0.25% gelatin and 0.06% Triton X-100. Finally, the sections were rinsed three times for 5 min in 0.05 M Tris buffer, and peroxidase activity was visualized with diaminobenzidine and hydrogen peroxide. Alkaline phosphatase was developed using freshly made solutions of 200 mg Fast Blue RR Salt in 200 ml 0.05 M Tris and addition of 20 mg naphthol AS-MX Phosphate in 500 µl dimethylformamide.
The same immunocytochemistry protocol was applied in six rats from each of the six groups (i.e., acute control, acute acidosis, acute alkalosis, chronic control, chronic acidosis, and chronic alkalosis) and six randomly selected sections from each animal were taken for one analysis. This procedure was repeated on different days at least 10 times. In total, at least 2160 sections were analyzed. Additional procedures were also performed using serial sections from adjacent tissue. Antibodies to the 31-kD and 70-kD subunits of the vacuolar H+-ATPase gave identical immunolabeling patterns. Control incubations without primary antibody, with preimmune serum, or preincubation with 0.5 mM synthetic peptide against which the antibodies had been raised gave no detectable staining. Sections of rat kidney were used as positive controls.
Labeled sections were examined with a light microscope (Olympus; Tokyo, Japan) and photographed. For cell counting, only cells with a distinct nucleus were included. The distribution of the immunoreactivity was classified into absent, apical, basolateral, or diffuse localization. At least 500 cells were counted in each section. Granular duct cells were subclassified with respect to their morphology into wide dark granular, light granular, and narrow agranular cells.
All values were expressed as mean ± SD. Unpaired Student's t-test was used for statistical analysis. p<0.05 was considered statistically significant.
Preparation of Cortical Brush Border Membranes, Endocytotic Vesicles, and Submandibular Gland Homogenate
Renal cortical brush border membrane vesicles (BBMVs) were isolated by the Mg2+/EGTA precipitation method described by
Endocytotic vesicles from renal cortical homogenates were isolated by differential and Percoll density gradient centrifugation as described by
SDS-PAGE and Western Blotting
Electrophoresis and blotting procedures were performed essentially as described earlier (
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Results |
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Systemic Acid-Base Parameters
NH4Cl administration produced acute metabolic acidosis, with a blood pH of 6.95, a base excess of -24.50 mM, and a urine pH of <6.0, compared to control values of 7.39, -2.13, and 6.46, respectively. NaHCO3 administration produced acute metabolic alkalosis with a blood pH of 7.47, a base excess of 12.48 mM, and a urine pH of 7.95. Chronic acidotic animals had a blood pH of 7.10, a base excess of -15.80 mM, and a urine pH of <6.0, compared to control values of 7.31, -3.90, and 6.30, respectively. Chronic alkalotic animals had a blood pH of 7.37, a base excess of -0.79 mM, and a urine pH of 7.98.
Figure 1 shows a Western blot analysis of endosomal vesicles, cortical brush border membrane vesicles (BBMV), and submandibular gland homogenate (Ho) with the polyclonal antibodies against the 31-kD and 70-kD H+ pump subunits raised as described in Materials and Methods. Similarly to endosomal and renal cortical brush border membranes, salivary gland homogenate showed immunoreactivity to V-ATPase subunits. However, reactivity of submandibular gland homogenate was less intense than in endosomal and brush border membranes.
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When immunocytochemistry was carried out, both 31-kD and 70-kD subunits could be localized in rat submandibular ducts but not in acinar cells. However, the pattern and the intensity of labeling varied among different duct segments.
V-ATPase Distribution in Controls
Figure 2 Figure 3 Figure 4 show the labeling pattern of V-ATPase in the different morphological segments of rat submandibular gland. A quantitative analysis of the results is provided in Table 1 and Table 2.
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In the main excretory duct cells (MD) of control animals, most cells showed a diffuse intracellular staining of weak to moderate intensity (Figure 2). Apical labeling was also observed in certain cells (Figure 2, arrowheads), representing approximately 3% of main duct cells (Table 1 and Table 2). In addition, V-ATPase-containing cells were separated from each other by nonreactive cells (Figure 2; arrows, Table 1 and Table 2).
In contrast to main excretory duct cells, V-ATPase immunoreactivity was present in all cells of the striated ducts (S) in control animals (Figure 3A and Figure 3B). Striated ducts showed homogeneous, diffuse intracellular labeling of moderate to strong intensity (Figure 3A and Figure 3B). Apical V-ATPase distribution was also detected in 5-6% of striated duct cells (Figure 3B, arrows; and Table 1 and Table 2). Control incubations using preimmune serum gave no detectable staining (Figure 3C).
Granular ducts, which are believed to be modified striated ducts and form the proximal segment of striated ducts, showed a complex pattern of V-ATPase immunoreactivity (G, Figure 4A and Figure 4B). Granular duct cells without any V-ATPase immunoreactivity (G0, Figure 4A) as well as granular duct cells showing diffuse labeling of weak intensity (G1, Figure 4A) were observed in the same section. Moreover, as can be seen from Figure 4A, close to the weakly stained segments, segments with intense diffuse (G2) or apical staining (arrow) were found. This variability was present in different granular ducts in all animals and also within the same duct, which reflects the morphological heterogeneity of these ducts. In wide dark granular cells (G0, Figure 4A), no V-ATPase immunoreactivity was found. In light granular cells, in which the granules occupy less total volume than in dark granular cells, diffuse intracellular staining of weak intensity was observed (G1, Figure 4B). In contrast, agranular cells (Figure 4B) always showed V-ATPase immunolocalization of moderate to strong intensity. In 88-95% of narrow agranular cells V-ATPase distribution was diffuse intracellularly, with 5-10% of cells being apically stained (Figure 4B , arrows). Staining was eliminated by incubation of the antiserum with 0.5 mM peptide antigen (Figure 4C).
Intercalated ducts, which represent the most proximal part of the duct tree, connect the secretory endpieces with the granular ducts. Intercalated ducts showed no detectable H+-ATPase immunoreactivity (data not shown).
V-ATPase Distribution During Acidosis
During acute and chronic metabolic acidosis, no major changes in the pattern of H+-ATPase labeling or staining intensity in main excretory duct cells were observed, compared to controls (Table 1 and Table 2). As an example, Figure 5A shows V-ATPase distribution in main excretory duct cells during acute metabolic acidosis. In contrast, both acute and chronic metabolic acidosis affected the distribution of V-ATPase in striated duct cells (Figure 5B-D). In >90% striated duct cells (S), V-ATPase was apically located, suggesting that a redistribution of V-ATPase from the interior of the cell to the apical cell side had occurred. Statistical analysis of the data revealed significant differences between control and acidotic animals (Table 1 and Table 2; p<0.0001). The intensity of immunoreactivity was moderate in acute and strong in chronic metabolic acidosis. Granular duct cells showed a selective sensitivity to acidosis (G, Figure 5D and Figure 5E). In narrow agranular cells of the granular ducts, a redistribution of V-ATPase similar to that in striated ducts occurred (Figure 5D and Figure 5E, arrows), away from diffuse distribution towards apical localization (p<0.002; Table 1 and Table 2). In contrast, no changes in the staining pattern were observed in wide dark and light granular cells compared to controls. Wide dark granular cells remained unstained (Figure 5D, arrowhead), whereas light granular cells showed a diffuse cytoplasmic V-ATPase distribution (Table 1 and Table 2; Figure 5E, arrowhead).
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V-ATPase Distribution in Alkalosis
During acute and chronic metabolic alkalosis, no significant changes in V-ATPase immunoreactivity were observed compared to controls (see Table 1 and Table 2).
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Discussion |
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In the present morphological study a vacuolar H+-ATPase (V-ATPase) was detected in rat submandibular gland. Immunocytochemical methods were applied by which localization of the V-ATPase could be determined in different duct segments. Using antibodies against cytoplasmic domains of the 31-kD and 70-kD subunits of V-ATPase, we were able to find immunoreactivity in striated, granular, and main excretory duct cells. The distribution of V-ATPase was found to be diffuse, i.e., intracellularly located, in all duct segments. In contrast, secretory endpieces showed no detectable V-ATPase. In addition, we investigated the distribution of V-ATPase in acute and chronic metabolic acidosis or alkalosis to see if body acid-base disturbances might influence the distribution of this H+ transporter, similarly as reported in the kidney (
From studies of ion transport mechanisms (
In the main excretory duct, electron microscopy has shown the presence of three morphologically different cell types: dark cells, light cells, and basal cells (
The kidney plays a vital role in acid-base homeostasis and several studies have established the involvement of a vacuolar H+-ATPase in acid-base regulation (
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