LOCALIZATION OF CARBONIC ANHYDRASE ACTIVITY IN TURTLE AND TOAD URINARY BLADDER MUCOSA

Transcription

1 Ti JOURNAL OF HISTOCHEMISTRY AND CYTOCHEM1STRY Copyright 1972 by The Histochemical Society. Inc. Vol. 20, No. 9. pp , 1972 Printed in U.S.A. LOCALIZATION OF CARBONIC ANHYDRASE ACTIVITY IN TURTLE AND TOAD URINARY BLADDER MUCOSA SEYMOUR ROSEN Department of Pathology, Beth Israel Hospital, and Harvard Medical School, Boston, Massachusetts Received for publication June 18, Carbonic anhydrase activity was demonstrated in a distinct population of mucosal cells of whole stretched bladder of turtle and toad. The apical portions of the cells containing the enzyme were discretely stained as noncontiguous polygonal forms and allowed an estimation of the luminal surface representation of this epithelium. The slight luminal surface (O.8 or less) occupied by cells with carbonic anhydrase activity in the toad bladder contrasted with a greater representation in turtle bladder ( %) and correlated with the relative capacity of these tissues to acidify. After completion of the enzyme staining reaction the tissues of the turtle bladder were directly processed for electron microscopic observations without osmium tetroxide postflxation. The deposition of the electron-opaque cobalt sulfide compound was selective and was found only in cells with luminal representation and small apical granules, the recently described third cell type ; cobalt sulfide was mainly present in the cytoplasm, an observation consonant with the known localization of carbonic anhydrase primarily in supernatant fractions. Recently carbonic anhydrase activity was demonstrated histochemically in a distinct population of the urinary bladder mucosal cells of turtle and toad (9). Biochemical investigations confirmed the presence of the enzyme in the turtle bladder mucosa (9, 11, 13) and are consonant with physiologic studies indicating that the secretion of H by the turtle bladder is inhibited by acetazolamide and is dependent on the enzymatic hydration of CO2 (12, 14, 15). The occurrence of carbonic anhydrase in the toad bladder, a tissue initially thought to have no mechanism of acidification (9), is of interest since the acidifying capacity of toad bladder is currently being reexamined (2). It is the purpose of this study to report the distribution of cells with carbonic anhydrase activity in whole bladder preparations of toad and turtle. The surface area of cells that reveal enzyme activity has been determined quantitatively and it is suggested that there may be a correlation between the luminal representation of cells with carbonic anhydrase activity and the capability of these urinary epithehia to acidify. In addition, this communication describes the adaptation of the histochemical 1 This study was supported in part by U.S. Public Health Service Grant RO1AM Revised manuscript received September 27, method for electron microscopic observations and its use in the specific identification of carbonic anhydrase-active cells. MATERIAL AND METHODS Bladders were obtained from pithed toads (Bufo marinus) of Dominican Republic origin and decapitated turtles (Pseudemys scripta). They were mounted between two halves of a Lucite chamber, washed with Ringer s solution, fixed in situ with 0.17 M cacodylate-buffered 3% glutaraldehyde for 1-2 hr and then washed three times in saline. The whole bladder was then divided into 1-2-cm2 portions, picked up on Millipore filters (25 p thick, pore size 0.45 p) and briefly stored in Petri dishes containing saline-soaked gauze until the histochemical reaction was carried out. The technique described by Hansson (3, 4) was used. The sections were floated on the surface of a freshly prepared incubation mixture containing 1.75 x 10 M CoSO4, 5.3 x 10 M H2S04, 1.57 x 101 M NaHCO3 and x 102 M KH2PO4. The reaction is dependent on the loss of CO2 at the surface, local hydroxyl accumulation and precipitation of a cobalt compound. The incubation solutions were allowed to stand for 20 mm at room temperature and then the filter paper was gently dropped onto the surface. Tissues were always facing up; if the paper was permitted to sink below the surface of the medium, there was no reaction. Incubation time was 12 mm at high concentrations of KH2PO4 and 30 mm at low concentrations. The control solutions contained acetazolamide at a concentration of 10-#{176}M. The tissues were then 696

2 CARBONIC ANHYDRASE 697 washed in a 6.7 x 10 M solution of KH2PO4, ph 5.9, exposed to a 0.6% ammonium sulfide solution and rinsed in saline. The tissue was either mounted with glycerol jelly or dehydrated and mounted with Permount. Bladders were photographed and the surface area represented by cells showing enzyme activity was estimated by cutting out and weighing individual stained cells from the print. Some bladders, after the histochemical procedure, were frozen with Dry Ice onto cryostat chucks using Tissue-tek (Ames) as an embedding medium. Sections were cut in an International Harris cryostat and picked up on a Millipore filter which was subsequently dehydrated and mounted with Permount. For electron microscopic observations, four whole bladders were washed three times with saline after the completion of the histochemical staining reaction. The tissues were then directly dehydrated and embedded in Epon 812 (6). Postfixation with osmium tetroxide solubilized the precipitate and therefore could not be employed. Survey sections were cut at 1 p and observed both stained with 1% methylene blue and unstained. The blocks were trimmed further and ultrathin sections were exam- FIG. 1. Whole toad bladder, fixed with buffered 3% glutaraldehyde after mounting between two halves of a Lucite chamber: a, a distinct population of cells with carbonic anhydrase activity can be seen; the dark lines represent blood vessels; b, at higher magnification, densely stained geometric areas (arrows) and faintly stained nuclei (n) are seen as cellular constituents. Figure la, xloo; b, x640.

3 698 ROSEN med either unstained or stained with uranyl acetate (16) in a Philips EM-200 electron microscope. RESULTS Light microscopic observations: Toad (Fig. 1): In whole bladder preparations, a distinct group of noncontiguous stained ovoid cells was noted. The stain was prominent in peripheral areas of these cells; more centrally, the staining took the form of a sharply demarcated triangular or rectangular region which was considerably smaller than the occasionally stained nucleus. Because this geometric area was in the plane of focus of the superficial aspect of adjacent cells whose hexagonal forms could sometimes be faintly discerned, it was considered to represent the surface region of the cell. In three bladders, these regions formed, respectively, I S. I S a,; S. S a. #{149} 0. 4 S : lb. #{149}w_. #{149} #{149}IUI - V,l a #{149} / -- O lbs N I. a b- a 0. fr FIG. 2. Whole turtle bladder, fixed with buffered 3% glutaraldehyde after mounting between two halves of a Lucite chamber: a, a distinct population of cells with carbonic anhydrase activity can be seen; b, at higher magnification these cells are represented as intensely stained polygonal areas. Figure 2a, x 140; b, x640.

4 -.. S. b....., : FIG. 3. Whole turtle bladder, fixed with 3% glutaraldehyde, stained for carbonic anhydrase activity and processed for electron microscopic observations without osmium tetroxide postfixation. a, the apical areas of the cell contain electron-dense precipitate. The inset shows a methylene blue-stained section of turtle bladder mucosa. Note the distinctly stained upper aspect of the center cell. b, higher power reveals the relative restriction of the electron-dense material to cytoplasmic sap. In particular, the mitochondria and small apical granules contain little reaction product. Note the portion of adjacent cell, unstained and sharply demarcated. The large clear ovoid areas in this cell represent the mucin granules characteristic of the usual superficial cell. Figure 3a, x ; inset, x800; b, x47,

5 700 ROSEN 0.3, 0.4 and 0.8% of the total surface area. Sections of the whole bladder after staining revealed either complete cell staining or, in well oriented sections, a dense superficial aspect sometimes accompanied by nuclear staining. Turtle (Fig. 2): The stained cells in this preparation were in the form of geometric shapes, i.e., triangular, rectangular and pentagonal. They were commonly separated but occasionally touched at angles or, more rarely, were immediately adjacent. Unstained cells, frequently in a hexagonal form delineated by a fine, apparently intercellular precipitate, were sometimes noted. Occasionally, the subjacent portion of a cell stained faintly. When this occurred, the densely stained, geometric configuration was seen to represent only the superficial aspect of the cell. Sections of whole bladder, after staining, confirmed precipitate localization in the superficial aspect of the epithelial cells. In five bladders, the active cells constituted, respectively, 6.4, 8.3, 9.0, 9.3 and 12.2% of total surface area. Histograms, constructed from 691 stained cells, showed a complete range of individual cell surface area in all bladders. In two, the curves were skewed to large surfaced Ti; FIG. 4. Electron micrographs of portions of two cells stained for carbonic anhydrase activity. The electrondense precipitate is mostly localized to the apical aspect of the stained cell (a) but extends into lateral cell processes that interdigitate with adjacent processes containing little precipitate. b, the luminal plasma membrane can be faintly seen (arrows) in this high magnification and appears to delineate the precipitate. Figure 4a, x27,500; b, x135,600.

6 CARBONIC ANHYDRASE 701 cells; the one with the smallest stained surface area was skewed to the smaller surfaced cells; the other two bladders had a bimodal distribution. Electron microscopic observations: The turtle bladder mucosa consists of two major kinds of cells: a superficial cell fronting the lumen and containing many mucin granules and a basal cell distinguished by a relatively high nuclear to cytoplasmic ratio. Less commonly, a third cell type was observed, characterized by a more restricted surface area, smaller granules and a bulky basilar portion extending to the basement membrane (8). In unstained cross-sections of turtle bladder embedded in Epon after the histochemical procedure, the precipitate appeared brown-black and in preparations stained with methylene blue was seen localized to the superficial areas of certain cells (Fig. 3a, inset). Using the small apical granules as the most important criterion for identification, electron microscopic observations revealed that these cells were of the third cell type, with considerable variation in luminal representation (Figs. 3-5). The finely granular electron-dense material was present in the apical regions of such cells and could be seen in both stained and unstained thin sections. This material appeared almost entirely confined to the cytoplasmic sap; in particular, mitochondria, the small granules and lysosomes contained only slight amounts. The sharp limitation of the material to the cytoplasm was particularly evident at cell junctions and in areas where the plasma membranes could be observed. The lack of osmium postfixation resulted generally in poor visualization of membranes (10). Occasional intercellular deposits were noted. The controls using 10 M acetazolamide showed no precipitate. FIG. S. Sections of a portion of the same cell stained for carbonic anhydrase. In a the section is unstained. b has been stained with uranyl acetate. The precipitate is distinctly noted in both electron micrographs. Figure 5a, x 19,000; b, x27,soo. DISCUSSION The relation of the stained cell to mucosal elements of toad and turtle bladder as delineated in fine structural studies (1, 8) is of great interest. The toad bladder has four kinds of epithelial cells: granulated cells that form most of the bladder surface, mitochondria-rich cells, goblet cells and basal cells. The mitochondria-rich cells, characterized by a flask shape and relatively limited surface representation, are the most likely to correspond to the cells with carbonic anhydrase activity. In the turtle bladder, these cells are identified in this report as the so-called third cell type, which may represent the homologue of the mitochondna-rich cell (8). Although restriction of the activity to the apical portion of the cell allows calculation of surface representation, the meaning of this localization must be interpreted with caution. In fact, the staining of the entire cell in sections (9) and the known localization of carbonic anhydrase activity in supernatant fractions (7) suggest that this discrete apical staining may be the result of incomplete substrate penetration. Nuclear staining, sometimes particularly prominent in the toad bladder, may be related to the use of the whole bladder in the histochemical procedure; distinct nuclear staining was not usually present in sections incubated for the demonstration of enzyme activity. Recent physiologic studies have prompted speculations on the special importance of carbonic anhydrase activity in H ion formation, especially under circumstances of limited CO2 supply (11, 12). Simple alteration of cellular luminal representation may be a further factor in the varying capability of turtles to secrete H ions-the latter averaging about 1.0 tmole/8 cm2 or 14 mg dry weight (14, 15). In addition, the low levels of secretion in the toad bladder, averaging approximately 0.1 tmolefhr for equal amounts of tissue (calculated from Frazier and Vanatta (2)), correlate with the limited surface representation of the carbonic anhydrase-active cells in this tissue. Carbonic anhydrase activity has been demonstrated electron histochemically in two studies. In an abstract, Hopsu, Arstila and Helminen (5) reported its localization at the surface of erythrocytes, in the brush border of

7 702 ROSEN the proximal tubular cells of the kidney and in the apical plasma membrane of distal renal tubules. Yokota (17), studying mouse liver, found hepatocytic, endothelial and erythrocytic plasma membrane localization. In the light microscopic method for carbonic anhydrase, the reaction is dependent on a catalyzed surface CO2 loss, local hydrozyl accumulation and precipitation of a cobalt compound containing phosphate which is then converted to sulfide for visualization (3). Both studies (5, 17) converted the cobalt phosphate compound to lead phosphate for electron microscopic visualization. The present investigation employs the material stained for light microscopic observations and directly processes it for electron microscopic studies without osmium postfixation. The advantage of this technique is that it permits initial light microscopic visualization of the staining reaction and the substitution of an anion rather than a heavy metal cation. The presence of the stain in the cytoplasmic sap in this study is consonant with the major localization of carbonic anhydrase in supernatant fractions (7). This finding, however, differs from Hansson s light microscopic observations of distinct staining of membranes bordering extracellular channels in a number of other epithelial tissues noted for active transport (4). The stained cells were different from other luminal cells and consistently contained small granules characteristic of the third cell type (8). The localization of carbonic anhydrase activity to one cell type suggests a distinctive function and, in particular, a capacity on the part of these cells for high rates of acidification. ACKNOWLEDGMENTS I wish to thank Drs. Philip Steinmetz, John Schwartz and Richard Cohen for their advice and criticism. The technical assistance of Mrs. Doris Hayes is gratefully appreciated. REFERENCES 1. Choi JK: The fine structure of the urinary bladder of the toad Bufo maninus. J Cell Biol 16: 53, Frazier LW, Vanatta JC: Excretion of H and NH4 by the urinary bladder of Bufo marinus in metabolic acidosis. Fed Proc 30:365, Hansson H: Histochemical demonstration of carbonic anhydrase activity. Histochemie 11:112, Hansson H: Histochemical demonstration of carbonic anhydrase activity in some epithelia noted for active transport. Acta Physiol Scand 73:427, Hopsu VK, Arstila AU, Helminen H: Studies on the electron microscopy of arylsulphatase and carbonic anhydrase activities. J Histochem Cytochem 14:762, Luft JH: Improvement in epoxy resin embedding methods. J Biophys Biochem Cytol 9:409, Maren TH: Carbonic anhydrase: chemistry, physiology, and inhibition. Physiol Rev 47:595, Rosen 5: The turtle bladder. I. Morphological studies under varying conditions of fixation. Exp Mol Pathol 12:286, Rosen 5: Localization of carbonic anhydrase in transporting urinary epithelia. J Histochem Cytochem 18:668, Sabatini D, Bensch K, Barrnett RJ: Cytochemistry and electron microscopy. J Cell Biol 17:19, Schwartz JH, Rosen 5, Steinmetz PR: Carbonic anhydrase function and the epithelial organization of H secretion in the turtle bladder. J Clin Invest, in press 12. Schwartz JH, Steinmetz PR: CO2 requirements for H secretion by the isolated turtle bladder. J Physiol 220:2051, Scott WN, Shamoo YE, Brodsky WA: Carbonic anhydrase content of turtle urinary bladder mucosal cells. Biochim Biophys Acta 219:248, Steinmetz PR: Characteristics of hydrogen ion transport in urinary bladder of water turtle. J Clin Invest 46:1531, Steinmetz PR: Acid-base relations in epithelium of turtle bladder: site of active step in acidification and role of metabolic CO2. J Clin Invest 48: 1258, Watson ML: Staining of tissue sections for electron microscopy with heavy metals. J Biophys Biochem Cytol 4:475, Yokota 5: Electron microscopic demonstration of carbonic anhydrase activity in mouse liver cells. Histochemie 19:255, 1969