Amount and Distribution of Carbonic Anhydrases CA I and CA II in the Gastrointestinal Tract

GASTROENTEROLOGY 1985;88:1151-61 Amount and Distribution of Carbonic Anhydrases CA I and CA II in the Gastrointestinal Tract GUDMAR LONNERHOLM, ORJAN SELKING, and PER J. WISTRAND Department of Medical Pharmacology. University of Uppsala, Uppsala, Sweden and Department of Surgery. University Hospital, Uppsala, Sweden The levels of carbonic anhydrase (CA) activity and the amounts of carbonic anhydrase isoenzyme CA I and CA II proteins in the human gastrointestinal tract were determined by kinetic assays and radioimmunoassays. Cellular distribution of carbonic anhydrase activity in the various segments of the gastrointestinal tract were studied by the histochemical method of Hansson and the distribution of CA I and CA II by an immunohistochemical method. The stomach and the colon showed high carbonic anhydrase activity, the jejunum had intermediate activity, and the ileum had low activity. In the stomach CA II was the dominating isoenzyme, whereas the jejunum and the colon contained considerable amounts of both forms. Small amounts of both isoenzymes were found in the ileum. Carbonic anhydrase II immunofluorescence was demonstrated in the surface epithelium and in the parietal cells of the gastric mucosa, and in the epithelium of the jejunal villi. The surface epithelium of the colon contained both CA I and CA II. Carbonic anhydrase I was found in many superficial capillaries in all regions studied. The histochemical method demonstrated enzyme activity also at the cell membranes of gastric chief cells, intestinal crypt cells, and a subpopulation of ileal surface cells. This probably indicates presence of the membrane-bound isoenzyme CA IV. Received August 14, 1984. Accepted November 19, 1984. Address requests for reprints to: Gudmar Liinnerholm, M.D., Department of Medical Pharmacology, Box 593, Biomedical Center, University of Uppsala, S-751 24 Uppsala, Sweden. This work was supported by the Swedish Medical Research Council grants No. 2874 and 5413. The authors thank Mona Schenholm for skillful technical assistance. 1985 by the American Gastroenterological Association 0016-5085/85/$3.30 The presence of carbonic anhydrase (CA) in the mammalian gastric mucosa was an early discovery by Davenport and Fisher (1) and its distribution and function in this tissue have been extensively studied in many species including humans (ef. 2). The enzyme is thought to playa role in the acid secretion of the gastric glands as acetazolamide, a specific inhibitor of the enzyme, reduces the output of hydrogen ions (3). Carbonic anhydrase is present also in the surface epithelium (4-12), and recent findings suggest that it takes part in alkaline secretion, which may protect these cells from damage caused by the low gastric intraluminal ph (13). The distribution of CA in the intestine has been studied in the dog (14), rat (15-17), mouse (15), guinea pig (6,18,19), monkey (11), and human fetus (12). Striking differences in enzyme activity and isoenzyme content between the various portions of the intestine were found in these species, but the physiologic significance of the findings is poorly understood (19,20). Some data on the presence of CA in the human stomach (4,8,), appendix (8,21), and colon (21).have been reported, but the distribution of the various forms of the enzyme in the adult human intestinal tract has not been studied systematically. We have recently reported on the distribution of the isoenzymes in the human fetal intestinal tract (12). The aim of the present study was to clarify the distribution of CA isoenzymes in these tissues of the adult human, as such data are necessary for a further discussion of the role of the enzyme. For this purpose histochemical, immunocytochemical, and biochemical (kinetic, immunologic) methods have been used to study tissue specimens obtained at surgical procedures. Abbreviations used in this paper: CA, carbonic anhydrase; anti CA, carbonic anhydrase antibody; PBS, phosphate-buffered saline.

1152 LONNERHOLM ET AL. GASTROENTEROLOGY Vol. 88, No.5, Part 1 Table 1. Carbonic Anhydrase Activities in the Human Gastric and Intestinal Mucosa Protein in Wet weight of whole Protein in % CA activity tissue homogenate supernatant %CA in supernatant No. of (enzyme (enzyme (enzyme activity in caused by Tissue samples units/g) units/mg) units/mg) pelleto RBC-CA b Stomach (fundus, corpus) Whole mucosa 3 282 (230-332) 3.00 (2.65-3.23) 4.47 (3.77-5.01) 6.5 (2.1-11) 5 (1-14) Jejunum C Whole mucosa 3 80 (55-119) 0.74 (0.55-1.07) 1.41 (0.45-2.54) 7.8 (4.3-10) 18 (11-28) Mucosal scraping 2 235 (202-268) 1.02 (0.92-1.12) 2.28 (1.59-2.97) d 12 (9-15) Ileum c Whole mucosa 2 23 (20-26) 0.22 (0.19-0.24) 0.40 (0.23-0.56) 7.5 33 (10-55) Mucosal scraping 3 21 (19-24) 0.22 (0.15-0.27) 0.39 (0.25-0.46) d 23 (5-32) Colon c ' Whole mucosa 5 157 (77-249) 1.79 (1.01-2.56) 3.15 (1.66-4.92) 3.7 (3.2-4.1) 5 (2-9) Mucosal scraping 3 405 (320-540) 4.07 (3.01-5.62) 5.21 (2.37-8.32) 8.1 (2-17) 5 (2-12) Values represent mean. Range is given in parentheses. CA, carbonic anhydrase; RBC, red blood cell. Assuming homogenate to have 100% activity. b Assuming CA activity in blood to be 1320 enzyme units/ml (unpublished data from this laboratory). The enzyme activity caused by RBC-CA in whole homogenate and pellet could not be calculated because turbidity prevented determination of the hemoglobin concentration. C Mucosal scraping and whole mucosa homogenates were always taken from different patients. d Not enough material to measure activity. Materials and Methods Tissue Preparation Tissue specimens were taken during surgical procedures, necessitated by neoplasms (ventricle, colon) or shunt operation for extreme obesity (small intestine). Only tissues that appeared normal macroscopically and microscopically were used. The number of tissue specimens examined by biochemical assays is shown in Table 1. For histochemical and immunohistochemical work tissues from at least 10 patients were obtained for each region studied. The tissue specimens were briefly rinsed in 0.9% NaCI solution immediately after removal and the mucosa was dissected free from other tissue layers. Parts of each specimen were fresh frozen in isopentane cooled to -70 C for histochemical and biochemical analysis. The remainder was divided into small (2-3 mm) tissue cubes. For histochemical studies the tissues were immersed in 2.5% glutaraldehyde + 0.1 M phosphate buffer (ph 7.4) at 4 C for about 24 h. After a brief rinse in 0.2 M sucrose + 0.05 M phosphate buffer (ph 7.4) some cubes were frozen in isopentane, whereas others were embedded in the watersoluble resin JB-4 (Polysciences, Warrington, Pa.) as described by RidderstriUe (22). Frozen tissues were stored at -70 C until studied. For immunofluorescent studies tissues fixed in 4% formaldehyde with 0.1% glutaraldehyde or Bouin's fluid were tested. Fixation in Bouin's fluid for 6 h, followed by dehydration through graded ethanols and embedding in paraffin, was found to yield the best material for immunofluorescence staining. Histochemical Staining Procedure Tissues were stained for CA activity according to Hansson (23,24). In this method sections are floated on a medium containing 157 mm NaHC0 3, 1.75 mm CoS0 4, 11.7 mm KH 2 P0 4, and 53 mm H 2 S0 4 The ph of the medium is 5.8 immediately after mixing. Carbonic anhy- Table 2. Immunochemical Levels of Carbonic Anhydrases I and II in the Human Gastric and Intestinal Mucosa a Tissue b No. of samples Stomach (fundus, corpus) Whole mucosa 3 Jejunum Whole mucosa 3 MU,cqsal ss;raping 2 Ileum Whole mucosa 2 Mucosal scraping 3 Colon Whole mucosa 5 Mucosal scraping 3 CAl (ng/mg protein in supernatant) 207 (0-620) 480 (282-800) 432 (93-770) 288 (217-358) 130 (20-289) 2477 (1225-4780) 1221 (693-1888) CA II (ng/mg protein in supernatant) 3434 (2314-4458) 1002 (370-1947) 1138 (700-1576) 84 (71-97) 90 (52-150) 2445 (1704-3678) 3270 (1556-5027) IIII (ratio in supernatant) Values represent mean. Range is given in parentheses. CA, carbonic anhydrase. Corrected for blood contamination assuming erythrocyte content of CA I and CA II to be 12.1 and 1.5 J,tg/mg hemoglobin, respectively (36). b Same tissues as in Table 1. 0.06 0.48 0.38 3.4 1.45 1.01 0.37

May 1985 CASTRClIJ\:TESTIJ\:AL CAEBCl0:IC l\nhyljj{l\se j j ')\ tor of CA. This concentration of acetazolamide completely abolished visible staining, whereas the presence of 10 /-LM of the inactive control substance CI 13850 (26), a N -tbutyl analogue of acetazolamide (American Cyanamid Company), did not interfere with the staining. Sections incubated in the medium without any substrate, that is, sodium bicarbonate, remained unstained. Muther (27,28) has questioned the specificity of the method. This disparity has, however, been resolved as several different laboratories have presented strong evidence supporting specificity of the method (10,25,29-33). Immunohistochemical Demonstration of Carbonic Anhydrase I and Carbonic. Anhydrase II The method was developed in our laboratory (34). Sections 3-6 /-Lm thick of fixed tissues were put onto gelatin-coated glass slides, deparaffinized, and rehydrated through xylol and graded ethanols. Sections 6 /-Lm thick of unfixed tissue were cut in a cryostat at - 20 C, put onto slides, and fixed in 99% methanol at 4 C for 2 min. The sections were exposed to specific or nonspecific (control) rabbit antiserum for 30 min at room temperature (22 C). Figure 1. Gastric surface epithelium. Immunofluorescence staining with anti-ca II (dilution 1: 10). Intense staining is seen in the cytoplasm of the surface cells, whereas parietal cells are weakly stained (lower left). x 250. drase catalyzes the dehydration of HC0 3 -- toco z and OH-. Continuous local formation of OH- at sites of enzyme activity causes deposition of a basic cobalt-phosphate complex, which is converted to CoS. Thus, a black precipitate is formed where enzyme is present. Sections of frozen tissue were cut in a cryostat at - 20 C and incubated for 1-15 min in the Hansson medium. Sections 8 /-Lm thick of fixed frozen tissue were used. Thin (4 /-Lm) unfixed sections were supported on Millipore filters (Millipore Corp., Bedford, Mass.) (24), whereas thick (15 /-Lm) unfixed sections were freeze-dried before staining to counteract disintegration (25). Sectiolls 1-2 /-Lm thick of resin-embedded tissue were incubated for 1-8 min in a slightly modified Hansson medium containing 3.5 mm CoS0 4 (22). Some sections were counterstained with hematoxylin and eosin. Excellent tissue preservation was achieved in plasticembedded sections, whereas frozen sections, especially unfixed ones, allowed considerably less detailed observations. The results of the staining for CA activity in the different sections were generally in good agreement, although cytoplasmic staining tended to be less intense in resin-embedded than in fixed frozen sections. Throughout the work the specificity of the staining procedure was checked by incubation of sections in the presence of 10 /-LM acetazolamide (Diamox, American Cyanamid Company, Pearl River, N.Y.), a specific inhibi- Figure 2. Gastric surface epithelium. Histochemistry. The basal part of the cells is heavily stained, whereas the apic,!l part of the cells with densely packed secretory granules is unstained or only weakly stained. Note one stained erythrocyte at the luminal side of the epithelium (arrow). Fixed, 8-J.Lm-thick section. Incubation time 5 min. X875.

1154 LONNERHOLM ET AL. GASTROENTEROLOGY Vol. 88, No.5, Part 1 The slides were examined with use of incident-light excitation with a filter system of type BP 450-490/FT 510/LP 520 (Zeiss, Oberkochen, Federal Republic of Germany). Biochemical Assays After thawing and gentle blotting on absorbent paper the tissue was homogenized in nine parts of distilled water, which contained 1 mm ethylenediaminetetraacetic acid (sodium salt) to protect the enzyme from inactivation by heavy metal ions. In some experiments the whole mucosa was used. In others, mucosal scrapings were taken with a knife blade (Table 1). An aliquot of the homogenate was taken for analysis of protein and for assay of CA activity. The homogenate was centrifuged at 100,000 g for 60 min. The supernatant was assayed for CA activity and content of protein, hemoglobin, and CA I and CA II. The pellet was resuspended and assayed for CA activity. A blood sample from each patient was assayed for hemoglobin and CA activity. These data were used to correct for blood contamination of the tissues. Carbonic anhydrase activity of the tissue homogenates was assayed by a changing-ph method (35). This involves the determination of the time taken to lower the ph (from 10 to 7.4) of 1 ml of carbonate Figure 3. Gastric glands. Histochemistry. The whole parietal cells are clearly stained (two are denoted by arrows). Intracellular canaliqlli are sometimes visible. Chief cells (arrowheads) are stained at lateral and basal cell borders. Fixed, 8-p.m-thick section. Incubation time 5 min. X875. For each section exposed to specific antiserum there was a corresponding control section. Antisera against the human carbonic anhydrase isoenzymes CA I and CA II (anti-ca I and anti-ca II) raised in rabbits were purchased from Behring Institut, Marburg-Lahn, Federal Republic of Germany. The specificities and affinities of the antisera for their respective isoenzymes were determined by immunodiffusion and radioimmunosorbent techniques. The antisera were diluted in phosphate-buffered saline (PBS) (0.8% NaCI in 0.01 M phosphate buffer at ph 7.2) and tested as serial dilutions from 1: 10 to 1: 320. Cross-reactivity between anti-ca I and anti-ca II was sometimes observed, but only when the concentration of the nonspecific antiserum was at least 20 times higher than that of the specific antiserum. The dilutions of the antisera were therefore adjusted so that staining occurred in sections treated with specific antisera, while there was little or no staining with nonspecific antiserum. After incubation, the sections were rinsed in PBS for 10 min and then exposed to a 1: 10 dilution of goat antirabbit immunoglobulin labeled with fluorescein isothiocyanate (Behring Institut) for 30 min. After a final wash in PBS for 30 min, followed by rinsing in distilled water, the sections were mounted under glass coverslips in PBS-glycerin (1 part PBS, 9 parts glycerin). No counterstaining was used. Figure 4. Jejunum. Immunofluorescence staining with anti-ca II (dilution 1: 10), showing presence of the enzyme in the surface epithelium of the villi, except for goblet cells. X320.

May 1985 GASTROINTESTINAL CARBONIC ANHYDRASE 1155 homogenate or in supernatant (Table 1). This tallied well with the high amounts of CA II found in the supernatant (Table 2). Small amounts of CA I were also found, which probably originated from capillary endothelial cells and blood contamination (see below). Only about 2%-11% of the activity in the whole homogenate was recovered in the particulate fraction after ultracentrifugation, so most of the enzyme appears to be cytoplasmic or only loosely bound to cell membranes. Immunohistochemistry. Intense fluorescence staining for CA II was found in the surface epithelial cells, except for the apical cell region (Figure 1). The parietal cells showed a considerably weaker staining for CA II (visible in the microscope but hardly discernible in Figure 1), whereas the chief cells appeared unstained. The erythrocytes also reacted with antibodies against CA II. No cells contained CA I except for erythrocytes and capillary endothelium (not shown). As in all other tissues studied, the erythrocytes were more intensely stained for CA I than for CA II. This is what one might expect, as erythrocytes have a CA I1CA II concentration ratio of about 8: 1 (36). Histochemistry. The surface epithelium displayed heavy staining in the cytoplasm and at the Figure 5. Jejunum. Immunofluorescence staining with anti-ca I (dilution 1 :40). Only capillaries and erythrocytes are positively stained. Same tissue as in Figure 4. x 320. buffer, as seen by change in color of phenol red at O C in a 7-ml volume. One enzyme unit is the amount of enzyme that reduces the reaction time by half. Carbonic anhydrase proteins were assayed by a radioimmunosorbent technique (36) using antibodies selective against the human erythrocyte isoenzymes CA I (anti-ca I) and CA II (anti-ca II). The antibodies were produced as previously described (36). The sensitivity of this method is 0.2 ng enzyme protein/ml tissue fluid and the precision is 5% in duplicate determinations. Recovery experiments, where known amounts of pure CA I and CA II were added to tissue homogenates, showed that all CA II was recovered but about 30% of CA I was lost. The reasoh for the loss of CA I is not known, and no correction for it was made in the data for CA I in Table 2. Protein was assayed by the method of Lowry et al. (37) and hemoglobin by the cyan-methemoglobin method (38). Results Stomach Only the corpus-fundus region was studied. Amount of enzyme. The gastric mucosa showed the highest CA activity of all tissues tested, both when measured as enzyme units per gram wet weight of tissue, or per milligram protein in whole Figure 6. Jejunum. Immunofluore&:ence staining with nonspecific antiserum (dilution 1: 10). Same tissue as in Figures 4 and 5. x320.

1156 LONNERHOLM ET AL. GASTROENTEROLOGY Vol. 88. No.5. Part 1 Immunohistochemistry. Fluorescence staining for CA II was found in the cytoplasm of the epithelial cells of the villi, except for the goblet cells (Figure 4). Carbonic anhydrase I was found in many capillaries (Figure 5). None of the isoenzymes was found in the crypt cells. Figure 6 shows an unstained control section. Histochemistry. In most of the enterocytes on the villi, moderately intense CA staining was seen in the cytoplasm, at the lateral cell borders, and in the nuclei, whereas the brush border was unstained (Figure 7). A small number bf epithelial' cells, however, also displayed intensely stained brush border (Figure 8). These cells otherwise appeared similiar to the neighboring enterocytes. The goblet cells were unstained (Figures 7 and 8). The crypt tells were distinctly stained at the lateral cell borders, whereas the cytoplasm showed no or faint staining (Figure 9). The Paneth cells in the bottom of the crypts were unstained (Figure 9). Heavily stained capillaries were seen in the villi and between the crypts (Figures 7 and 9). Figure 7. Jejunum. Histochemistry. The surface epithelium on the villi shows clear carbonic anhydrase staining except for goblet cells. Crypt cells (below) are mainly stained at lateral cell borders. Note heavily stained capillaries in the villi. L. lumen. Fixed. 2-p,m-thick section. Incubation time 8 min. x 175. lateral cell borders (Figure 2). The apical part of the cells, which contains tightly packed mucosal granules, was either unstained or showed faint staining at the apical cell membrane. The whole parietal cells appeared heavily stained in frozen sections (Figure 31, whereas there was a netlike cytoplasmic staining pattern of less heavy precipitates in resin-embedded sedions (not shown). In the chief cells the staining was re!>tricted to the basal and lateral cell borders (Figure 3). Stained capillaries were seen close to the surface epithelium and between the glands, whereas other vessels were unstained. Jejunum Amount of enzyme. The CA activity of the jejunal mucosa was ~25% of that of the gastric mucosa. Most of the enzyme activity of the; whole homogenate was found in the supernatant fraction (Table il. The concentration of CA II was about twice that of CA I (Table 2). Figure 8. Jejunum. Histochemistry. Detail of two neighborillg intestinal villi. Most enterocytes show staining in the whole cell. except for the brush border. One cell has distinctly stained brush border (arrow). Goblet cells (GJ are unstained. Fixed, 2-p,m-thick section. Incubation time 8 min. Weak counterstaining with H & E. x 1400.

May 1985 GASTROINTESTINAL CARBONIC ANHYDRASE 1157 cell membranes, but with longer times the whole cells took heavy stain, including the brush border region (Figure 11). The identity of the CA-rich cells has not been clarified yet. The staining of the crypt cells varied. In some specimens they were unstained and in others a weak staining was found at lateral cell borders (not shown). Paneth cells were always unstained. The capillaries in the villi and between the crypts were intensively stained (Figures 10 and 11). Colon 1 Amounts of enzyme. The whole mucosa showed roughly half the CA activity of the gastric' mucosa. Most of the enzyme was found in the supernatant fraction. The mucosal scraping showed about twice as high CA activity as the whole mucosa, indicating that the enzyme is most abundant in the surface epithelium. Although the concentrations of the isoenzymes were about equal in the supernatant fraction from whole mucosa, the mucosal scraping, Figure 9. Jejunum. Histochemistry. Intestinal crypt. Epithelial cells are stained at lateral cell borders. whereas the granulated Paneth cells (arrow) in the bottom of the crypt remain unstained. Fixed. 2-Mm-thick section. Incubation time 8 min. x 700. Ileum Amount of enzyme. The CA activity and the concentrations of CA isoenzymes in the ileal whole mucosa were quite low (Tables 1 and 2). Most of the enzyme activity was found in the supernatant fraction. The mucosal scrapings showed low but consistant CA activity, and a CA IICA II concentration ratio of 1.45: 1. This argues for the presence of small amounts of CA in the ileal mucosa not caused by blood contamination, as the CA IICA II ratio in erythrocytes is about 8: 1 (36). Immunohistochemistry. No consistent staining was found in the epithelial cells. Similar to the jejunum, many capillaries reacted strongly with antibodies against CA I. Histochemistry. Only a few stained epithelial cells were found on each villus (Figures 10 and 11); most of the cells, including the numerous goblet cells, were unstained. The stained cells were tall and slender, and they were morphologically similar to the neighboring, unstained enterocytes. With short incubation times most of the stain was found at the Figure 10. Ileum. Histochemistry. Intestinal villus. Only three clearly stained epithelial cells are seen (arrows). Two of them are cut so that only the apical part of the cell is visible. Note heavily stained capillaries. Arrowhead indicates stained erythrocytes within an unstained blood vessel. Fixed, 2-Mm-thick section. Incubation time 8 min. Weak counterstaining with H & E, X350.

1158 LONNERHOLM ET AL. GASTROENTEROLOGY Vol. 88, No.5, Part 1 staining in the cytoplasm (Figure 13). Longer incubation times produced heavy staining of the whole surface cells, precluding any detailed observations (Figure 14). The goblet cells always remained unstained. Staining of the glandular epithelium was similar to that of the surface epithelium in the upper part of the glands. It gradually became weaker, however, and the lower one-third to one-half of the glands was unstained (Figure 14). Capillary staining was most prominent under the surface epithelium, but stained capillaries were also found between the glands (Figures 13 and 14). Figure 15 demonstrates complete inhibition of histochemical staining in the colon by addition of 10-5 JLM acetazolamide to the incubation medium. Discussion The immunoassayable amounts of the enzyme proteins CA I and CA II corresponded well with the catalytic activities in all parts of the gastrointestinal tract. This indicates that CA I and CA II probably constitute the major forms of the enzyme in the mucosa of these tissues. About 4%-8% of the activity, however, was found in the particulate fraction of Figure 11. Ileum. Histochemistry. Detail of intestinal villus. One stained epithelial cell and the apical part of another one are seen, surrounded by unstained enterocytes and goblet cells (G). Note heavily stained capillaries in the lamina propria. Fixed, 2-fLm-thick section. Incubation time 8 min. Weak counterstaining with H & E, Xll00. contained higher concentrations of CA II than of CA I, probably due to high concentration of CA II in the brush border (see below). Immunohistochemistry. Tissues from all regions of the colon except the ascending part were studied by immunocytochemical and histochemical techniques. Carbonic anhydrase I and CA II were both located at the surface epithelium and the upper part of the glands, except for the goblet cells. Thus, the same individual cells of colonic epithelium contained both isoenzymes. Carbonic anhydrase II, however, seemed to be especially abundant in the brush border region of the surface epithelium. The CA staining of the surface epithelium varied slightly between neighboring cells for both isoenzymes. Carbonic anhydrase I was also found in the capillaries adjacent to the surface epithelium and the upper part of the glands (Figure 12). Histochemistry. The surface epithelium already took stain after very short incubation times of 1-2 min. The apical brush border region was most intensely stained in such sections, with a weaker Figure 12. Colon. Immunofluorescence staining with anti-ca I (dilution 1 :40). Note staining in the surface epithelium and the upper part of the glands, and in the walls of capillaries located close to the surface epithelium. X250.

May 1985 GASTROINTESTINAL CARBONIC ANHYDRASE 1159 subpopulation of ileal surface cells. This is explained by the fact that the histochemical method detects all catalytically active forms of CA, whereas the immunocytochemical technique only can detect the isoenzyme(s), against which the antibodies used are directed. Because antibodies against the membrane-bound isoenzyme CA IV were not available to us, it is conceivable that this form of the enzyme caused the histochemical staining at the locations mentioned previously. In mammalian species the distribution of CA in the intestinal tract has been most extensively investigated in the monkey (11) and in the guinea pig (6,18). Monkey tissues have only been studied by the histochemical technique, and the staining was similar to that in human tissues (11). The CA activities found in guinea pig mucosal homogenates (18) generally agreed well with those of the human tissues. The only clear-cut difference was that the activity of the guinea pig jejunum was much lower than that of humans. Histochemically, human and guinea pig tissues appeared similar except for the guinea pig jejunum, which showed only a few stained cells in Figure 13. Colon. Histochemistry, short incubation time (2 min). Surface epithelium. The staining is most intense at the brush border of the epithelial cells, which also show a weak cytoplasmic and nuclear staining. Goblet cells are unstained. Two stained capillaries are seen to the lower right. L, lumen. Fixed, 2-lLm-thick section. X1100. the homogenates. It is possible that the CA activity of the particulate fractions originated from a membrane-bound form designated CA IV (see further below), which has been found in human kidney microsomal fraction and bovine lung (ef. 39). The biochemical and histochemical findings agreed well. Thus, the high enzyme activities found in homogenates of the stomach and colon corresponded to the histochemical demonstration of a large number of intensely stained cells in these tissues. Homogenates of the jejunum were less active, and the jejunal epithelial cells correspondingly showed less intense histochemical staining. The low activity of ileal homogenates tallied with the histochemical finding that only a few cells contained the enzyme, i.e., capillary endothelium and a small number of surface epithelial cells. Generally, there was excellent agreement between the histochemical and immunocytochemical staining patterns. The histochemical method, however, demonstrated presence of enzyme at some sites where immunofluorescence did not: basolateral cell membranes of gastric chief cells, lateral cell membranes of intestinal crypt cells, and cell borders of a Figure 14. Colon. Histochemistry. The surface epithelium (except goblet cells) and the upper part of the glands show intense staining. It gradually weakens and the lower part of the glands (below) is unstained. Stained capillaries are seen. Fixed, 2-lLm-thick section. Incubation time 6 min. x 175.

1160 LONNERHOLM ET AL. GASTROENTEROLOGY Vol. 88, No.5. Part 1 of CA in the intestinal tract, as found here, clearly call for further research to clarify this matter. In all tissues studied the walls of many capillaries demonstrated clear staining for CA activity. The stained capillaries were generally located close to surface epithelia or glands. Capillary enzyme activity in gastrointestinal tissues has previously been detected in the guinea pig (6) and in the monkey (11). The function of capillary CA in the gastrointestinal tract is not known. It might be to promote CO 2 exchange between tissue and plasma, as suggested for capillary CA in skeletal muscles (41). Other possibilities are that the enzyme has an effect on the transfer of HC0 3 -, H+, or CI- across the capillary wall, or that it allows rapid equilibration of all species of CO 2 within the endothelial cells, thereby stabilizing ph (ef. 34). Anyhow, the close spatial relationship between CA-containing capillaries and the epithelia of the gastrointestinal tract suggests that there is a functional interplay between epithelia active in transport of water or electrolytes, or both, and their neighboring capillaries, which requires the presence of the enzyme in these capillaries. Figure 15. Colon. Histochemistry. All staining is inhibited by the presence of 10-0 ~M acetazolamide in the incubation medium. Fixed. 2-~m-thick section. Incubation time 6 min. Same tissue as in Figure 14. Counterstaining with H & E. X140. the epithelium of the villi (6). Yet another staining pattern for CA has been reported for the rat jejunum (17). Thus. there are clear species differences between mammals in the localization of the enzyme in the small intestine. which contrasts to the stomach and colon, where species differences have not been described [Spicer et al. (15) reported lack of CA II in the rat colonic mucosa, but our unpublished data show presence of both CA I and II in the rat colon]. In the gastric mucosa CA is thought to take part both in acid (glands) and alkaline (surface epithelium) secretion (40) (see introductory section). We can show, as other authors have (8,9,14), that the enzyme involved is CA II, which is found in most epithelia that perform acid-base work. It can be postulated that CA II is used also by the epithelia of the jejunum and colon to facilitate electrolyte transfer, e.g., reabsorption of sodium chloride and excretion of bicarbonate (20). The function of CA I in the intestine, however, remains obscure and should be further studied. Unfortunately, very little is known about the role of the enzyme at its various sites in the intestine. The striking differences in the distribution References 1. Davenport HW. Fisher RB. Carbonic anhydrase in the gastrointestinal mucosa. J Physiol (Lond) 1938;94:16. 2. Helander HF. The cells of gastric mucosa. Int Rev Cyto11981; 70:217-89. 3. Janowitz HD. Colcher J, Hollander F. Inhibition of gastric secretion in dogs by carbonic anhydrase inhibitor 2-acetylamino-l.3.4 thiadiazol-5-sulfonamide. Am J Physiol 1952; 171 :325-30. 4. Vollrath L. Ober Entwicklung und Funktion der Belegzellen der Magendrusen. Z Zellforsch 1959;50:36-60. 5. Boass A. Wilson TH. Cellular localization of gastric intrinsic factor in the rat. Am J Physiol 1964;206:783-6. 6. Uinnerholm G. Carbonic anhydrase in the intestinal tract of the guinea-pig. Acta Physiol Scand 1977;99:53-61. 7. O'Brien p. Rosen S. Trencis-Buck L. Silen W. Distribution of carbonic anhydrases within the gastric mucosa. Gastroenterology 1977;72:870-4. 8. Kumpulainen T. Human carbonic anhydrase isoenzyme C. Histochemistry 1981;72:425-31. 9. Kumpulainen T. Immunohistochemical localization of human carbonic anhydrase isozymes. Ann NY Acad Sci 1984; 429:359-68. 10. Sugai N. Ito S. Carbonic anhydrase. ultrastructural localization in the mouse gastric mucosa and improvements in the technique. J Histochem Cytochem 1980;28:511-25. 11. Li:innerholm G. Carbonic anhydrase in the monkey stomach and intestine. Acta Physiol Scand 1983;117:273-9. 12. Li:innerholm G. Wistrand PJ. Carbonic anhydrase in the human fetal gastrointestinal tract. Bioi Neonate 1983;44:166-76. 13. Flemstri:im G. Gastric secretion of bicarbonate. In: Johnsson LR. ed. Physiology of the gastrointestinal tract. Voir. New York: Raven. 1981:603-16. 14. Kuriaki K. Magee DF. On the carbonic anhydrase activity of the alimentary canal and pancreas. Life Sci 1964;3:1377-82.

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