Acta physiol. scand. 1977. 99. 53-61 From the Department of Medical Pharmacology, University of Uppsala, Sweden
Carbonic Anhydrase in the Intestinal Tract of the Guinea-pig BY
GUDMAR LONNERHOLM Received 24 May 1976
Abstract LONNERHOLM, G. Carbonic anhydrase in the intestinal tract of the guinea-pig. Acta physiol. scand. 1977. 99. 53-61. The distribution of carbonic anhydrase activity in the intestinal tract of the guinea-pig was studied by the histochemical method of Hansson. Enzyme activity was demonstrated in epithelial cells, erythrocytes and capillary walls. In the gastric mucosa parietal cells, surface mucous cells and neck mucous cells were highly active. In the small intestine only a few epithelial cells o n the villi and in the upper part of the crypts showed enzyme activity. They seemed to be randomly scattered among inactive ones. It is not clear a t present if they represent a distinct cell type or specialized absorptive cells. In the proximal colon most surface epithelial cells were highly active (goblet cells were inactive), whereas the surface cells in the distal colon showed less activity with a more varying degree of staining. In the cecum enzyme activity was found in the surface epithelium and in the upper part of the crypts, the staining being most marked at the luminal border of the surface cells. The staining reaction was completely inhibited in all tissues by 10 p M acetazolamide, except for the luminal staining of the cecum, which was inhibited only by 100 p M acetazolamide. This indicates the presence of high concentrations of carbonic anhydrase, probably of the “low activity” form, at this locus. Mucosal scrapings were taken from the intestinal tissues, homogenized and assayed for carbonic anhydrase activity by a changing-pH indicator method. The results confirm those of previous studies and correlate well with the histochemical findings. Key words: Carbonic anhydrase, histochemistry, stomach, intestine
The presence of carbonic anhydrase in the gastric mucosa is well known, and its distribution and function in this tissue have been extensively studied (see Maren 1967). However, much less is known about the enzyme in the other parts of the intestinal tract. Carter and Parsons (1971) found that homogenates of the stomach, proximal colon and cecum of the guinea-pig all possess high carbonic anhydrase activity, whereas the activity of the small intestine and distal colon is low. Similar findings have been reported for the dog and rat (Kuriaki and Magee 1964, Maren 1967). The physiological significance of these findings is poorly understood, however (see Carter 1972). The aim of the present study was to clarify the detailed distribution of the enzyme in the intestinal tract of the guinea-pig, since such data are necessary for a further discussion of the role of the enzyme. For this purpose Hansson’s (1967, 1968) cobalt-phosphate method for histochemical demonstration of carbonic anhydrase activity has been used. The specificity of this method has been described in another paper (Lonnerholm 1974). Except for 53
54
GUDMAR LONNERHOLM
the stomach, previous histochemical studies are scarce (Korhonen et a/. 1966, Cassidy and Lightfoot 1974). The enzyme activity of mucosal homogenates of the intestinal tissues was also determined. using a changing pH indicator method.
Methods Ti.\sue prrpprrrution. Nonfasted, female guinea-pigs, weighing 300-600 g, were used. They were fed pellets
(EWOSa\eIsfoder kanin och marsvin, Astra-Ewos AB, Sodertalje. Sweden) and water, to which ascorbic acid had been added, ~ r t llihitroir. They were killed by a blow on the head. The abdomen and the thorax were rapidly opened, and a cannula was introduced into the ascendent aorta. Perfusion with cold 0.9”, NaCI started after the portal vein had been opened to secure free flow, and lasted until the abdominal vessels were macroscopically blood-free. The stomach and cecum uere remoLed. opened, and the contents washed away by agitating the tissue in a large \ o h m e of 0.9:,, NaCI. Samples were taken from the gastric corpus and from the middle part of the cecum. intestinal segments were used with or without previous perfusion of the intestinal lumen with 0.9”,, NaCI. Jejunal samples were taken 5 to 20 cm distal to the duodenojejunal flexure and ileal sampler 5 to 20 cm proximal to the ileocolic \al\e. Proximal colonic samples were taken 5 t o 10 cm distal to the ileocolic Lalve and distal colonic samples 5 to 10 cni proximal to the anus. H i ~ t o ~ ~ / r i(11 ~ isrcrinitrg ~iic pro( r h r e
Fixed and unfixed tissues from 10 animals were used. Unfixed tissues were immediately frozen in isopentane cooled with a mixture of ethanol and solid CO,. They were stored in small plastic bags at 70°C for dajs or weeks before use. Fixed tissues were immersed in 2.5”,, glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 4°C f o r 4-5 hrc. The glutaraldehyde was prepared from a 50
Carbonic Anhydrase in the Intestinal Tract of the Guinea-pig BY
GUDMAR LONNERHOLM Received 24 May 1976
Abstract LONNERHOLM, G. Carbonic anhydrase in the intestinal tract of the guinea-pig. Acta physiol. scand. 1977. 99. 53-61. The distribution of carbonic anhydrase activity in the intestinal tract of the guinea-pig was studied by the histochemical method of Hansson. Enzyme activity was demonstrated in epithelial cells, erythrocytes and capillary walls. In the gastric mucosa parietal cells, surface mucous cells and neck mucous cells were highly active. In the small intestine only a few epithelial cells o n the villi and in the upper part of the crypts showed enzyme activity. They seemed to be randomly scattered among inactive ones. It is not clear a t present if they represent a distinct cell type or specialized absorptive cells. In the proximal colon most surface epithelial cells were highly active (goblet cells were inactive), whereas the surface cells in the distal colon showed less activity with a more varying degree of staining. In the cecum enzyme activity was found in the surface epithelium and in the upper part of the crypts, the staining being most marked at the luminal border of the surface cells. The staining reaction was completely inhibited in all tissues by 10 p M acetazolamide, except for the luminal staining of the cecum, which was inhibited only by 100 p M acetazolamide. This indicates the presence of high concentrations of carbonic anhydrase, probably of the “low activity” form, at this locus. Mucosal scrapings were taken from the intestinal tissues, homogenized and assayed for carbonic anhydrase activity by a changing-pH indicator method. The results confirm those of previous studies and correlate well with the histochemical findings. Key words: Carbonic anhydrase, histochemistry, stomach, intestine
The presence of carbonic anhydrase in the gastric mucosa is well known, and its distribution and function in this tissue have been extensively studied (see Maren 1967). However, much less is known about the enzyme in the other parts of the intestinal tract. Carter and Parsons (1971) found that homogenates of the stomach, proximal colon and cecum of the guinea-pig all possess high carbonic anhydrase activity, whereas the activity of the small intestine and distal colon is low. Similar findings have been reported for the dog and rat (Kuriaki and Magee 1964, Maren 1967). The physiological significance of these findings is poorly understood, however (see Carter 1972). The aim of the present study was to clarify the detailed distribution of the enzyme in the intestinal tract of the guinea-pig, since such data are necessary for a further discussion of the role of the enzyme. For this purpose Hansson’s (1967, 1968) cobalt-phosphate method for histochemical demonstration of carbonic anhydrase activity has been used. The specificity of this method has been described in another paper (Lonnerholm 1974). Except for 53
54
GUDMAR LONNERHOLM
the stomach, previous histochemical studies are scarce (Korhonen et a/. 1966, Cassidy and Lightfoot 1974). The enzyme activity of mucosal homogenates of the intestinal tissues was also determined. using a changing pH indicator method.
Methods Ti.\sue prrpprrrution. Nonfasted, female guinea-pigs, weighing 300-600 g, were used. They were fed pellets
(EWOSa\eIsfoder kanin och marsvin, Astra-Ewos AB, Sodertalje. Sweden) and water, to which ascorbic acid had been added, ~ r t llihitroir. They were killed by a blow on the head. The abdomen and the thorax were rapidly opened, and a cannula was introduced into the ascendent aorta. Perfusion with cold 0.9”, NaCI started after the portal vein had been opened to secure free flow, and lasted until the abdominal vessels were macroscopically blood-free. The stomach and cecum uere remoLed. opened, and the contents washed away by agitating the tissue in a large \ o h m e of 0.9:,, NaCI. Samples were taken from the gastric corpus and from the middle part of the cecum. intestinal segments were used with or without previous perfusion of the intestinal lumen with 0.9”,, NaCI. Jejunal samples were taken 5 to 20 cm distal to the duodenojejunal flexure and ileal sampler 5 to 20 cm proximal to the ileocolic \al\e. Proximal colonic samples were taken 5 t o 10 cm distal to the ileocolic Lalve and distal colonic samples 5 to 10 cni proximal to the anus. H i ~ t o ~ ~ / r i(11 ~ isrcrinitrg ~iic pro( r h r e
Fixed and unfixed tissues from 10 animals were used. Unfixed tissues were immediately frozen in isopentane cooled with a mixture of ethanol and solid CO,. They were stored in small plastic bags at 70°C for dajs or weeks before use. Fixed tissues were immersed in 2.5”,, glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 4°C f o r 4-5 hrc. The glutaraldehyde was prepared from a 50
