2.2. Functions of carbonic anhydrases in the alimentary tract

2.2.1. Carbonic anhydrases in the major digestive glands

Two CA isoenzymes, CA II and VI, are known to be expressed in the mammalian salivary glands (Kivelä et al. 1997). In the serous acinar cells of the parotid and submandibular glands CA II has been suggested to participate in the production of bicarbonate into the saliva (Parkkila S et al. 1990, 1991b, 1994, Ogawa et al. 1993). CA VI is a secretory isoenzyme produced by the serous acinar and demilune cells of the parotid and submandibular glands (Parkkila S et al. 1990). CA VI and II may together form a complementary system regulating the acid-base balance in the mouth and upper alimentary tract (Parkkila S et al. 1990, 1996, Kivelä et al. 1997). CA II in the salivary glands may supply the saliva with HCO₃^- and the CA VI secreted into the saliva would then accelerate the removal of bacterially produced acid in the mouth in the form of CO₂. CA VI may protect teeth by catalyzing the bicarbonate-based buffering system in the oral cavity and thus accelerate the removal of acid from the local microenvironment of the tooth surface. It is located at the optimal site on dental surfaces for catalyzing the conversion of salivary bicarbonate and microbe-delivered hydrogen ions to carbon dioxide and water (Leinonen et al. 1999). This hypothetical model of CA VI function was supported by Kivelä et al. (1999), who showed that low salivary CA VI concentrations are associated with increased caries prevalence, particularly in subjects with neglected oral hygiene.

Compared with other secretory organs, the mammalian liver contains relatively low levels of total CA activity. A basic physiological function of CA II in the liver is to produce HCO₃^- for the alkalization of the bile (Swenson 1991).

The mammalian liver expresses high levels of mitochondrial CA V. Physiologically, CA VA has been implicated in two metabolic processes in the mitochondria of hepatocytes: ureagenesis and gluconeogenesis, supplying bicarbonate for the first urea cycle enzyme, carbamyl phosphate synthetase I in ureagenesis and for pyruvate carboxylase in gluconeogenesis (Dodgson 1991). CA inhibitors have been observed to retard both of these processes in the livers of guinea pigs and rats (Dodgson et al. 1983, Metcalfe et al. 1985, Dodgson 1991). CA V is the first CA isoenzyme to be found to participate in intermediary metabolism, but it is conceivable that it may also have other functions, as it is contained in both periportal and perivenous hepatocytes, while urea and glucose synthesis occur only in the periportal region.

The presence of low activity, hormonally regulated CA III in hepatocytes has aroused interest in its specific function. Cabiscol & Levine (1995) have demonstrated that it functions in an oxidizing environment and that it is the most oxidatively modified protein in the liver known so far. These and other recent results have suggested that CA III may provide protection from oxidative damage and CA III may serve as a useful marker protein to investigate in vivo the mechanisms, which contribute to oxidative damage in the liver. It has also been suggested that lower levels of free radicals in cells overexpressing CA III may affect growth signalling pathways (Räisänen et al. 1999).

Earlier studies have indicated that both CA IV and CA IX are expressed in the biliary epithelial cells, whereas hepatocytes are devoid of immunoreactivity for these isoenzymes (Parkkila S et al. 1996, Pastoreková et al. 1997). Interestingly, previous histochemical studies have indicated CA enzymatic activity also in the hepatocyte plasma membrane (Ridderstråle et al. 2000). CA XIV could be the most promising candidate protein to represent the hepatocyte isoenzyme, since its mRNA has recently been reported in both mouse and human liver (Mori et al. 1999, Fujikawa-Adachi et al. 1999c).

In pancreas, the role of CA II in the secretion of bicarbonate into the pancreatic juice by the epithelial duct cells is well documented (Swenson 1991). CA I is expressed in the α-cells of the endocrine Langerhans islets (Parkkila S et al. 1994). However, the physiological role of CA I in α-cell function has remained unclear. CA V is the second isoenzyme described in the endocrine pancreas where its expression is solely confined to the -cells (Parkkila A-K et al. 1998). The suggestion that CA V may have a role in the regulation of insulin secretion was based on its cellular distribution and the observation that the CA inhibitor acetazolamide inhibited glucose-stimulated insulin secretion (Parkkila A-K et al. 1998).

2.2.2. Carbonic anhydrases in the gastrointestinal canal

CA II has been located in the squamous epithelial cells of the oesophagus, where it may contribute to HCO₃^- secretion (Meyers & Orlando 1992, Parkkila S et al. 1994, Christie et al. 1997). The presence of this isoenzyme in the oesophagus is physiologically important, because endogenous HCO₃^- secretion is capable of raising the pH of the gastro-oesophageal reflux-derived residual acid from 2.5 almost to neutrality. The immunohistochemical evidence for the presence of CA II in the human oesophagus is thus in accordance with the biochemical evidence that the oesophagus disposes of an endogenous mechanism for protecting the mucosa against acidity, but suggests that it is the stratified oesophageal epithelium rather than the submucous glands that is responsible for HCO₃^- secretion.

Immunohistochemical techniques have revealed cytosolic CA II in the parietal cells of the gastric glands, where it regulates the acidity of the gastric juice by proton secretion (Davenport & Fisher 1938, Davenport 1939, O"Brien et al. 1977, Sato et al. 1980, Lönnerholm et al. 1985, Parkkila S et al. 1994, Parkkila S & Parkkila A-K 1996). On the other hand, in the gastric surface epithelial cells CA II is involved in the secretion of mucus and HCO₃^- to form a bicarbonate containing mucous gel layer covering the epithelium and protecting it from digestion. This gastroduodenal HCO₃^- secreted by the surface epithelial cells neutralizes the gastric acid (Richardson 1985).

Membrane-associated CA IX is another major CA isoenzyme expressed in the gastric epithelium. Both parietal and surface epithelial cells contain CA IX at the basolateral plasma membrane (Pastoreková et al. 1997). Evolutionary conservation in vertebrates and the abundant expression of CA IX in the normal human gastric mucosa have indicated its physiological importance. CA IX may participate in physiological processes via the activity of its CA-like domain. On the other hand, basolateral localization of CA IX suggests its possible involvement in intercellular communication and/or cell proliferation.

It is widely known that both CA I and II are expressed in the non-goblet epithelial cells of the mammalian colon (Lönnerholm et al. 1985, Parkkila S et al. 1994), in which these isoenzymes have been implicated in the regulation of electroneutral NaCl reabsorption via the synchronous operation of apical Na⁺-H⁺ and Cl^--HCO₃^- exchange (Charney & Egnor 1989). In addition to cytosolic CA I and II, the intestinal enterocytes express at least two membrane-associated isoenzymes, CA IV (Fleming et al. 1995) and IX (Pastoreková et al. 1997, Saarnio et al. 1998a). The location of CA IV in the apical brush border of the colonic epithelium has suggested a functional role in the regulation of colonic ion homeostasis (Fleming et al. 1995). Recent studies have demonstrated that the distribution of CA IX in the gut has several unique features when compared to the other CA isoenzymes (Pastoreková et al. 1997, Saarnio et al. 1998a). One of them was its localization on the basolateral surfaces of proliferating cryptal enterocytes, suggesting that it may serve as a ligand or receptor for another protein that regulates intercellular communication or cell proliferation. Furthermore, CA IX has a completely conserved active site domain of CAs (Opavsk et al. 1996), suggesting that it could also participate in CO₂/ HCO₃^- homeostasis in the colon.

The most novel CA isoenzymes, CA XII and XIV, may also be expressed in the gut. Positive signals have been obtained in the human colon using mRNA blots (Ivanov et al. 1998, Türeci et al. 1998, Fujikawa-Adachi et al. 1999c). However, their cellular localisation was not reported in the gastrointestinal tract prior to the completion of the present study.