A distinct carbonic anhydrase in the mucus of the colon of humans and other mammals

Abstract

SUMMARY We have collected gastrointestinal, mainly colonic, mucus from humans,guinea pigs, rats, and normal and carbonic anhydrase II (CAII)-deficient mice. In the mucus of all species, substantial CA activity was present. Using antibodies against human CA isoforms we found that the human mucus CA differs from cytosolic CAI and CAII, membrane-bound CAIV, and the secreted CAVI of saliva. The high sensitivity of mucus CA to acetazolamide rules out its identity with cytosolic CAIII. Partial sequences obtained from the purified human mucus CA show similarity, but not identity, with human CAI, and clear differences from the other known CAs. Additional evidence concerning the CA isoform present in mucus was obtained for the mucus CA of other species and was derived from: (1) the mucus of CAII-deficient mice, whose high CA activity confirms that it is not CAII that is responsible; (2) the inhibitory effect of iodide, which shows that mucus CA from mice, guinea pig and humans does not have the high anion sensitivity of CAI; (3) the inactivating effect of 0.2%SDS on guinea pig, mouse and human mucus CA, ruling out the SDS-resistant CAIV; and (4) the partitioning of guinea-pig mucus CA into the water phase in Triton X114 phase separation experiments, which also argues against its identity with membrane-bound CAs, such as CAIV. A comparison of colonic mucus CA activity in normal and germ-free rats indicates that the mucus CA is not of bacterial origin but is produced by the gastrointestinal tissues. We conclude(from its immunoreactivity, from iodide inhibition and from partial amino acid sequences) that mucus CA of human origin probably represents an isozyme, which is specific for mucus and is not identical with the known CA isozymes. The results obtained for mucus CA of other species collectively point in the same direction.

Segregation of Na/H exchanger-3 and Cl/HCO3exchanger SLC26A3 (DRA) in rodent cecum and colon

“…Intimate proximity would also allow for efficient regulatory coupling, as each component would be allosterically stimulated, via changes in regional intracellular pH, by the transport activity of the other; thus the intracellular acidifying effect of Cl/HCO 3 exchange would tend to stimulate NHE3 (84) and the alkalinizing effect of NHE3 would tend to stimulate DRA (17,37). Like other segments, the rodent midcolon exhibits high CA activity, with contributions from various CA isoforms situated in the cytosol, the inner and outer surfaces of the apical membrane, and the luminal mucus layer (49). The anion exchange activity of DRA is known to be enhanced by cytosolic CA-II, presumably by furnishing H ϩ and HCO 3 Ϫ for exchange with Na ϩ and Cl Ϫ , although this does not appear to involve a direct interaction (78).…”

Contractile properties of skeletal muscle fibre bundles from mice deficient in carbonic anhydrase II

“…The latter represents CA III [10,30] and is identical with the former in the case of CAD mouse muscle, as expected. The third column in Table 1 represents an estimate of total cytosolic CA activity caused by the red cells present in the muscle tissue during homogenization; it has been estimated from the concentration of hemoglobin determined in the supernatant from the absorbance at 410 nm, where hemoglobin exhibits an absorbance maximum, and from the intraerythrocytic CA activities in LM (71,100 U) and in CAD (887 U), as reported by Kleinke et al [16]. It should be noted that, due to the presence of other heme proteins in the supernatants such as myoglobin, the numbers in Table 1 for the contamination of the supernatant with erythrocytic CA activity represent upper limits.…”

“…The CA activity remaining after treatment with 0.2% SDS approximates the membrane-bound isoform CA IV. An upper limit of cytosolic CA activity due to the presence of red cells in the muscle tissue was calculated from the heme concentration in the supernatants and the intra-erythrocytic CA activity of LM and CAD mice (Kleinke et al [16]). Hemoglobin concentrations were calculated from absorbances of the undiluted supernatants at 410 nm and averaged 6.1×10 −5 M for LM and 4.6×10 −5 M for CAD.…”

“…It should be noted, however, that it has been shown, for example by Swenson and Maren [32], that a near-complete (with acetazolamide doses ≫ than clinical doses) systemic carbonic anhydrase inhibition will impair maximal physical performance, an effect that is likely to be essentially due to impaired CO 2 exchange in the tissues and in the lung. It is conceivable, although it has not been studied, that the CAD mice exhibit a similar limitation of maximal exercise in vivo, especially as they possess rather low red cell activity of CA I [16].…”

“…However, a mouse model deficient in CA II has been developed via mutation induced by administration of N-ethyl-N-nitrosourea [19]. We have previously confirmed that the mice lack CA II but still express CA I in their red cells [16]. However, the toxin employed to induce this mutation might affect other genes besides that of CA II.…”