Rat renal and erythrocyte carbonic anhydrases. Purification and properties.

Rat renal and erythrocyte carbonic anhydrases (carbonate hydro-lyase, EC 4.2.1.1) were isolated by affinity chromatography. The erythrocytes contain two major forms of the enzyme. One of the forms has a specific activity (towards CO2) 30 times higher than the other and constitutes the major part of the total cellular carbonic anhydrase. The amino acid compositions of this high-activity type and of the low-activity type are similar to the compositions reported for these types in other species. The kidney appears to have only one high-activity form of carbonic anhydrase which is very similar to and probably identical with the erythrocyte high-activity form.

Membrane specific carbonic anhydrase (CAIV) expression in human tissues.

Membrane-bound carbonic anhydrase IV (CAIV) expression has been evaluated in a range of fetal and adult human tissues and in cell culture. All tissues tested showed expression of CAIV, assessed by Western blotting, with a single immunodetected band at 55 kDa. The levels varied in fetal lung and liver during development and in various zones of the fetal brain. CAIV was clearly expressed in lung, pancreatic tumour and skin cell cultures.

Membrane-associated carbonic anhydrase activity in the kidney of CA II-deficient mice.

Carbonic anhydrase II-deficient mice offer a possibility to study the localization along the nephron of membrane-associated carbonic anhydrase (CA) activity without interference from the cytoplasmic enzyme. We studied the localization of CA in kidneys from CA II-deficient and control mice by immunocytochemistry (CA II) and histochemistry. Cytoplasmic staining was found in convoluted proximal tubule, thick limb of Henle, and principal and intercalated cells of collecting duct in the control animals but was absent in the CA II-deficient mice. In cells with cytoplasmic staining the cell nuclei were stained. Intense histochemical activity was associated with apical and basolateral membranes of convoluted proximal tubule, first part of thin limb, thick limb, and basolateral membranes of late distal tubule. In collecting ducts of control animals, the basolateral cell membranes of intercalated cells were the only clearly stained membranes. In CA II-deficient animals one type of intercalated cell was stained most intensely at the apical membranes and another only at the basolateral. We suggest that the former corresponds to Type A intercalated cells secreting H+ ions to the luminal side and the latter to Type B cells secreting H+ ions to the basolateral side.

Evidence of a high-activity C type of carbonic anhydrase in human ciliary processes.

Carbonic anhydrase activity was found in the ciliary process of fresh human donor eyes, originating from an enzyme antigenically similar to the erythrocyte high-activity isoenzyme HCA C. It was sensitive to inhibition by acetazolamide and resistant to inhibition by halides like HCA C. The enzyme is probably identical with HCA C. Its tissue concentration was one fifth to one tenth of that in the human kidney. The erythrocyte low-activity isoenzyme HCA B was also found in the processes as a contaminant.

Carbonic anhydrase isoenzymes CA I and CA II in the human eye.

The distribution of carbonic anhydrase was studied in human donor eyes by the cobalt-phosphate histochemical method of Hansson and by immunofluorescence and immunoperoxidase techniques using antisera specific against the human cytoplasmic isoenzymes CA I and CA II. Corneal endothelium displayed specific immunological staining for CA I and CA II. Distinct enzyme activity was observed histochemically in the plasma membranes and cytoplasm of the endothelium. In the ciliary processes immunological evidence for the presence of CA II was found both in pigmented (PE) and in nonpigmented (NPE) epithelium. Activity was observed in the cytoplasm and basolateral membranes of NPE, but only in the basal membranes of PE. In the lens the plasma membranes of both the epithelium and fibers displayed intense activity, whereas cytoplasmic enzyme activity was seen only in the epithelium. There was no activity in the lens capsule. Immunofluorescence studies were difficult because of autofluorescence, but the immunoperoxidase technique indicated the presence of both CA I and CA II in the lens. In the central retina, Müller cells stained for CA II. Histochemically, enzyme activity was seen in the cytoplasm and at the plasma membranes. Activity was also observed in some but not all cones. Electron microscopy revealed this to be located in the cristae and plasma membranes adjacent to the pigment epithelium. Activity was also found in PE. Neurons and rods lacked both immunological staining and activity. Endothelial cells of capillaries in ciliary processes and in the choroid stained for CA I and exhibited histochemical activity, particularly those which faced neighboring epithelial cells containing the enzyme. The isoenzyme CA III, which is resistant to inhibition by sulfonamides, did not appear to be present in these ocular tissues, since the histochemical staining of enzyme activity was completely abolished by 10(-6) M acetazolamide.

Membrane-associated CA activity in the eye of the CA II-deficient mouse.

PURPOSE: Membrane-associated carbonic anhydrase (CA) activity is probably of great importance for transepithelial transport of ions and fluid. Histochemical studies have indicated its presence in the eye, but such histochemical data are difficult to evaluate because of interference from cytoplasmic CA isozymes, of which CA II is predominant. CA II-deficient mice offered the possibility to study the localization of membrane-associated CA activity, without influence from CA II: METHODS: The localization of CA in the eyes of CA II-deficient mice and of normal mice was studied by the cobalt-phosphate histochemical method. RESULTS: In both types of mice, intense histochemical CA activity was associated with the apical and basolateral membranes of the pigmented and nonpigmented ciliary epithelium, of the corneal endothelium, and of the pigmented epithelium of the retina. It also was localized at the cell borders of the Müller cells and of the lens epithelium and fibers. There also was CA activity in the endothelium of the capillaries of the choroid and retina but not in that of the larger vessels. CONCLUSIONS: Membrane-associated CA activity is found in many ocular cells known to transport fluid and ions. Inhibition of the CA activity of the basolateral membranes of the ciliary nonpigmented epithelium probably explains the reduction of aqueous humor flow seen after the administration of CA inhibitors.

The incidence and time-course of latanoprost-induced iridial pigmentation as a function of eye color.

Latanoprost, a phenyl-substituted analogue of prostaglandin F2 alpha administered as eye drops, induces increased melanogenesis in the iridial melanocytes of monkeys. Similar effects were seen in 12, 23 and 11% of patients in the USA, United Kingdom (UK) and Scandinavia, respectively, during one year of treatment. The highest incidence of induced pigmentation was seen in green-brown, yellow-brown and blue/grey-brown eyes, in that order. The relatively high proportion of patients with green-brown eyes in the UK explains the larger number of affected patients in this country. Typically, a concentric increase of the iris pigmentation appeared after six months (range: 3-17) and was judged to be noticeable by the patient in about 2/3 of the cases. After cessation of latanoprost, no change of the induced pigmentation has been seen in patients followed for two years, and there have been no signs of dispersion of pigment into the anterior chamber. Irides, homogeneously blue, grey, green or brown, were seldom affected. Naevi or freckles on iris, conjunctiva, or eye lids were not affected. It is intriguing that many patients with mixed eye color, particularly the blue-brown eyes, have not developed increased pigmentation even during two years of treatment. This could be due to a relatively slow melanogenesis or to refractory melanocytes in these individuals.

Serum carbonic anhydrase III in neuromuscular disorders and in healthy persons after a long-distance run.

A radioimmunosorbent technique was used for the assay of the skeletal muscle specific enzyme, carbonic anhydrase III (CA III). The usefulness of serum CA III determinations for detecting skeletal muscle damage was evaluated by comparing the serum levels of this enzyme and of myoglobin and creatine kinase in 64 patients with neuromuscular disorders and in 13 healthy volunteers before and after a long-distance run. Increased serum CA III levels were found in all patients with muscular dystrophy, chronic polymyositis and amyotrophic lateral sclerosis and in many with myasthenia gravis. In patients with polymyositis who were followed up with repeated blood sampling, the time courses of serum CA III levels, myoglobin levels and clinical symptoms were closely related. In all the runners the serum CA III level immediately after the run was increased. In the present study serum CA III and myoglobin seemed to be equally sensitive as biochemical markers of muscular damage and more sensitive than creatine kinase.

Inhibition of carbonic anhydrase in the lens does not induce myopia in cynomolgus monkeys.

Refraction was measured in eyes of cynomolgus (Macaca irus) monkeys, before and during continuous intravenous infusion of large doses of the carbonic anhydrase (CA) inhibitors acetazolamide and ethoxzolamide. No changes of refraction were seen. Therefore, inhibition of CA in lens, cornea and retina does not appear to be the cause of the transient myopia and associated symptoms, occasionally observed in patients treated with CA inhibitors.

Carbonic anhydrase C in the human renal medulla.

Soluble carbonic anhydrase (carbonate dehydratase, EC 4.2.1.1) from the papillary and inner medullary regions of thoroughly perfused human donor kidneys was isolated by affinity chromatography. The purified enzyme was homogenous with respect to sedimentation in the ultracentrifuge and iso-electric focusing. It had an amino-acid composition and behaved chromatographically, kinetically, electrophoretically and immunochemically like the high-activity (with respect to CO2) erythrocyte carbonic anhydrase HCA-C, and the renal enzyme previously isolated from extracts of the whole kidney. The results suggest that all regions of the human kidney contains one soluble form of carbonic anhydrase similar to and probably identical with HCA-C. Small immunoassayable amounts (1/30 of total enzyme protein) of the low-activity erythrocyte isoenzyme HCA-B were also found, but are considered to be a contaminant. There was no indication for the presence of sulfonamide-resistant isoenzymes.

Human renal cytoplasmic carbonic anhydrase. Tissue levels and kinetic properties under near physiological conditions.

The cytoplasmic form of human renal carbonic anhydrase, CA-C (carbonate dehydratase, EC 4.2.1.1), purified by affinity chromatography, was characterized kinetically at 37 degrees C in 25 mM N-methyl imidazole buffer, I = 0.15, pH 7.1. using a pH-indicator stopped-flow technique. Under these conditions the rate constants for the uncatalyzed hydration of CO2 and dehydration of H2CO3 were 0.12 . s-1 and 0.60 . s-1, respectively. The kinetic parameters for CA-C were found to be: Hydration reaction, Km = 11.8 mM, V/[E]0 = 10.6 x 10(5) . s-1, dehydration reaction Km = 70 mM, V/[E]0 = 5 x 10(5) . s-1. In the hydration reaction CA-C was non-competitively inhibited by acetazolamide, Ki = 16 nM, sulfanilamide, Ki = 8 micrometer, and chlorothiazide, Ki = 1 micrometer. The levels of immunoassayable CA-C in cortex, medulla and papilla of perfused donor kidneys were 1.3, 1.0 and 0.6 mg enzyme protein/g tissue protein respectively which corresponded well with the levels measured catalytically. The erythrocyte form, HCA-B, was also detected immunochemically (approximately 0.1 mg/g protein) but is thought to be a contaminant. Calculations indicated that the uncatalyzed hydration of CO2 in the tubular cells can support 17 or 0.7% of the rate of urine acidification, dependent on whether the cellular alkaline pH-disequilibrium during acid secretion is 0.1 or 0.01 pH units, respectively. CA-C accelerates the hydration rate 6800-fold which enables the cell to sustain high rates of proton generation, while maintaining near CO2-equilibrium and maximal buffering capacity. Even at an assumed pH-disequilibrium of only 0.01 pH-unit, CA-C is present in 50-fold excess of apparent physiological needs. When the enzyme is inhibited the rate of the uncatalyzed reaction increases, which partly overcomes the effect of inhibition.

Human lens carbonic anhydrases. Purification and properties.

PURPOSE: To isolate and characterize carbonic anhydrase (CA) isozymes of human lenses. METHODS: Affinity chromatography was used to separate CA isozymes, as monitored by an immunosorbent assay. Amino acid analysis was done on the antigenic CAII. CA catalytic activity and its sensitivity to inhibition was measured on soluble and membrane-bound CA isozymes. RESULTS: The lens contains 0.25, 9, and 2 microg/g wet weight of CAI, II, and III, respectively. Almost all of the CA catalytic activity originates from CAII. Plasma membranes had a CA activity that was inhibited like the membrane-bound isozyme CAIV CONCLUSIONS: CA activity in human lenses originates from CAI, II and III in the cytoplasm, and from CAIV at plasma membranes of lens epithelium and fibres. CA probably functions like CA in erythrocytes, by facilitating CO2 transport. CAII and CAIV are probably also involved in translenticular ion transport. Chronic intake of CA inhibitors does not seem to induce cataract formation.

The importance of carbonic anhydrase B and C for the unloading of CO2 by the human erythrocyte.

Carbonic anhydrase (carbonate dehydratase, EC 4.2.1.1) isoenzymes HCA B and HCA C from human erythrocytes were purified by affinity chromatography and characterized kinetically at 37 degrees C in 25 mM N-methylimidazole buffer, I = 0.15, pH 7.1, using a pH-indicator stopped-flow technique. The rate constants for the uncatalyzed hydration of CO2 and dehydration of H2CO3 were 0.12 . s-1 and 58 . s-1, respectively, Km and turnover numbers in the hydration reaction were 14.0 mM and 19.1 . 10(5) . s-1 for HCAC and 3.3 mM and 0.56 . 10(5) . s-1 for HCA B. Km and turnover numbers in the dehydration reaction were 70 mM and 5 . 10(5) . s-1 for HCA C and 16.8 mM and 0.27 . 10(5) . s-1 for HCA B. Ki for chloride was 14 mM for HCA B and greater than 200 mM for HCA C. Ki for acetazolamide was 0.9 microM against HCA B and 16 nM against HCA C. The rates of hydration of CO2 in hemolysates with known concentrations of HCA B and HCA C, and in mixtures of purified HCA B and HCA C with concentrations near those in the erythrocyte, were similar to the rates calculated from the kinetic parameters of each isoenzyme. HCA B is 86% inhibited by chloride in vivo. This strong inhibition, together with the low specific activity, explains why HCA B only accounts for 10% of the total carbonic anhydrase activity of the erythrocyte, en spite the cellular concentration HCA B is 8 times higher than that of HCA C. HCA B and HCA C can together accelerate the intracellular CO2-reaction 23 000-fold, which gives a margin of 25-fold over physiological needs in hard work and 50-fold at rest. Perceptible physiological effects on respiration should therefore be seen when total carbonic anhydrase activity of erythrocytes is 96 to 98% inhibited. These degrees of inhibition are achieved when plasma concentrations of acetazolamide reach 2 and 5 microM, respectively.