Localization of a protein inhibitor of carbonic anhydrase in pig tissues.

The protein inhibitor of carbonic anhydrase (CA), pICA, was localized in pig tissues by an immunohistochemical technique, using rabbit antipICA IgG. Staining for pICA was found in liver sinusoids and kidney glomeruli, where phagocytic cells are located, i.e. Kupffer and mesangial cells, respectively. pICA was not found inside parenchymal cells, or in tissues from striated muscle, heart, eye or lung. It is concluded that the function of pICA is perhaps to bind the carbonic anhydrase isozymes CA I, II, and III, released from erythrocytes into the blood circulation by intravascular haemolysis. The complex of CA-pICA in plasma may then be transported to the reticuloendothelial system, for degradation and reclamation of CA-bound zinc. This would be similar to the fate of the haemoglobin-haptoglobin complex for the recycling of iron.

Biochemical, histological, and inhibitor studies of membrane carbonic anhydrase in frog gastric acid secretion.

Gastric acid secretion is dependent on carbonic anhydrase (CA). To define the role of membrane-bound CA, we used biochemical, histochemical, and pharmacological approaches in the frog (Rana pipiens). CA activity and inhibition by membrane-permeant and -impermeant agents were studied in stomach homogenates and microsomal fractions. H(+) secretion in the histamine-stimulated isolated mucosa was measured before and after mucosal addition of a permeant CA inhibitor (methazolamide) and before and after mucosal or serosal addition of two impermeant CA inhibitors of differing molecular mass: a 3,500-kDa polymer linked to aminobenzolamide and p-fluorobenzyl-aminobenzolamide (molecular mass, 454 kDa). Total CA activity of frog gastric mucosa is 2,280 U/g, of which 10% is due to membrane-bound CA. Membrane-bound CA retains detectable activity below pH 4. Histochemically, there is membrane-associated CA in surface epithelial, oxynticopeptic, and capillary endothelial cells. Methazolamide reduced H(+) secretion by 100%, whereas the two impermeant inhibitors equally blocked secretion by 40% when applied to the mucosal side and by 55% when applied to the serosal side. The presence of membrane-bound CA in frog oxynticopeptic cells and its relative resistance to acid inactivation and inhibition by impermeant inhibitors demonstrate that it subserves acid secretion at both the apical and basolateral sides.

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.

Androgen-linked control of carbonic anhydrase III expression occurs in rat perivenous hepatocytes; an immunocytochemical study.

Carbonic anhydrase (CA) isozymes CAII and CAIII were assayed by a radioimmunosorbent technique in liver cytosolic fractions and in isolated hepatocytes of adult male and female rats. Male livers contained 0.16 mg of CAII and 57 mg of CAIII per g cytosolic protein. Corresponding values for female livers were 0.34 mg CAII and 4 mg CAIII. Similar values and differences between CAII and III were found in isolated hepatocytes. Neonatal and adult castration of males reduced the CAIII levels to those of the females. Treatment with testosterone for three weeks restored the copulatory behaviour in the males castrated at adult age, but restored only partially the levels of CAIII. No significant effects of the endocrine manipulations were seen on CAII. Oophorectomy, with or without testosterone substitution, had no significant effect on CAII and CAIII levels in female rats. Immunohistochemistry and histochemistry showed that the regulation of CAIII is confined to perivenous hepatocytes. CAIII can therefore serve as a useful marker in the separation of these cells. CAIII appears to belong to the proteins and enzymes of the rat liver, known to be regulated via the hypothalamo-pituitary-liver axis. It may be used as a model of gene regulation in perivenous hepatocytes.

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

Membrane-associated carbonic anhydrase (CA) activity is of importance for transepithelial transport of ions and fluid. Histochemical studies have indicated its presence in the brain, but the data are difficult to evaluate because of interference from cytoplasmic CA isozymes, of which CA II is the predominant one. CA II-deficient mice offer a possibility to study the location of membrane-associated CA-activity, without interference from CA II. The location of CA activity in the brain of CA II-deficient and normal mice was studied by the cobalt-phosphate histochemical method, and that of CA I, CA II and CA III by an immunocytochemical method. The brains of both types of mice lacked cytoplasmic isozymes CA I and CA III, and the CA II-deficient mice also lacked CA II. In the normal mice, oligodendrocytes and choroid epithelium stained for CA II in the cytoplasm. In normal and CA (II)D-mice there was an intense membrane associated histochemical CA activity in neuronal processes. Neuronal perikarya were not stained. Endothelial membranes of brain capillaries showed strong histochemical CA-activity. Choroid epithelial cells had histochemical CA activity in the cytoplasm and along apical and baso-lateral cell membranes. The results suggest that membrane-associated CA-activity found along neuronal processes probably modulates pH of the extracellular fluid and thus neuronal activity. CA II and the membrane-associated CA of choroidal epithelium are probably involved in the secretion of cerebrospinal fluid.

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.

Carbonic anhydrase IV activity is localized on the exterior surface of human erythrocytes.

Carbonic anhydrase (CA) cytoplasmic isozymes CA I and CA II were found in human erythrocyte membrane ghosts, when prepared at pH 5.4 and pH 7.4, but not in ghosts prepared at pH 8.2. These findings could indicate that previously reported CA activity of ghosts was owing to contamination by CA I and CA II during the preparation of the ghosts. However, using a sensitive micro-assay, CA activity was also found in ghosts prepared at pH 8.2. This activity constitutes 0.2% of the erythrocytes' total CA-activity, and originates from a membrane-associated isoform of CA, located at the exterior membrane surface. It has immunochemical and kinetic properties like those of the membrane-bound CA IV, previously isolated from kidney, lung and small blood vessels. Its function is possibly to interact with the red cell membrane anionic transport protein, band 3, for the bicarbonate/chloride exchange.

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.

Colour vision and side-effects during treatment with methazolamide.

The retina contains Na+K(+)-ATPase and carbonic anhydrase (CA), enzymes that regulate ion fluxes across cell membranes of photoreceptors. Since inhibition of retinal Na+K(+)-ATPase by digitalis impairs colour vision, we wanted to find out whether this also occurs after inhibition of CA. In a double-masked cross-over study with placebo, 14 male volunteers were given 50 mg q.i.d. of the CA inhibitor methazolamide for 2 weeks. A disturbance of colour discrimination was observed in 8 of the 14 subjects, in the classification phase of Lanthony New Color Test. The presence of the disturbance was not significantly correlated to the degree of acidosis or to other side-effects. Its mechanism could be interpreted as a specific effect of CA inhibition in the retina (or the visual cortex) calculated to more than 99.9%.

Renal handling and plasma elimination kinetics of carbonic anhydrase isoenzymes I, II and III in the rat.

Using a radio-immunosorbent technique, the levels of the carbonic anhydrase (CA) isoenzymes CA I, II and III in plasma (1-3 micrograms ml-1), lymph (0.5-1.6 micrograms ml-1) and urine (0.03-0.06 micrograms ml-1), were determined in the rat. The renal clearances of CA I, II and III were 11 +/- 3, 42 +/- 11 and 35 +/- 4 nl min-1 (g kidney wet wt)-1 (n = 4-5), respectively. After a single i.v. injection of purified native or 125I-labelled isoenzymes, the elimination of CA I, and CA III from plasma followed a bi-exponential decline, with half-times of 7 and 9 min for the rapid phase and 112 min for the slow phase, respectively. Nephrectomy decreased the rapid phase and the build-up of catabolites. Therefore, the rapid phase of CA I and III elimination is probably explained by filtration of unbound isoenzyme at the glomeruli and subsequent degradation by the proximal tubules. The plasma elimination curve for CA II was different and followed a mono-exponential decline, with a half-time of 210 min both in normal and nephrectomized animals. This indicates that CA II is not filtered at the glomeruli. However, in acute renal failure, with leaking tubular cells, CA II was excreted into the urine. The slow elimination of the major part of the isoenzymes from plasma is explained by the binding of CA I, II and III to a plasma protein, immunochemically similar to transferrin, forming a macromolecular complex with a mol wt of 114 +/- 2 kDa.

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.

Chemical properties of carbonic anhydrase IV, the membrane-bound enzyme.

The carbonic anhydrase (CA) isozyme (IV) in microsomes is thought to have a dominant role in secretory processes. Using microsomes from bovine kidney and lung (which had the same activity), we have measured the Km and kcat for CO2 hydration and compared these numbers with those for CA I (red blood cells and gut), CA II (red blood cells and secretory cells), and CA III (muscle). For kidney CA IV, Km is 10 mM and kcat is 170,000 sec-1 at 0 degree, approaching the rate for CA II but much greater than those for CA I or III. The Ki values for 11 sulfonamides with CA IV were measured and in all cases showed less binding (averaging 17-fold) than to CA II. This is the result of reduction of the association rate constants (k(on)), whereas the dissociation constants of the drug-enzyme complexes (k(off) are similar between CA II and IV. Based on these data, full physiological effects may be expected when inhibition of CA IV is about 99%. Anion inhibition of CA IV is similar to that of CA II and less than that of CA I or CA III. Data are compatible with the proposed role of CA IV in physiological events, i.e., HCO3- formation and secretion at one cell border and H+ separation and excretion at the other.

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.

Leakage of carbonic anhydrase III from normal and denervated rat skeletal muscle following contractile activity.

Skeletal muscle extracellular carbonic anhydrase III was investigated in anesthetized rats by a microdialysis technique. A small dialysis probe was inserted into the tibialis anterior (TA) muscle and perfused continuously. Perfusates were collected before and during muscle contraction, induced by electrical stimulation of the muscle or of the sciatic nerve. In the perfusate of resting normal and denervated muscle, the concentration of CA III was 10 to 12 ng/mL, as measured by a radioimmunosorbent technique. During contractile activity, the concentrations of CA III increased markedly in the normal and denervated muscle. A TA muscle suspended in physiological saline behaved similarly, even though the leakage before and during contraction was higher than in vivo. The results show that skeletal muscle leaks CA III both in vivo and in vitro, a leakage which was markedly increased by contractile activity. The microdialysis technique should also be useful in humans to study the efflux of various proteins from different kinds of diseased or fatigued muscles.

Muscle ATP and lactate and the release of myoglobin and carbanhydrase III in acute lower-limb ischaemia.

Serum-myoglobin and carbanhydrase III (S-CAIII), a specific muscle enzyme, were measured on admission, during surgery and in the postoperative period in 23 patients with acute lower-limb ischaemia and in 21 patients with chronic limb ischaemia in order to evaluate these molecules as possible markers of the degree of clinical ischaemia. The muscle contents of ATP and lactate were determined in muscle biopsies from patients with acute ischaemia on admission. Unlike S-myoglobin and S-CAIII they discriminated between cases which required subsequent amputation from those which did not. Clinical signs of ischaemia were, however, of no value in this respect and there were no correlations between clinical signs or the duration of ischaemia and S-myoglobin or S-CAIII or the contents of ATP or lactate in muscle tissue. The levels of S-myoglobin and S-CAIII correlated well (r = 0.95, p less than 0.0001). In patients who subsequently required an amputation S-myoglobin increased ten-fold (i.e., from 24-48 h postoperatively in acute arterial ischaemia and from 3 h postoperatively in patients with chronic limb ischaemia). In patients with a successful revascularisation S-myoglobin returned to normal levels. It is concluded that in this investigation S-myoglobin had no prognostic value on admission and that S-myoglobin analyses in the postoperative course may be useful for making clinical decisions concerning impending recirculation failures.

Membrane-bound carbonic anhydrase CA IV in the human kidney.

The distribution of membrane-bound carbonic anhydrase, CA IV, was studied in human kidneys by an indirect immunoperoxidase method using a rabbit polyclonal antibody directed against human kidney CA IV. Clear staining of CA IV was found in the apical cell borders of some cells in the cortical and medullary segments of the collecting ducts, presumably the A type of intercalated cells. Weak staining for CA IV was located in the interior of a number of collecting duct cells and in the basolateral regions of the proximal convoluted tubules. However, no staining was found in the brush border of the same tubules. This is a surprising finding, since evidence for carbonic anhydrase activity has been found biochemically and histochemically both in isolated brush-border and baso-lateral membranes. Further work is needed to clarify this matter. The endothelium of the peritubular capillaries also stained for CA IV.

The excretion of carbonic anhydrase isozymes CA I and CA II in the urine of apparently healthy subjects and in patients with kidney disease.

Carbonic anhydrase isozymes CA I and CA II were assayed by a radio-immunosorbent technique in the plasma and urine of apparently healthy subjects and of patients with renal disease. The concentrations (mean +/- SD, n = 8) of CA I and CA II in the plasma of healthy subjects were 2.3 +/- 2.3 and 0.8 +/- 0.5 mg/l, respectively. The urinary excretion values were 3.8 +/- 2.0 and 3.5 +/- 1.9 micrograms/24 h, and the apparent renal clearances were 21 +/- 17 and 52 +/- 44 microliters/min, respectively, values that are similar to those of other low molecular weight proteins. CA I and CA II have mol. wt of 28,850 and 29,300, respectively, they are globular in shape and have a Stoke-Einstein radius of 25 A. They could, therefore, be expected to be filtered at the glomeruli and thereafter reabsorbed by the proximal tubules. CA II is also present in the cytoplasm of renal proximal and distal tubular cells. A study of the pattern of urinary excretion of CA I and CA II could permit detection of damage to renal tubular cells in two ways--either from defective reabsorption of filtered CA I and CA II by the proximal tubular cells, or from leakage of CA II from the proximal or distal tubules into the urine. Some patients with hypercalcuria and renal tubular acidosis showed increased excretion of these enzyme proteins and of beta 2-microglobulin (BMG) into the urine, but the prevalence was rather low (27%). Further studies of patients with more severely damaged kidneys are required.