Does DDT inhibit carbonic anhydrase?

At a concentration of 50 to 100 micrograms per milliliter, p,p'-DDT (and p,p'-DDE) did not inhibit the rate of hydration or dehydration of carbon dioxide by carbonic anhydrase. At concentrations greater than 500 micrograms per milliliter, partial inhibition of the rate of dehydration of carbonic acid was observed, but this involved precipitation of drug in the reaction vessel. This degree of inhibition suggests that DDT may not inhibit carbonic anhydrase effectively at the usual concentrations found in tissue after exposure of organisms to DDT in the environment.

Inhibition by anions of human red cell carbonic anhydrase B: physiological and biochemical implications.

The hydration rate of CO2 catalyzed by human red cell carbonic anhydrase B is 92 percent reduced by the normal concentrations of chloride and bicarbonate in red cells. This reflects a general sensitivity of this reaction to halides and other anions, up to 87 times greater than the effect on red cell carbonic anhydrase C. The catalytic hydration of CO2 is generally more (up to 24 times) sensitive to inhibition by anions and sulfonamides than the dehydration of HCO3-, probably reflecting different mechanisms. The sensitivity of enzyme B to anion inhibition also depends upon the substrate, being much greater for CO2 than for certain esters. On the basis of the very low catalytic activity of B for CO2 in the presence of physiological concentration of chloride, and the fact that carbonic anhydrase C is effective for CO2 hydration (in the presence of chloride) at a rate 340 times greater than that of CO2 output from tissues, it appears that the biological role of enzyme B is not that of a carbonic anhydrase.

A search for the function of human carbonic anhydrase B.

Because of the very high activity and abundance of human red cell carbonic anhydrase C (carbamate hydrolase, EC 4.2.1.1), it seemed likely that the second isozyme, B, might not be essential for CO2 metabolism. It was then found that physiological concentrations of Cl- inhibited catalysis of CO2 hydration by the B enzyme (but not by type C), suggesting further that type B does not function in vivo as a carbonic anhydrase. The versatility of the catalytic activity of carbonic anhydrase for a number of 'artificial' substrates suggested that enzyme B may be utilized in reactions of intermediary metabolism. A number of hydration, dehydration, decarboxylation, kinase, and phosphatase systems were tested to determine a possible physiological function for the enzyme. Results with eighteen possible substrates were negative and the possibility is discussed that mammalian carbonic anhydrase B is an evolutionary accident.

Kinetics and inhibition of carbonic anhydrase-catalyzed hydrolysis of 2-hydroxy-5-nitro-alpha-toluenesulfonic acid sultone. Comparison with reactions of other substrates.

The catalytic activities of human red cell carbonic anhydrase (EC 4.2.1.1) isozymes B and C for the hydrolysis of 2-hydroxy-5-nitro-alpha-toluenesulfonic acid sultone have been compared with their activities towards three other substrates. The substrate specificity (measured as kcat/Km) for either isozyme decreases in this order: CO2 greater than 2-hydroxy-5-nitro-alpha-toluenesulfonic acid sultone greater than acetaldehyde greater than p-nitrophenyl acetate. Unlike CO2 hydration, enzyme B is slightly more active towards sultone hydrolysis than C. Despite these widely differing activities of both isozymes with regard to different substrates, the inhibition constants for anion and sulfonamide inhibitors are nearly independent of the substrate used. This suggests that the binding sites of these substrates in the enzyme are the same or nearly the same. Earlier studies on 2-hydroxy-5-nitro-alpha-toluenesulfonic acid sultone from this and other laboratories had underestimated both the intrinsic activity and the susceptibility to anion inhibition of human carbonic anhydrase B. We now find that this was due to the use of acetonitrile as the substrate solvent, which is often contaminated with cyanide, a powerful inhibitor of carbonic anhydrase. The inhibition of human carbonic anhydrase B by several industrial batches of acetonitrile agrees completely with the spectrophotometrically determined cyanide content of these batches.

Kinetics and inhibition of membrane-bound carbonic anhydrase from canine renal cortex.

The membrane-bound carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) in the canine renal cortex has been characterized in terms of its CO2 hydration kinetics and inhibition by sulfonamides and inorganic anions. Comparing these properties with those of the renal cytoplasmic and the human red cell B and C isozymes, it appears that the membrane enzyme is quite different from the soluble carbonic anhydrases. The turnover number of the particulate enzyme is about 3-times lower than that of the cytoplasmic enzyme. The membrane-bound enzyme is also different from its cytoplasmic counterpart in being more resistant against inhibition, particularly against Cl-. Microsomes from the renal cortex were purified to yield luminal and anti-luminal fractions. Carbonic anhydrase activity was found in both. The luminal and anti-luminal carbonic anhydrases appeared similar in terms of their kinetic properties and susceptibility to inhibition.

Localization and activity of renal carbonic anhydrase (CA) in CA-II deficient mice.

A null allele at the mouse Car 2 locus was induced by ethylnitrosurea; mice homozygous for the new allele lack the carbonic anhydrase (CA)-II isoenzyme. The expression of this genetic lesion was investigated by: (1) using tissue fractionation techniques to determine localization and activity of CA in the kidney, and (2) examining renal response to CA inhibition in CA-II deficient mice (CAD), in normal (N) mice and in heterozygous litter mates (LM). N and LM mice had CA activity in proximal tubule brush border membranes and cytosol. CA activity was also localized to membranes and cytosol of the outer medullary region. CAD mice lacked cytosolic activity but had normal CA activity in all membranes examined. All membrane associated CA had 2-8-fold lower sulfonamide sensitivity than cytosolic CA. These inhibition characteristics suggest that the membrane enzyme is CA-IV. Baseline urinary excretion of Na+, K+, and HCO3- was similar in all groups. Urine pH and Cl- excretion were higher and titratable acid output was lower in CAD mice. Inhibition of CA (methazolamide, 25 mg/kg) led in all groups to equivalent increments of urine pH, urine flow, and HCO3-, Na+, and K+ excretion. Cl- excretion was unchanged. Thus the extent of the genetic deficiency of CA-II mice extends to the kidney cytosol but does not alter membrane localization or levels of CA, probably CA-IV. The similar response to CA inhibition in CAD mice suggests that CA-IV, the membrane bound isoenzyme is the important isoenzyme in proximal tubule HCO3- reabsorption.

The effect of acidosis in hypokalemic periodic paralysis.

Metabolic acidosis was produced in two patients with hypokalemic periodic paralysis by the administration of ammonium chloride over a period of three days. The challenging test of glucose and insulin produced a substantially smaller reduction of both serum potassium concentration and muscle strength than when the patients were tested in normal acid-base balance. The findings agree with earlier work on acetazolamide, suggesting that metabolic acidosis provides protection against episodes of muscle weakness in periodic paralysis.

Acetazolamide treatment of hypokalemic periodic paralysis. Probable mechanism of action.

Following administration of glucose and insulin to three patients with hypokalemic periodic paralysis, serum K+ fell 1.9 mM. After administration of acetazolamide, 250 mg four times daily, serum K+ fell 0.9 mM, a substantial difference. In normal persons glucose and insulin lowered serum K+ 0.5 mM, and this was not changed substantially by acetazolamide. The metabolic acidosis induced by the drug appears to be responsible for the change in decrement of serum K+ and for the amelioration of symptoms in the patients. The findings agree with earlier reports that metabolic acidosis lowers the rate of entry of K+ into muscle, thus opposing the heightened or pathological entry of K+ into muscle cells during attacks of the disease.

Synthesis and physicochemical properties of thiadiazolo[3,2-a]pyrimidinesulfonamides and thiadiazolo[3,2-a]triazinesulfonamides as candidates for topically effective carbonic anhydrase inhibitors.

A series of bicyclic 1,3,4-thiadiazolo[3,2-a]pyrimidine- and 1,3,4-thiadiazolo[3,2-a]triazine-7-sulfonamides were synthesized from 5-amino-1,3,4-thiadiazole-2-sulfonamide and evaluated for topical efficacy as ocular hypotensive agents. The compounds were tested for the physicochemical properties of sulfonamide pKa, free acid water solubility, CHCl3/buffer partition, and transcorneal penetration (kin), as well as for activity against carbonic anhydrase (I50). A number of these compounds exhibited lower sulfonamide pKa and higher water solubility than those of acetazolamide (1) and methazolamide (2), and one, 12, brought about a small reduction in IOP in the normal rabbit eye.

Synthesis and physicochemical properties of sulfamate derivatives as topical antiglaucoma agents.

Several imidazolylphenyl sulfamate and (imidazolylphenoxy)alkyl sulfamate derivatives were synthesized and evaluated as topically active carbonic anhydrase inhibitors. Water solubility, pKa, carbonic anhydrase inhibition, and partition coefficient for the compounds were measured. Sulfamic acid 2-[4-(1H-imidazol-1-yl)phenoxy]ethyl ester monohydrochloride (16) has the best combination of properties and showed excellent topical activity in lowering the intraocular pressure in New Zealand white rabbits.

pH and drug ionization affects ocular pressure lowering of topical carbonic anhydrase inhibitors.

PURPOSE: To evaluate the effect of drug ionization on the ocular hypotensive activity of topical carbonic anhydrase inhibitors. METHODS: Ocular normotensive New Zealand albino and ocular hypertensive Dutch Belted pigmented rabbits were used. Tonometric intraocular pressure levels were taken after topical application of 50 microliters of drug (at various concentrations and pH values) to one eye with the contralateral eye used as an untreated control. The drugs tested were MK-927, L-662,583, and AHR-16329. Eye tissues were analyzed for drug by our enzymatic methods. RESULTS: In all cases, the more ionized the applied drug the greater the ocular hypotensive activity. Tissue distribution studies showed that there was more drug found in the eye after the ionized form of a drug was applied than that found after application of the less ionized forms. CONCLUSIONS: Increasing the ionization of three ampholyte topical carbonic anhydrase inhibitors increases their ocular hypotensive activity. These data taken with ocular disposition data suggest that ionized compounds of this type are more readily sequestered in the cornea, which serves as a drug depot for prolonged drug delivery and activity.

The relations between ionic and non-ionic diffusion of sulfonamides across the rabbit cornea.

We studied the transcorneal permeability of five carbonic anhydrase inhibitors, with particular attention to the passage of these acidic compounds in their ionized and nonionized forms. Their pKs varied from 5.9 to 8.0, and CHCl3/pH 7.2 buffer partition coefficients from 10(-4) to 25. Solutions of appropriate pH of each compound were applied to the cornea in steady state, and the rates of passage to the anterior chamber for each compound in each form were measured. The rate constants for the ionized forms were surprisingly high, only four- to sevenfold less than those for the uncharged species. Thus these compounds penetrate the cornea in both forms, and the data show that by increasing the pH of solutions, and thereby solubility, the overall rates of accumulation in the eye are increased.

The effects of carbonic anhydrase inhibitors on aqueous humor chemistry and dynamics.

The effects of topical application of the carbonic anhydrase inhibitor trifluormethazolamide (TFM) on intraocular pressure (IOP), ascorbate and CO2 concentrations in aqueous humor, and aqueous humor flow were studied in rabbits. These effects were compared with those produced by systemic treatment with methazolamide. The decrease in IOP observed after TFM was accompanied by changes in the composition of the aqueous humor. Posterior aqueous ascorbate concentration showed a marked increase (up to 1.7-fold), whereas the anterior aqueous ascorbate did not change significantly. Similar changes were found in rabbits after systemic treatment with methazolamide. A small but statistically significant decrease in the CO2 content of both posterior and anterior aqueous was observed after topical TFM application. Methazolamide yielded a more profound lowering in the CO2 content of the aqueous humor, a reflection of the significant decrease in plasma CO2 content. For topical TFM or systemic methazolamide doses yielding complete inhibition of carbonic anhydrase in the eye, a 55-59% reduction of aqueous flow was calculated from the ascorbate data using the Kinsey and Palm equation. However, a 31-42% reduction in aqueous flow was obtained from the same data using an equation based only on posterior chamber data. The reasons for using only posterior aqueous ascorbate data for calculating the changes in aqueous humor flow are discussed.

Timolol decreases aqueous humor flow but not Na+ movement from plasma to aqueous.

PURPOSE: To determine whether the well-known effect of timolol in reducing ocular pressure and aqueous humor (AH) flow is a function of reduced Na+ movement from plasma to aqueous. Previously, the authors have shown this to be the case for carbonic anhydrase inhibitors. METHODS: The rate of appearance of 22Na in rabbit posterior aqueous was measured 1 to 3 minutes after the intravenous injection (time T) of the isotope. One hour before this, the animals received one of the following: two drops of 0.5% timolol, two drops of 3.5% pilocarpine, or 25 mg/kg intravenous methazolamide. At 1 minute (T + 1), a posterior chamber sample was taken; 2 minutes later (T + 3) a second sample was removed from the fellow eye. The rate constant of sodium accession is simply the difference between the two counts/2 minutes. Aqueous flow was measured by dilution of sulfacetamide marker as described previously. RESULTS: The rate constant (k(in)) for sodium entering the posterior chamber was 0.036 +/- 0.004 minute-1 (n = 17). Corresponding to previous findings, methazolamide (25 mg/kg intravenous) reduced this to 0.023 +/- 0.003 minute-1 (n = 14). Conversely, timolol (two drops of 0.5% solution) had no effect on kin, which measured 0.037 +/- 0.004 minute-1 (n = 12). Similarly, as expected, pilocarpine had no effect on k(in) (0.035 +/- 0.003 minute-1). Control flow was 3.9 microliters/minute +/- 0.4; after timolol, 2.5 microliters/minute +/-0.1; after methazolamide, 2.4 microliters/minute +/-0.2; after pilocarpine, 3.6 microliters/minute +/- 0.2. These are converted to rate constants by dividing by volume of posterior aqueous (60 microliters). The control rate constant for fluid entry was 0.065 minute-1, 1.8-fold higher than for sodium. CONCLUSIONS: A central dogma of the formation of AH (and cerebrospinal fluid) is that fluid moves isotonically from plasma to AH or cerebrospinal fluid and, therefore, that rate constant k(in) for fluid and for sodium are approximately the same. In the authors' hands, the fluid constant was modestly higher than for sodium. This holds for normal function and also for the reduced k(in) for fluid and sodium after carbonic anhydrase inhibition. The k(in) for neither flow nor sodium was affected by pilocarpine. Surprisingly, however timolol, which reduces flow, had no effect on Na+ entry.

Permeability of human cornea and sclera to sulfonamide carbonic anhydrase inhibitors.

Corneal penetration of sulfonamide carbonic anhydrase inhibitors for topical treatment of glaucoma has been tested in human eye bank and rabbit tissue. Paired corneas, with the epithelia intact or removed, and excised sclera were perfused in vitro. Corneal permeability (Kp) to methazolamide and ethoxzolamide was similar in both species, but for benzolamide and bromacetazolamide the Kp was greater in humans. Human corneas without epithelium had Kp the same as scleral Kp. Topical methazolamide (6 mmol/L) was studied in vivo in rabbits and in ten humans before cataract surgery. The mean (+/- SE) concentration in the rabbit aqueous was 3.2 +/- 1.4 mumol/L at eight minutes and 1.2 +/- 0.16 mumol/L at one hour. In humans, less than 0.2 mumol/L was detected at eight minutes; at one hour none was detected in three cases, and 0.4 +/- 0.08 mumol/L was detected in four cases. Lower permeability in humans than rabbits may result from a fourfold greater blinking rate, a twofold greater tear turnover, and a twofold lower corneal/conjunctival area.

Current status of membrane-bound carbonic anhydrase.

We have studied the kinetic properties, and susceptibility to inhibition, of cytoplasmic and membrane carbonic anhydrase from dog kidneys, and attempted to place the data in the context of earlier work on this subject. The cytoplasmic enzyme thus far seems the same as human red cell carbonic anhydrase C, on the basis of kinetics, inhibition, amino acid composition and immunochemistry. On the other hand, the membrane enzyme is quite a different protein from either the cytoplasmic, or human red cell B or C. This enzyme is found in both luminal (brush border) and antiluminal (basolateral) fractions, and there appear no differences between the two. The turnover number (kcat) lies between those of B and C, and susceptibility to sulfonamide inhibition is two to 135-fold less than for the cytoplasmic enzyme, depending on the drug used. The usual difference is about fivefold. The KI for acetazolamide against the membrane enzyme is 10(-7) M, so that at the renal concentrations achieved at the usual in vivo doses (approximately 20 mg/kg) or used in current in situ perfusion work (both 10(-4) M) the enzyme is 99.9% inhibited. A striking difference between the membrane carbonic anhydrase and cytoplasmic or red cell B or C is its resistance to inhibition by halions. At 0.5 M chloride, there is no effect, whereas for the other three types inhibition ranges from 70%-99%. The membrane renal enzyme is also immunologically distinct from the other three types. The membrane enzyme has activity in its native state, but can be solubilized without loss of activity by treatment with Triton or sodium dodecyl sulfate. The actions of the renal carbonic anhydrases are depicted in a scheme that takes into account the protolysis of water, the attraction of H+ and of OH (and HCO3-) to the luminal and antiluminal membranes respectively, and the catalytic hydration and hydroxylation of CO2.

The effect of topically administered carbonic anhydrase inhibitors on aqueous humor dynamics in rabbits.

Repeated topical administration of 2.5% trifluormethazolamide, a halogenated derivative of methazolamide, resulted in a unilateral decrease in intraocular pressure in rabbits. Mean (+/- S.E.M.) baseline intraocular pressure (19.8 +/- 2.1 mm Hg) was significantly (P less than .05) decreased 30 minutes (16.1 +/- 2.2 mm Hg) and 60 minutes (15.8 +/- 2.7 mm Hg) after drug administration. Trifluormethazolamide did not alter outflow facility. Aqueous humor flow calculated from the tonographic data was reduced 44% and flow measured by fluorophotometry was reduced 29%. Topical delivery of trifluormethazolamide decreased the level of carbon dioxide in the aqueous humor in the treated eye in a manner similar to that observed after systemic administration of carbonic anhydrase inhibitors. Topical administration of 10% acetazolamide did not decrease intraocular pressure. However, topical administration of either trifluormethazolamide or acetazolamide before oral administration of water resulted in a blunting of the water-induced ocular hypertensive response.