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From the Departments of Pediatrics, Pharmacology and Therapeutics, Physiology, and Environmental Health, University of CincinnatL College of Medicine, and from the Children's Hospital Research Foundation. Supported in part by United States Public Health Service grant HD06337 and by the Children "s Hospital Research Foundation. *Part I appeared in the April, 1975, issue o f THE JOURNAL; Part l l I will appear in the June, 1975, issue of THE JOURNAL. **Reprint requests: Division of Clinical Pharmacology, Children "s Hospital Research Foundation, Elland A re., Cincinnati, Ohio 45229. tion will also be given to those diuretics which are considered useful as adjunctive therapy (e.g., spironolactone) and to some agents which have been of historical importance in the development of our current concepts of diuretics and diuretic therapy. At the outset, it should be said that diuretic therapy is almost always adjunctive and that every attempt should always be made to influence the course of the primary, underlying disease which is producing the edematous state

The saluretic effect of the thiazide diuretics has been attributed to inhibition of sodium reabsorption in the distal nephron of the kidney. Recent micropuncture studies have shown, however, that chlorothiazide administration can also inhibit sodium reabsorption in the proximal convolution. To clarify the site of the saluretic effect of chlorothiazide, these micropuncture studies examined the effect of chlorothiazide on chloride transport in the nephron. The effect of chlorothiazide on chloride transport was studied because chlorothiazide's effectiveness as a saluretic is largely due to its ability to enhance sodium chloride excretion; if only changes in sodium transport are examined, it would be then difficult to determine if sodium as bicarbonate or as chloride is affected, since chlorothiazide can inhibit carbonic anhydrase. One group of rats was studied before and after 15 mg/kg per h chlorothiazide. For comparison, another group of rats was studied before and after 2 mg/kg per h benzolamide, a carbonic anhydrase inhibitor. Fractional chloride delivery from the proximal tubule was similarly increased in both groups from 59.4 to 71.0% by chlorothiazide administration, Pless than 0.0001, and from 54.3 to 68.2% by benzolamide administration, P less than 0.001. The increased delivery very of chloride from the proximal tubule was largely reabsorbed before the early distal tubule as fractional chloride delivery to this site increased only from 5.08 to 7.40% after chlorothiazide administration, P less than 0.001, and from 4.50 to 6.29% after benzolamide administration, P less than 0.01. Benzolamide had no effect on chloride reabsorption in the distal convoluted tubule. However, chlorothiazide administration resulted in a marked decrease in distal tubular chloride reabsorption, the fraction of filtered chloride present at the late distal tubule incresing from 1.24 to 6.25%, P less than 0.001. Fractional chloride excretion in the urine increased from 0.29 to 3.44%, P less than 0.001, after chlorothiazide, but did not change after benzolamide. The influence of chlorothiazide on proximal chloride transport presumably is related to its ability to inhibit renal carbonic anhydrase. However, it is not the effect of chlorothiazide in the proximal convolution but rather its effect in the distal convoluted tubule which is primarily responsible for its ability to be an effective saliuretic.

The ability to excrete an oral water load and the renal diluting mechanism were studied in hypothyroid rats and in age-matched euthyroid controls. Hypothyroid animals excreted a significantly smaller fraction of a 50-ml/kg oral water load than controls, demonstrating the same limited ability to excrete free water as thyroid-deficient man. During hypotonic (0.45%) saline infusion, absolute sodium delivery to the diluting segment and free water clearance were markedly lower in hypothyroid rats. However, both fractional distal sodium delivery and fractional free water clearance were similar in hypothyroid and control animals, suggesting that the reduced absolute free water formation in hypothyroid rats was due to decreased net distal delivery. In support of this hypothesis was the observation that fractional distal sodium reabsorption was equal or higher in thyroid-deficient rats, which indicates that the sodium reabsorptive capacity of the diluting segment was preserved in these animals. The results cannot be attributed to incomplete suppression of antidiuretic hormone (ADH) since they were identical in diabetes insipidus rats, nor to different rates of non-ADH-dependent backflux of filtrate since tissue osmolality and solute concentrations in the cortex, medulla, and papilla were similar in hypothyroid and control rats of both Sprague-Dawley and Brattleboro strains. The functional integrity of the diluting segment in hypothyroid rats was further demonstrated in experiments in which distal delivery was increased by contralateral nephrectomy or by administration of carbonic anhydrase inhibitors which decrease proximal sodium reabsorption. In both studies, fractional free water clearance increased markedly reaching levels significantly greater than in euthyroid controls. These results demonstrate that the impaired ability of the hypothyroid rat to excrete a water load is not due to incomplete suppression of ADH or decreased reabsorptive capacity of the diluting segment but results from decreased filtrate delivery to this site secondary to reduced GFR.

Carbonic anhydrase activity was studied in stomach and kidney homogenates, and isoenzymes were purified from erythrocytes and livers of male and female rats. Two liver isoenzymes of male and female rats and one erythrocyte isoenzyme had low CO 2 hydration activity. The enzymes of stomach and kidney, one isoenzyme of erythrocytes and one of female rat liver had high CO 2 hydration activity. The esterase activity toward p -nitrophenyl acetate paralleled the CO 2 hydration activity of all the isoenzymes. However, the esterase activity toward β-naphthyl acetate was either absent or did not show any correlation with the CO 2 hydration activity of isoenzymes. Male rat liver carbonic anhydrases were 1000 times less sensitive to sulfonamides than female rat liver carbonic anhydrases for the inhibition both of CO 2 hydration and esterase activity. Male rat liver carbonic anhydrases were as sensitive to inhibition by monovalent anions as were the female rat liver carbonic anhydrases. It is concluded that the active site of carbonic anhydrases from male rat liver is more hydrophilic than the active site of carbonic anhydrase from female rat liver or other tissues of the rat.

In contrast to previous concepts, the noradrenergic mechanism required for the anticonvulsant action of inhibitors of carbonic anhydrase in mice is extracerebral on the basis of the following: (1) the l-aromatic amino acid decarboxylase inhibitor Ro 4-4602/1 abolished the restorative action of d,1-dopa in reserpine- and phenoxy-benzamine-treated mice without evidence of significant inhibition of 1-aromatic amino acid decarboxylase in brain, (2) depletion of extracerebral stores of norepinehrine by the i.v. administration of 6-hydroxydopamine abolished the anticonvulsant effect of methazolamide, and (3) the anticonvulsant action of the carbonic anhydrase inhibitor in 6-hydroxydopamine-treated mice was restored by the administration of d,1-dopa. The noradrenergic mechanism appears to be of the α-type. Consistent antagonist action was shown by the α-blockers varied in action. The antagonist action of the α-blockers studied appeared to be specifically related to α-adrenergic blockade because (1) potency and duration of antagonist action correspond with potency and duration of α-adrenergic blockade, (2) phenoxybenzamine failed to antagonize the anticonvulsant action of diphenylhydantoin, (3) the anticonvulsant action of methazolamide was not abolished when phenoxybenzamine was given after the carbonic anhydrase inhibitor, (4) d,1-dopa administration reversed the antagonist action of phenoxybenzamine, and (5) the dopamine β-hydroxylase inhibitor U-14624 annulled the reversal of phenoxybenzamine's antagonist action by d,1-dopa. The anticonvusant action of methazolamide in rats was not affected by depletion of cerebral or extracerebral stores of norepinephrine by 6-hydroxydopamine. Anticonvulsant action was abolished by the administration of reserpine to rats injected i.vent. with 6-hydroxydopamine. The data suggest that norepinephrine and serotonin both are involved in the anticonvulsant action of inhibitors of carbonic anhydrase in rats.

The addition of Zn2+ to human carbonic anhydrase B holoenzyme was shown to enhance the protein fluorescence, and this enhancement was correlated with the inhibition of the p-nitrophenyl acetate esterase activity. The affinity for the inhibitory Zn2+ was increased when the ionic inhibitors, acetate or chloride, were added, suggesting that the inhibitory Zn2+-binding site is within the region of the protein that undergoes an anion-induced conformational change. A similar fluorescence enhancement was observed when Zn2+ was added to human carbonic anhydrase C and to bovine carbonic anhydrase, demonstrating that the binding site is not a thiol group. Circular-dichroism studies showed that the C isoenzyme but not the B isoenzyme underwent a major conformational change in the presence of Zn2+. A mechanism for the Zn2+-induced fluorescence enhancement was suggested on the basis of studies with simple compounds.

Suppression of a conditioned avoidance response in rats by 2 mg/kg chlorpromazine HCl (CPZ) was enhanced (230%) after treatment with 50 mg/kg acetazolamide. This enhanced pharmacological response in the acetazolamide-treated rats was accompanied by increased brain levels of 35 S after administration of chlorpromazine. In parallel experiments, an analogue of chlorpromazine, CL-13,850. which possesses no carbonic anhydrase inhibitory activity, had no influence on the pharmacological effect or the brain levels of 35 S after administration of chlorpromazine. The data suggest the enhanced pharmacological response following acetazolamide treatment is the result of increased brain levels of chlorpromazine brought about by carbonic anhydrase inhibition.

Canine peripheral venous or suprarenal aortic injections of sodium or meglumine iothalamate produced a significant swing towards an alkaline urine only in individual dogs injected with meglumine salts. When mean values were compared for the sodium or meglumine groups as a whole, however, no significant differences could be established for pH change or induced diuresis. With both salts, ATPase inhibition could play a role in urinary electrolyte excretion. Carbonic anhydrase inhibition does not seem to play a significant role in urinary pH changes induced by contrast media.

The role of bicarbonate in proximal tubular sodium chloride transport. Microperfusion studies of single proximal tubules in rat kidney cortex slices show a progressive increase in the rate of reabsorption of isosmotic fluid and NaCl as the bathing medium bicarbonate concentration is raised from 0 to 12 mEq/liter while the pH is maintained at 7.3 to 7.4. When bathing medium bicarbonate was 25 mEq/liter, tubules maintained a significantly lower intraluminal NaHCO 3 concentration: 15 mEq/liter. At the same time luminal NaCl concentration was equivalently elevated above that of the bathing medium. Reabsorptive rates were decreased when acetazolamide was added either to the bathing medium or to the tubular perfusate. Intratubular carbonic anhydrase reversed the effects of acetazolamide. Active transport of NaHCO 3 stimulates NaCl reabsorption by generating and maintaining concentration gradients favoring passive reabsorption of this salt. The proximal tubule also possesses mechanisms for active NaCl reabsorption.

The binding of acetate ions to bovine carbonic anhydrase (carbonate hydrolyase, EC 4.2.1.1) was investigated by NMR and by inhibition measurements. Two acetate ions were found to interact with the protein but only one is linked with the inhibition of the esterase activity of the enzyme. The NMR spectrum of the anion is sensitive to the stronger acetate binding, which is to a noninhibitory binding site. This acetate ion could be titrated with monovalent anions as N 3 − but not with p- toluenesulfonamide . The noninhibitory acetate ion performs an isotropic random motion faster than that of the enzyme. The correlation time for the tumbling of the methyl group was calculated from the T 1 /T 2 ratio, as well as from the NMR line-broadening ratio at two NMR frequencies and was found to be (2.3·10−9 0.8)·10−9 s. The binding of the two ligands decreases with pH and is probably dependent upon two different groups on the enzyme with p K a values of 7.5 and 7.4 for the binding of the inhibitory and noninhibitory acetate ions, respectively.

The distribution of carbonic anhydrase in the kidney of the frog (Rana temporaria) was studied by the histochemical method of Hansson, using light and electron microscope. In all cells showing enzyme activity, the staining deposits seemed to be diffusely distributed without any consistent local accumulation. The proximal tubule contained no or little enzyme. Some part of the distal tubule showed high enzyme activity, whereas the remainder was inactive. In the initial collecting tubule a mosaic pattern of stained and unstained cells was found, the stained cells being identical with the so called canaliculi cells. The collecting duct contained some stained cells, too. The renal corpuscle, the neck and the intermediate segment were inactive. Apparently the enzyme is less widely distributed along the kidney tubules of the frog compared to the tubules of man and rat, where enzyme activity is found in the convoluted part of the proximal tubule, the whole distal tubule and in many cells in the collecting duct. These differences in enzyme distribution as well as certain physiological data might indicate differences in the localization of the urinary acidification between the frog and these mammals.

The avian respiratory system is a crosscurrent gas exchange system. One of the aspects of this type of gas exchange system is that end-expired PCO2 is greater than arterial PCO2, the highest possible value being equal to mixed venous PCO2. We made steady-state measurements of arterial, mixed venous, and end-expired PCO2 in anesthetized, spontaneously breathing chickens during inhalation of room air or 4–8% CO2. We found end-expired PCO2 to be higher than both arterial and mixed venous PCO2, the sign of the differences being such as to oppose passive diffusion. The observation that end-expired PCO2 was higher than arterial PCO2 can be explained on the basis of crosscurrent gas exchange. However, the observation that end-expired PCO2 exceeded mixed venous PCO2 must be accounted for by some other mechanism. The positive end-expired to mixed venous PCO2 gradients can be explained if it is postulated that the charged membrane mechanism suggested by Gurtner et al. (Respiration Physiol. 7: 173–187, 1969) is present in the avian lung.

The active site of various carbonic anhydrases is not blocked by DDT. DDE, or dieldrin. Impairment of catalytic efficiency can only be documented in opalescent test solutions in which the insecticides are present in excess of their solubility limit. These slowly forming precipitates occlude enzyme from solution and furnish a physical explanation of the supposed inhibition. The amounts occluded depend, among other things, on the conformational and topographical characteristics of the particular carbonic anhydrase molecules.

Studies of the immediate and long-term effects of the ingestion of 2.2 to 6.3 mg./kg. diamox, given every six hours, on acid-base balance of the blood, plasma electrolyte levels, urinary electrolyte excretion rates, respiration and metabolic rate were carried out in five patients suffering from chronic lung disease and respiratory acidosis, and in five patients without lung, heart and kidney disease. Within four hours after the ingestion of a single dose of diamox both groups of patients exhibited a fall in the level of plasma bicarbonate and alkalinization of the urine with a diuresis of sodium, potassium and bicarbonate ions. The alterations in the level of arterial pCO 2 were variable and arterial pH either remained unchanged or fell moderately. The intensity of these changes bore no relationship to the size of the dose of diamox, the initial levels of plasma electrolytes, arterial pH or oxygen saturation. Alkalinization of the urine and diuresis of sodium, potassium and bicarbonate no longer occurred after the first few days in all but one patient, who suffered from heart failure associated with chronic pulmonary emphysema and cor pulmonale. He exhibited inhibition of acidification of the urine without electrolyte diuresis throughout an eight and one-half week study period. Prolonged administration of the drug was accompanied in all the patients by a significant fall in the level of plasma bicarbonate. Arterial pCO 2 fell significantly in three and arterial pH in two of the patients without heart, lung or kidney disease. Two of the emphysema patients exhibited a significant decrease in arterial pCO 2 and one an intensification of acidosis. Arterial pH did not rise to a normal level in the emphysema patients, nor to control values in the patients without lung, heart or kidney disease. Arterial pCO 2 often fluctuated considerably, without apparent cause, in the course of a morning and from day to day in control and diamox periods in three of the chronic emphysema patients. Hyperventilation or an increase in tidal air and a moderate slowing in rate of respiration, while minute volume of respiration remained relatively unchanged, accounted for the instances of sustained decrease in arterial pCO 2 , rise in alveolar pO 2 and alveolar ventilation. The ventilatory adjustments to prolonged administration of diamox appear to involve a stimulus or stimuli other than those included in Gray's multiple chemical factor theory of the regulation of ventilation. Intensification of acidosis by diamox-induced fall in plasma bicarbonate in the chronic emphysema patients did not alter their degree of respiratory depression during inhalation of 100 per cent oxygen and the response of one such patient was not less depressed when there was a concomitant reduction in the level of arterial pCO 2 . Potential disadvantages to the administration of diamox every six hours in some patients with chronic respiratory acidosis are intensification of the acidosis due to depletion of plasma bicarbonate and a delay of a few days in its return to control levels following discontinuance of the drug.

A procedure is described for evaluating drug effects on increased intraocular pressure (IOP) of the rabbit eye. The elevation of the IOP is obtained by evacuating the anterior chamber of the rabbit eye and replacing the aqueous humor with 0.5% methylcellulose solution in 0.9% saline. The resulting IOP increases steadily over the next two hours from a normal of 20.3 \m=+-\1.6 mm Hg to a peak of 29.8 \m=+-\1.8mm Hg as determined in 40 control rabbits. The IOP was measured with a modified Maklahov (Posner-lnglima) applanation tonometer. A dose dependent effect was obtained for the following carbonic anhydrase inhibitors: acetazolamide (regression coefficient [r] = \p=m-\0.954),methazolamide (r = \p=m-\0.767),and ethoxzolamide (r = \p=m-\0.886),when administered orally 30 minutes before the methylcellulose treatment.

Repeated urinary calculi developed in a patient who was treated with methazolamide for glaucoma. Previously, he had developed calculi while receiving acetazolamide.

The structural features of 4-ethylsulphonylnaphthalene-1-sulphonamide (ENS) responsible for the induction of DNA synthesis in the mouse bladder have been investigated using a method in which DNA synthesis is measured by the uptake of a thymidine analogue, [125I]5-iodo-2′-deoxyuridine (IUdR). The ability to stimulate DNA synthesis was unaffected by the nature of the alkyl group in 4-alkylsulphonylnaphthalene-1-sulphonamide. The sulphonamide group appeared to be essential to the activity of the molecule because naphthalene-1,4-disulphonamide was active whereas 1,4-diethylsulphonylnaphthalene was not. Maximum activity was found when the sulphonamide group was attached to an aromatic system (benzene or naphthalene) containing an alkylsulphonyl or a sulphonamide group. Bladder carcinogens other than ENS failed to stimulate the uptake of [125I]IUdR sufficiently to produce statistically significant results. The reasons for the large variation in response between individual mice are discussed, as are the implications of the structure activity relationships to the mode of action of ENS.

THE dependence of renal acidifying mechanisms on the carbonic anhydrase in the renal tubules is well recognized.1 The role of this enzyme in acidification of the urine, as well as its function in the production of acid and alkaline secretions in other organs such as the stomach and the pancreas, depends on the catalysis of the reversible reaction: H2O + CO2 = H2CO3.2 In 1940 Mann and Keilin3 discovered that sulfanilamide and other unsubstituted sulfonamides inhibit carbonic anhydrase activity, and subsequently the inhibition of urinary acidification by these compounds was demonstrated.4 , 5 Even before . . .

The influence of large doses of acetazolamide upon the regulation of the CSF pH in normocapnia and hypercapnia was studied in non-nephrectomized and nephrectomized rats. In non-nephrectomized animals studied over a 24 h period there was a decrease in the CSF HCO-3 concentration of 10 mEq/kg. Since the CSF acidosis was far in excess of that usually encountered with a comparable degree of plasma acidosis, the results indicate that acetazolamide inhibited a carbonic anhydrase-dependent transport of H+or HCO-3 between CSF and plasma. However, when the fall in plasma [HCO-3] was prevented by means of a bilateral nephrectomy, acetazolamide did not affect the CSF pH under normocapnic conditions, nor did it retard the accumulation of HCO-3 in the CSF during hypercapnic conditions. The results do not support any of the existing hypotheses which try to explain the regulation of the CSF pH in both respiratory and non-respiratory acid-base disturbances in terms of a common mechanism.

With the histochemical method of Hausler, activity of carbonic anhydrase (CAH) in normal mouse kidney was found most concentrated in proximal tubules, while distal tubules showed less intense activity of CAH. Specificity of the reaction was confirmed by complete block of the reaction with either in vivo or in vitro application of Diamox. After in vivo administration of Diamox, even in a large dose, complete block of CAH in sections was detectable for about half an hour. Duration of this blocking effect could be sustained longer by simultaneous administration of sodium bicarbonate, but not when the alkali and Diamox had been given successively. Reduction of the effect of Diamox by acidosis was also confirmed in the mice in a state of artificial acidosis which had been induced by oral administration of ammonium chloride.

Measurements were made of PCO2 and PO2 in alveolar gas of dog lungs rebreathing in situ and in the pulmonary arterial blood. The PCO2 in alveolar gas was almost always found to be higher than that of the blood under a variety of experimental conditions. The Po2 was also usually higher in alveolar gas than in blood, but less consistently than in the case of CO2. The magnitude of the observed gradients showed no consistent relationship to duration of experiment or rate of blood flow through the lung. Respiratory acidosis increased the CO2 gradient, but reduced that for O2. Inhibition of blood carbonic anhydrase enhanced the CO2 gradient. When normalized with respect to plasma bicarbonate concentration, the CO2 gradient is inversely related to plasma pH. No definitive explanation is offered for these observations.

Summary 1. Carbonic anhydrases from vertebrates, plants and bacteria have molecular weights of 30,000 (or multiples thereof), contain one zinc atom per 30,000 molecular weight, and are inhibited by acetazolamide and related compounds. 2. In mammals, there are two major isoenzymes of carbonic anhydrase. The so-called ‘high activity’ carbonic anhydrase possesses a carbon dioxide-hydratase activity many times (in the case of the guinea-pig isoenzymes, 18 times) that of the ‘low activity’ isoenzyme. 3. The mammalian isoenzymes differ from one another in their amino-acid compositions (the difference in serine contents being a consistent finding in a number of species), in their physical properties (isoelectric pH, retention by ion-exchange resins, electrophoretic mobility) and in their kinetic properties. 4. Mammalian carbonic anhydrases also catalyse the hydrolysis of some esters and the hydration of aldehydes. Their relative activities with these other substrates may be substantially different from their relative activities with carbon dioxide as substrate. The discovery that carbonic anhydrase may also catalyse other reactions raises the possibility that the enzyme may have other roles in metabolic pathways (e.g., in certain dehydrogenation reactions). 5. In plants, the role of carbonic anhydrase may be to catalyse the inter-conversion of bicarbonate and carbon dioxide, to provide ‘carbon dioxide’ in the form appropriate for carbon-fixing reactions. A similar role has been suggested for the carbonic anhydrase found in Neissariae. 6. The carbonic anhydrase of vertebrates (the ‘high activity’ isoenzyme of mammals) is found in many ion-transporting epithelia, but its role in them is still uncertain. It occurs in acid-transporting epithelia such as the stomach and kidney, and also in certain bicarbonate-transporting epithelia like the large intestine. There is, however, a poor correlation between the presence of the enzyme and the occurrence of acid or bicarbonate secretion. Thus there is little or no carbonic anhydrase in some tissues noted for their ability to secrete bicarbonate ions, such as the pancreas and ileum. Conversely, carbonic anhydrase-containing tissues like the avian salt gland and the elasmobranch rectal gland form concentrated sodium chloride solutions which are of nearly neutral pH. The correlation between the distribution of carbonic anhydrase and the occurrence of active chloride transport appears similar to that between carbonic anhydrase and bicarbonate ion transport. 7. While the red cells of most mammalian species so far studied contain both ‘high activity’ and ‘low activity’ isoenzymes, the latter is reported to be absent from the erythrocytes of sheep, ox and dog. Presumably the ‘low activity’ isoenzyme is not necessary for an adequate rate of carbon dioxide exchange to occur between tissues and lungs in these species. 8. The ‘low activity’ isoenzyme is present in tissues other than blood, for example, the colon, caecum and ox rumen, and probably also, the gall bladder and kidney medulla. Its distribution differs markedly from that of the ‘high activity’ isoenzyme and presumably it has a particular functional importance of its own. It is not possible to define the role of the ‘low activity’ isoenzyme at present, but attention is drawn to the possibility that it is concerned in the handling of the products of microbial fermentation, such as ammonia and organic acids, by the large intestines and ruminant forestomach. My work on the isoenzymes of carbonic anhydrase was supported by a Medical Research Council Scholarship, and by an equipment grant to Dr D. S. Parsons. I am also grateful to Dr D. S. Parsons for his comments on the manuscript.

Formation of 13 C-resonances attributable to carbamino derivatives has been observed in human erythrocyte hemolysate preparations equilibrated with 13 CO 2 at 33°. Carbamino formation is most marked in deoxyhemoglobin and at alkaline pH, and is very largely inhibited by the addition of 2,3-diphosphoglycerate or conversion to oxyhemoglobin. The prominent carbamino resonance at 30.0 ppm upfield of CS 2 is visible in the spectrum of packed, deoxygenated erythrocytes equilibrated in 13 CO 2 . This chemical shift falls close to that observed with sperm-whale myoglobin and within 2 ppm upfield of that seen with simple amino acids and peptides. The bicarbonate-carbonate resonance near 33 ppm is broad in the hemoglobin preparations, which always contain some carbonic anhydrase, but becomes narrow in the presence of the carbonic anhydrase inhibitor, acetazolamide. The nuclear magnetic resonance condition of intermediate exchange rate with dissolved CO 2 (68.4 ppm) obtains in the absence of inhibitor. The process has marked consequences in reducing the spin-lattice relaxation time, T 1 , of the bicarbonate resonance by more than 10 times. The deoxyhemoglobin carbamino resonance has a T 1 value of 700 msec, indistinguishable from that of the protein carbonyl resonance envelope.

1. 1.The activity of Forms B and C of human erythrocytic carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) has been studied at 1.6°. The catalytic effect of the enzymes on the dehydration of carbonic acid was measured by means of a pH-stat method. 2. 2.The dependence of the activity of Form C on the hydrogen ion activity has been studied between pH 6.9 and 7.9. The formation of the enzyme-substrate complex appears to be dependent on the acid form of a group which ionizes within this pH range, whilst the breakdown of the complex into free enzyme and the product, CO2, proceeds indepdendently of ionizing groups. 3. 3.Conversely, the breakdown of the B enzyme-substrate complex into CO2 and free enzyme appears to be dependent on the acid form of a group with pK = 7.4, whilst the formation of this enzyme-substrate complex appears to proceed independently of groups ionizing between pH 6.8 and 8.4. 4. 4.Monovalent anions act as inhibitors for both enzymes. When listed in order of increasing inhibitory effectiveness towards the C enzyme, the following series is obtained: Cl−

A study of the mechanism of exchange regulation between the different calcium pools in freshwater lamellibranch mantle shows: 1) that the substances concerned in this regulation, namely the calcium reserves and carbonic anhydrase, must be topographically in close proximity, 2) that the calcium reserves occur as small extracellular calcified granules localised in the interstitial tissue of the mantle (evidence both from experiments on homogenates and from histological examination), and 3) that observations on the sedimentation in mantle homogenates suggest that the enzyme is bound to the calcified granules. The role of such an enzyme-substrate unit is discussed.

The anticonvulsant effect of the carbonic anhydrase inhibitors methazolamide, acetazolamide and dichlorphenamide can be antagonized by reserpine. This antagonism was related to depletion of catecholamines and was reversed by α-methyldopa, DOPA, monoamine oxidase inhibitors and by the sympathomimetics amphetamine and methylphenidate. The ED 5 0 (mg/kg) values for restoring the anticonvulsant effect of methazolamide, 500 mg/kg, in reserpine-pretreated mice were: α-methyldopa, 15.4, DOPA, 259, and pheniprazine 13.5 mg/kg. The effects of α-methyldopa and DOPA were blocked by large doses of RO 4-4602, and partially by carbidopa (α-hydrazino-α-methyldopa), as well as by inhibitors of β-hydroxylase, disulfiram, picolinic acid, pyrimidinethiol and FLA-63. The effects of acetazolamide, 1000 mg/kg, and dichlorphenamide, 1500 or 3000 mg/kg, were partially restored by α-methyldopa; methylphenidate (ED 5 0 35.6 mg/kg) and amphetamine (ED 5 0 11.2 mg/kg) restored fully the anti-convulsant effect of dichlorphenamide. The anticonvulsant activity of carbonic anhydrase inhibitors involve largely noradrenergic mechanisms within the central nervous system.

Pancreatic flow and HCO3– and Cl– output were studied in dogs following 2 U/kg (i.v.) of secretin. Acetazolamide reduced flow and HCO3– output to half normal. HCl produced the same effect. NaHCO3 increased flow and HCO3– output. CO2 produced little change. Severe hyperventilation decreased flow and HCO3– output. Results are consistent with secretion dependent on the over-all reaction OH– + CO2 → HCO3–, with both a carbonic-anhydrase catalyzed and an uncatalyzed component. Reduction of either reactant or of enzyme decreased output: increase of the reactants increased output. From in vitro kinetics and analysis of rates in vivo, the role of carbonic anhydrase may be assessed. About half of HCO3– output normally depends on the uncatalyzed reaction, and half on the catalyzed. In metabolic alterations, only the uncatalyzed component is affected. Pancreatic carbonic anhydrase is greatly in excess of that needed for the observed catalyzed rate in vivo. Correspondingly, reduction of output by acetazolamide occurs only when >99% of pancreatic carbonic anhydrase is inhibited. It is suggested that the rate-limiting event in pancreatic HCO3– secretion is transport of HCO3– from cell to duct.

The systematic study of the magnetic circular dichroic (MCD) spectra of Co(II) model complexes allows recognition of hitherto unknown identities or dissimilarities of their geometries with those of Co(II) at the active site of metalloenzymes. Thus based on such comparisons, MCD spectra indicate that the overall geometry of Co(II) at the active site of Co-carboxypeptidase or Co-thermolysin is tetrahedral, and is not altered significantly by addition of inhibitors. On the other hand, the MCD spectra of Co-alkaline phosphatase and the alkaline form of Co-carbonic anhydrase are quite similar to but not identical with that of Co(Me6Tren)Br2, a pentacoordinated cobalt complex. Furthermore, addition of acetazolamide to Co-carbonic anhydrase markedly alters its MCD signal suggesting a possible change in geometry.

Summary The role of carbonic anhydrase in the regulation of siezure activity has been studied by comparing the brain enzyme levels of normal rats with those of rats found seizure resistant in the maximal electroshock test. These includeed rats (1) treated with acetazolamide, an anticonvulsant drug; (2) exposed to head X-irradiation, a procedure known to abolish the tonic extensor response to maximal electroshock; and (3) exposed to a combination of acetazolamide and head X-irradiation. In addition, rats which are congenitally seizure resistant in the maximal electroschock test as well as two strains of mice, one audiogenic seizure prone and the other seizure resistant, were also examined in this study. The enzyme determinations were done in different anatomical regions of the CNS. Our results show a regin specific effect. In all the conditions where seizure activity was suppressed, carbonic anhydrase activity was also suppressed with the greatest change occuring in the caudate nucleus. Since it is demonstrated in widely different experimental conditions and in two species, the association of a region specific inhibition of carbonic anhydrase in the caudate nucleus and seizure resitance (anticonvulsant action) is considered highly significant.

The CO2 hydration activity of human carbonic anhydrase B is rapidly inactivated by 1-fluoro-2,4-dinitrobenzene at neutral pH. The inactivation reaction is second order and is retarded by the presence of various enzyme inhibitors. The second order rate constant for inactivation is about 100 times the second order rate constant for the reaction of 1-fluoro-2,4-dinitrobenzene and α-N-acetylhistidine. The inactivated enzyme was purified and was found to contain one 2,4-dinitrophenyl group per molecule, at the 3' position of a histidine residue. While such a reacted enzyme has lost more than 95% of its CO2 hydration activity, it retains over half its esterase activity. The dinitrophenyl group can be quantitatively removed by thiolysis, regenerating a fully active native carbonic anhydrase. Dinitrophenyl carbonic anhydrase binds iodoacetate with somewhat greater affinity than the unmodified enzyme. However, iodoacetate does not react covalently with the dinitrophenyl enzyme, as it does with the native enzyme. Analysis of the tryptic peptides of dinitrophenyl carbonic anhydrase shows that reaction takes place with histidine residue 204, the same residue modified by iodoacetate. It is suggested that histidine 204, while clearly in the active site, may not play any direct role in enzyme catalysis. Modifications at this position may disrupt the normal substrate binding site.

Esterases found in rabbit erythrocytes and platelets were separated by zone electrophoresis in starch gels. Activity tests in various substrates were used to classify the esterase bands into four major categories. The bands of each category were further subdivided into groups by the effects of various inhibiting and activating agents. These experiments showed that the genetic loci which control the enzymes of systems 1, 2 and 3 control isozymes that respond identically with the test reagents in their respective systems. Diisopropyifluorophosphate and eserine sulfate inhibition indicated that the active sites of systems 1 and 2 enzymes are different. This supported the conclusion that the esterases of systems 1 and 2 are the products of closely linked loci and not multiple alleles at a single locus. Carbonic anhydrases which migrated in the cathodal portion of the gel were identified by their sensitivity to acetazolamide and more intense staining with $- rather than anaphthyl acetate as substrate. Carboxylic acid esters of naphthols, phenols and alcohols are hydrolyzed by esterases. In addition to their hydrolytic activity, some esterases are capable of catalyzing synthetic substrates. a- and �3-Naphthol substrates have been used as a method for distinguishing between the simple fatty acid esterases and true lipases (16).

A spin-labeled sulfonamide [1-oxy1-4-(p-sulfamylbenzoyl)-amino- 2,2,6,6-tetramethyl-piperidine] has been synthesized and its interaction with bovine erythrocyte carbonic anhydrase B studied by means of electron spin resonance. When free in solution, this sulfonamide exhibited a sharp three line spectrum due to hyperfine interaction between the unpaired electron and the nitrogen nucleus of the nitroxide group. However, in the presence of bovine carbonic anhydrase B, the spectrum of the spin-labeled sulfonamide resembled the powder spectrum of the nitroxide free radical. This indicated that when the sulfonamide binds to carbonic anyhydrase the nitroxide group is immobilized by interaction with the enzyme binding site. Ethoxyzolamide (a drug which has a very high affinity for the active site of carbonic anhydrase) specifically displaced the sulfonamide spin label from its binding site on carbonic anhydrase. These results suggest that the active site of bovine carbonic anhydrase B is a narrow crevice similar to that already described for human carbonic anhydrase C.