Simulataneous and independent versus antagonistic inhibition of muscle carbonic anhydrase (CA III) by acetazolamide and cyanate

The inhibition by cyanate and acetazolamide of pig muscle carbonic anhydrase III (CA III) CO₂ hydratase activity was studied in order to explore mechanistic features possibly unique to the muscle isoenzyme. The turnover number for CO₂ hydration was found to be 6000 sec^?1 with a K_m of 83 mM for CO₂. Cyanate inhibition (K_i, 3 μM) and acetazolamide inhibition (K_i, 44 μM) were both found to be noncompetitive with respect to CO₂. Significantly, acetazolamide and cyanate displayed non-exclusive binding to pig muscle carbonic anhydrase. The similarity of mode and degree of inhibition of muscle carbonic anhydrase by cyanate as compared with the inhibition of the erythrocyte isoenzymes suggests the existence of a similar metal environment. However, the observation that cyanate and acetazolamide bind simultaneously to CA III and the comparatively large K_i for acetazolamide per se appear to be more compatible with a different mode of coordination of the zinc with the sulfonamide, thus supporting a five-coordinant zinc in the catalytic mechanism of CO₂ hydration for CA III.     

Effects of omeprazole, famotidine, and ranitidine on the enzyme activities of carbonic anhydrase from bovine stomach in vitro and rat erythrocytes in vivo

In this study, the effects of omeprazole, famotidine, and ranitidine on bovine stomach carbonic anhydrase (EC 4.2.1.1.) isoenzymes have been investigated in vitro. Bovine stomach carbonic anhydrase (CA) was purified from four different cell localisations of bovine stomach using affinity chromatography by Sepharose 4B-L-tyrosine sulphanilamide. The inhibition or activation effects of three different medical drugs on CA isoenzymes were determined using esterase activity and the CO(2)-hydratase method by plotting activity % vs. [medical drug]. The K(i) values for omeprazole, famotidine, and ranitidine were determined in all localization CA, respectively. The I(50) values of the drugs exhibiting an inhibition effect were found by means of these graphs. It was observed that omeprazole, famotidine, and ranitidine showed inhibition of bovine stomach CA activity. In addition, in vivo studies were performed for these medical drugs in Sprague-Dawley rats. It was demonstrated that CA in erythrocytes was significantly inhibited by these drugs to 3 h.     

Examination of two independent kinetic assays for determining the inhibition of carbonic anhydrases I and II: structure-activity comparison of sulfamates and sulfamides

Enzyme inhibition assays often require deviations from physiological conditions. For carbonic anhydrases, procedures involving native CO(2) and non-native substrates have been used. We compared a native and a non-native substrate in the context of inhibition of human carbonic anhydrases I and II by examining various sulfamate and sulfamide compounds in two kinetic assays: hydration of CO(2) and hydrolysis of 4-nitrophenylacetate. For carbonic anhydrase II, the two assays consistently generated similar K(i) values, with the relative difference between the assays never exceeding 2.5-fold. However, for carbonic anhydrase I there was more variability between the two assays, with K(i) values for three compounds differing by more than 2.5-fold, up to eightfold. In the CO(2) hydration assay, some sulfamates and sulfamides exhibited mixed kinetics or partial inhibition. Our results indicate that K(i) or K(d) values from carbonic anhydrase assays involving non-native substrates should be confirmed by assays that use CO(2) (or HCO), to establish pharmacological relevance. From structure-activity comparisons, the sulfamate is more effective than the sulfamide in inhibiting carbonic anhydrase I and II, but the sulfamate does not confer selectivity. In contrast, the sulfonamide confers selectivity for carbonic anhydrase I (10- to 30-fold). Selectivity for carbonic anhydrase II occurred with the substituted fructose moiety, especially the d-enantiomer (>100-fold).     

Anticonvulsant/antiepileptic carbonic anhydrase inhibitors: a patent review

Introduction: An epileptic seizure is a transient occurrence of signs and/or symptoms due to abnormal excessive or synchronous neuronal activity in the brain. The International League Against Epilepsy classifies seizures in two broad categories: partial (localized to one cerebral hemisphere) and generalized (localized to both cerebral hemispheres). One indirect pathway for the treatment of epilepsy includes the inhibition of carbonic anhydrase (CA), thereby increasing CO₂ levels in the brain.

Areas covered: Carbonic anhydrases (EC 4.2.1.1) are ubiquitous metalloenzymes that catalyze the reversible hydration/dehydration of CO₂/HCO₃ ^-, respectively. CA inhibitors (CAIs) such as acetazolamide, methazolamide, topiramate, zonisamide, and sulthiame can reduce seizures through perturbation of the CO₂ equilibrium and/or the inhibition of ion channels. This review focuses on the mechanism of epilepsy, CA catalysis, and recent developments in the treatment of epilepsy using CAIs.

Expert opinion: Based on the observed active-site binding interactions of CAIs in crystal structures and their respective inhibition constants, structure–activity relationships can be mapped. Various CAIs along with novel techniques to administer them have been patented in the last four years. However, epilepsy continues to be a path less traveled when it comes to CAIs. A major area of research must focus toward the design of isoform-specific inhibitors using analogs of existing CAIs.     

Stimulatory effect of Coca-Cola<Superscript>TM</Superscript> on gastroduodenal HCO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack> secretion in rats

We examined the effect of various carbonated beverages, especially Coca-Cola^TM, on the HCO₃⁻ secretion in the rat stomach and duodenum. Under urethane anaesthesia, a chambered stomach or a proximal duodenal loop was perfused with saline, and HCO₃⁻ secretion was measured at pH 7.0 using a pH-stat method and by adding 2 mM HCl. The amount of CO₂ contained in these beverages was about 4–7 g/mL. Coca-Cola^TM topically applied to the mucosa for 10 min significantly increased the HCO₃⁻ secretion in both the stomach and the duodenum. The HCO₃⁻ response in the duodenum was totally abolished by indomethacin and also partially inhibited by acetazolamide, an inhibitor of carbonic anhydrase. Likewise, the response in the stomach was also markedly inhibited by either acetazolamide or indomethacin. The mucosal application of Coca-Cola^TM increased the PGE₂ contents in both the stomach and the duodenum. Other carbonated beverages, such as sparkling water, Fanta Grape^TM or cider, also increased the HCO₃⁻ secretion in these tissues. These results suggest that Coca-Cola^TM induces HCO₃⁻ secretion in both the stomach and the duodenum, and these responses may be attributable to both the intracellular supply of HCO₃⁻ generated via carbonic anhydrase, and endogenous PGs, probably related to the acidic pH of the solution. Received 4 August 2006; accepted 10 November 2006     

Inhibition of the archaeal beta-class (Cab) and gamma-class (Cam) carbonic anhydrases

Five independently evolved classes (alpha-, beta-, gamma-, delta-, zeta-) of carbonic anhydrases facilitate the reversible hydration of carbon dioxide to bicarbonate of which the alpha-class is the most extensively studied. Detailed inhibition studies of the alpha-class with the two main classes of inhibitors, sulfonamides and metal-complexing anions, revealed many inhibitors that are used as therapeutic agents to prevent and treat many diseases. Recent inhibitor studies of the archaeal beta-class (Cab) and the gamma-class (Cam) carbonic anhydrases show differences in inhibition response to sulfonamides and metal-complexing anions, when compared to the alpha-class carbonic anhydrases. In addition, inhibition between Cab and Cam differ. These inhibition patterns are consistent with the idea that although, alpha-, beta-, and gamma-class carbonic anhydrases participate in the same two-step isomechanism, diverse active site architecture among these classes predicts variations on the catalytic mechanism. These inhibitor studies of the archaeal beta- and gamma-class carbonic anhydrases give insight to new applications of current day carbonic anhydrase inhibitors, as well as direct research to develop new compounds that may be specific inhibitors of prokaryotic carbonic anhydrases.     

Uptake and Stereoselective Binding of the Enantiomers of MK-927, a Potent Carbonic Anhydrase Inhibitor,by Human Erythrocytes in Vitro

MK-927 [5,6-dihydro-4H-4(isobutylamino)thieno(2,3-B)thiopyran-2-sulfonamide-7.7 dioxide], a potent carbonic anhydrase inhibitor, contains a chiral center and exists as a racemate. In order to understand the kinetic behavior of the enantiomers of MK-927 in the body, the uptake and binding of these compounds were studied in human erythrocytes in vitro. Since no degradation or metabolism of the enantiomers occurred during incubation in blood, one can describe the equilibration of the drugs between plasma and erythrocytes by a closed two-compartment system. Erythrocytes were considered as a compartment composed of two parts: one in which free drug is exchangeable to plasma and the other in which drug is tightly bound to carbonic anhydrase in a Michaelis–Menten type binding. After the addition of the enantiomers individually to fresh blood, they were taken up by erythrocytes rapidly in a concentration-dependent manner. The time to achieve equilibrium decreased as the concentration increased, suggesting saturation of binding sites. With the assumption of simple diffusion, the binding and transfer kinetics were determined simultaneously by computer fitting. There were no Stereoselective differences in the transfer process of the enantiomers across the erythrocyte membrane, while binding of the enantiomers exhibited stereoselectivity. The penetration of the unbound enantiomer across the erythrocyte cell membrane was rapid, with a mean transit time of about 3 sec. The S-( + )-enantiomer was bound to the high-affinity carbonic anhydrase isoenzyme more strongly than the R-( – )-enantiomer by approximately 10-fold. For the low-affinity isoenzyme, the R-( – )-enantiomer was bound more strongly than the S-( + )-enantiomer.     

Differential in vitro inhibitory effects of anticancer drugs on tumor-associated carbonic anhydrase isozymes CA IX and CA XII

Carbonic anhydrase IX (CA IX) and, to a lesser extent, carbonic anhydrase XII (CA XII) are highly overexpressed in hypoxic tumors. In this study, the inhibitory effects of 11 different anticancer drugs including paclitaxel, amethopterin, etoposide, irinotecan, gemcitabine, 5-fluorouracil, oxaliplatin, epirubicin, cisplatin and carboplatin on the tumor-associated carbonic anhydrase isozymes CA IX and CA XII and cytosolic carbonic anhydrases I and II have been investigated. SX.18MV-R Applied Photophysics stopped-flow instrument was used for measuring the initial velocities for the CO2 hydration reaction catalyzed by different CA isozymes, by following the change in the absorbance of a pH indicator. CA IX and CA XII were the most affected by carboplatin and cisplatin amongst the panel of anticancer drugs. Moreover, the cytosolic carbonic anhydrases I and II can also be affected. Consequently, CA IX and CA XII are interesting targets for anticancer drug development, although more selective and powerful CA inhibitors could prove useful for elucidating the role of the protein in hypoxic cancers, for controlling the pH imbalance in tumor cells and for developing diagnostic or therapeutic applications for the management of hypoxic tumors, generally unresponsive to classical chemo- and radiotherapy.     

Design and development of novel series of indole-3-sulfonamide ureido derivatives as selective carbonic anhydrase II inhibitors

Indole is a privileged moiety with a wide range of bioactivities, making it a popular scaffold in drug design and development studies as well as in synthetic chemistry. Here, novel urea derivatives of indole, containing sulfonamide at position-3 of indole, were synthesized using a well-known tail approach, as carbonic anhydrases (CAs; EC 4.2.1.1) inhibitors. All the newly synthesized molecules were screened for their CA-inhibitory activity against four clinically relevant isoforms of human-origin carbonic anhydrase (hCA), that is, hCA I, hCA II, hCA IX, and hCA XII. These compounds were specifically active against hCA II, more than against hCA I, hCA IX, and hCA XII. Derivative 6l was found to be most active, with a K_i value of 7.7 µM against hCA II.     

Synthesis,characterization, and biological studies of chalcone derivatives containing Schiff bases: Synthetic derivatives for the treatment of epilepsy and Alzheimer's disease

In this study, first, Schiff base-containing chalcone derivatives were synthesized. The human carbonic anhydrase (hCA) isoenzymes I and II were then purified from human erythrocytes using Sepharose-4B-l -tyrosine-sulfanilamide affinity chromatography. In addition, the inhibitory effects of the newly synthesized compounds on the activities of hCA and acetylcholinesterase (AChE) were investigated in vitro, using the esterase and acetylcholine iodide method. The IC₅₀ values were determined and the K_i values of AChE and hCA activities were calculated from the Lineweaver–Burk graphs determined in this study. The hCA I isoform was inhibited by these chalcone derivatives containing Schiff bases ( 3a–j and 5a–f ) in low nanomolar levels, whose K_i values ranged between 141.88 ± 24.10 and 2,234.47 ± 38.11 nM. Against the physiologically dominant isoform hCA II, the compounds demonstrated K_i values varying from 199.31 ± 40.45 to 602.79 ± 263.22 nM. Also, these compounds effectively inhibited AChE, with K_i values ranging from 20.41 ± 6.04 to 125.94 ± 23.88 nM. According to these results, the newly synthesized molecules were found to be potent inhibitors of these enzymes.     

Which Carbonic Anhydrases are Targeted by the Antiepileptic Sulfonamides and Sulfamates?

Zonisamide, sulthiame, and topiramate, clinically used antiepileptics are inhibitors of mammalian carbonic anhydrase isoforms I–XIV, indiscriminately inhibiting with variable efficacy all the catalytically active isoforms present in mammals. However, it is not clear which carbonic anhydrase isozymes might be responsible for the anticonvulsant activity of such sulfonamide/sulfamate drugs. We examine here the full inhibition profile against all mammalian carbonic anhydrases of the above antiepileptic drugs together with two investigational, structurally related sulfonamides, one of which is and the other is not an anticonvulsant. No clear‐cut data allow us to propose which are the carbonic anhydrases involved in these processes, but strong carbonic anhydrase II, VII, IX, and XII inhibition, correlated with sufficiently high liposolubility may lead to effective anticonvulsants of this pharmacological class. Therefore, further studies are warranted for better understanding these phenomena. Whether the inhibition of such carbonic anhydrases present in the mammalian brain might be relevant for designing pharmacological agents useful in the management of neurological disorders or for understanding the multifactorial mechanism of action of some of these drugs is a topic which surely deserves further investigations.     

Carbonic anhydrase inhibitors and the management of cancer

Recent progress in understanding the role of catalytically active carbonic anhydrases in tumors has opened new possibilities for diagnostic and/or therapeutic applications of carbonic anhydrase inhibitors selectively blocking the enzyme activity of cancer-related isoforms, namely CA IX and CA XII. Different classes of inhibitors have been investigated in order to evaluate their usefulness as in vivo imaging tools, as modulators of intratumoral pH that influences uptake of conventional chemotherapeutics, or as drugs impeding survival of tumor cells exposed to physiological stresses including hypoxia and acidosis. Here we summarize the most important data related to expression, regulation and functional aspects of cancer-related carbonic anhydrases and discuss advances in synthesis and preclinical studies of isozyme-selective and highly efficient carbonic anhydrase inhibitors.     

In vivo effects of oral contraceptives on paraoxonase, catalase and carbonic anhydrase enzyme activities on mouse

Many effects that oestrogens and progestrogens used in oral contraceptive (OC) have on enzyme physiology are of importance on homeostasis. This study was carried out in order to determine the in vivo effect of three oral contraceptives containing ethinyl estradiol in combination with desogestrel and levonorgestrel on the paraoxonase (PON), catalase (CAT) and carbonic anhydrase (CA) activities in mice, which are model organisms for humans. Serum and liver paraoxonase activities were determined spectrophotometrically by using paraoxan as a substrate according to the methods of Gan et al. and Gil et al., respectively. Catalase and carbonic anhydrase activities were determined from erythrocytes used Aebi and Maren methods, respectively. For these studies, a group of ten mice (25+/-2 g) was selected for oral administration for 21 d of each drug (0.15 mg desogestrel+0.03 mg ethinylestradiol (A); 0.15 mg levanogestrel+0.03 mg ethinylestradiol (B) and 0.15 mg desogestrel+0.02 mg ethinylestradiol (C)). A group of ten mice was included in the study for a control group, which were not subject to drug administration. For each drug, a mean of the serum and liver paraoxonase activity and erythrocytes catalase and carbonic anhydrase activities were determined and compared to the control groups. While mouse liver PON activity showed a statistically significant decrease for all three drugs, serum PON activity increased. Erythrocytes catalase activity was significantly decreased by all contraceptives used. On the other hand, these contraceptives did not change the erythrocytes carbonic anhydrase activity.     

Carbonic anhydrase enzymes for regulating mast cell hematopoiesis and type-2 inflammation: a patent evaluation (WO2017/058370)

Introduction: Activity modulators of carbonic anhydrases hold great potential for several therapeutic applications against ophthalmologic and neurological disease, cancer, and infectious diseases. The involvement of carbonic anhydrase in the regulation of mast cell response opens new ways for the treatment of mastocytosis, allergic inflammation, and parasite infection.

Areas covered: The application claims the use of carbonic anhydrase activity modulators (inhibitors or activators) for treating allergic disease, bacterial infection, fungal infection, viral infection, mastocytosis, or mast cell–mediated inflammation.

Expert opinion: Although there is a lack of essential biological data, this patent proposes a new type of applications for carbonic anhydrase inhibitors and deserves further studies. This may lead to new advances in the field of carbonic anhydrase with potential therapeutic implications in the management of type-2 inflammation.     

Biomedical applications of prokaryotic carbonic anhydrases

Introduction: The hydration/dehydration of CO₂ catalyzed by carbonic anhydrases (CAs, EC 4.2.1.1) is a crucial physiological reaction for the survival of all living organisms because it is connected with numerous biosynthetic and biochemical pathways requiring CO₂ or HCO₃^?, such as respiration, photosynthesis, carboxylation reactions, pH homeostasis, secretion of electrolytes, transport of CO₂, bicarbonate, etc.

Areas covered: The bacterial genome encodes CAs belonging to the α-, β-, and γ-CA classes able to ensure the survival and/or satisfying the metabolic needs of the bacteria, as demonstrated by in vivo and in vitro experiments. The discovery of new anti-infectives that target new bacterial pathways, such as those involving CAs, may lead to effective therapies against diseases subject to the antibiotic resistance. This aspect is important in pharmaceutical and biomedical research but received little attention till recently.

Expert opinion: An overview of the potential use of CAs in biomedical applications, as drug targets, bioindicators, and within artificial organs is presented. The discovery of thermostable bacterial CAs allowed the use of CAs in biotechnological applications, but patents related to the use of bacterial CAs in the development of pharmacological agents are scarce.     

Differences in the kinetic properties and sensitivity to inhibitors of human placental, erythrocyte, and major hepatic aldehyde dehydrogenase isoenzymes

(i) The characteristics of the major human hepatic isoenzymes of aldehyde dehydrogenase (ALDH), ALDH I and ALDH II, were compared with the ALDH activities found in human placenta and erythrocytes, (ii) In human liver biopsies, the Km of ALDH I was approximately 7 mumol/L whereas it was 32 mumol/L for ALDH II. The Vmax for ALDH I was 2-3 times greater than the ALDH II Vmax. Human liver ALDH I and II also differed in their sensitivity in inhibitors. Namely, ALDH I was less sensitive to disulfiram than the ALDH II isoenzyme. (iii) ALDH activity in human placenta and erythrocytes was much lower than in liver tissue. Kinetic data showed that placental ALDH isoenzyme had a high Km (in the millimolar range) and increased its activity raising the pH from 7.4 to 8.8, more than the hepatic ALDH I and ALDH II isoenzymes did. Erythrocyte ALDH activity presented a dual component; the smaller one was characterized by a low Km (micromolar range), whereas most of the ALDH activity showed a high Km (millimolar range). (iv) Placental ALDH was resistant to nitrefazole inhibition and was inhibited by disulfiram in a manner similar to the hepatic ALDH I isoenzyme; erythrocyte ALDH was more sensitive to the inhibitory action of disulfiram and nitrefazole. (v) It is concluded that erythrocyte and placental ALDH isoenzymes are different from the hepatic ALDH I and ALDH II forms. It is also suggested that placental and erythrocyte ALDH isoenzymes are different high-Km isoenzymes.     

Bioavailability,tissue distribution,and excretion characteristics of the novel carbonic anhydrase inhibitor tolsultazolamide in rats

Aim： Tolsultazolamide, a novel carbonic anhydrase inhibitor, is designed for the prophylaxis and treatment of acute mountain sickness. The aim of this study was to investigate the pharmacokinetics, tissue distribution, and excretion characteristics of tolsultazolamide and the sex difference in pharmacokinetics in rats. Methods： For pharmacokinetic study, rats were intravenously injected tolsultazolamide at I and 2 mg/kg or orally administered tol- sultazolamide at 20, 40, or 80 mp=/kg） in a pharmacokinetic study. The concentrations of tolsultazolamide in plasma were determined with high-performance liquid chromatography, with a liquid-liquid extraction. For tissue distribution study, tolsultazolamide （80 mpJkg） was orally administered to overnight fasted rats （six per group and three per sex）. Samples were collected from the brain, heart, lung, liver, spleen, muscle, kidney, stomach, fat, intestines, pancreas and sexual gland. For excretion study, tolsultazolamide （40 mg/kg） was orally administered to 6 rats （three per sex）. The urine, feces, and bile samples were collected at 24, 48, and72 h. Results： After its intravenous administration, tolsultazolamide was rapidly eliminated from the plasma, with T~/2 of about 60-90 min. The AUCo_t and the initial concentration （CO） values were proportional to the intravenous doses. After its oral administration, tolsult- azolamide showed dose-independent pharmacokinetic characteristics, with Tmax and T~/2 of approximately 2 h and 5-7 h, respectively, and good oral absolute bioavailability of about 60%. Tolsultazolamide was distributed widely in various tissues. The highest tolsult- azolamide levels were detected in the stomach, intestine, spleen, lung, and kidney. Total excretion of unchanged tolsultazolamide in the urine, feces, and bile was less than 2%. The Cm,x and AUC of tolsultazolamide were significantly higher in female rats than those in male rats. Clearance and volume of distribution were greater in male rats than those in female rats. The oral absolute bioavailability was also significantly different between female rats （about 83%） and male rats （about 37%）. Conclusion： Tolsultazolamide was well absorbed and widely distributed in the rat, and very little of the unchanged form was excreted. Sex had a significant effect on the pharmacokinetics of tolsultazolamide.     

Binding of Sulfonamides to Carbonic Anhydrase: Influence on Distribution Within Blood and on Pharmacokinetics

Four new aromatic sulfonamides were synthesized and purified by standard techniques. Two were unsubstituted, primary sulfonamides and two possessed substituents on the sulfonamide nitrogen. The affinity of the inhibitors for the enzyme carbonic anhydrase was determined in terms of the inhibitory potency, which was found to be dependent on the presence of an unsubstituted sulfonamide group. Binding studies were performed in erythrocyte suspensions using a range of concentrations and the unbound, extracellular concentrations were determined by high-performance liquid chromatographic (HPLC) assay. The dissociation constant of binding and the total binding capacity of the erythrocytes were estimated by nonlinear regression using a two-site binding model. The affinity of the compounds for erythrocytes reflected their inhibitory potency against the enzyme. Binding to plasma proteins was more dependent on lipophilicity and pK _a and was stronger for the substituted sulfonamides. Pharmacokinetic studies in rats showed that the unsubstituted sulfonamides with a high affinity for carbonic anhydrase in erythrocytes have longer half-lives and lower clearance values than the substituted sulfonamides which were more strongly bound to plasma proteins. However, comparison of unbound clearance values showed that the variations in molecular structure, which produced differences in carbonic anhydrase binding and in distribution, also produced variations in susceptibility to elimination processes.     

Diuretics and the renal adenylate cyclase system

1 The relationship between the diuretic effectiveness and the effect on the renal adenylate cyclase of three diuretics, acetazolamide, frusemide and ethacrynic acid, was examined. The hypothesis that acetazolamide and parathyroid hormone (PTH), inhibit renal carbonic anhydrase by a cyclic adenosine 3′,5′-monophosphate (cyclic AMP)-dependent mechanism was also tested.

2 In vitro, acetazolamide, frusemide and ethacrynic acid at high concentrations (10^-3M) all produced some inhibition of basal and stimulated rat kidney plasma membrane adenylate cyclase. The effect of acetazolamide was much less than that of frusemide and ethacrynic acid. These plasma membrane effects were reproduced in studies of cyclic AMP formation in isolated kidney tubules of rats.

3 Intravenous injections of acetazolamide did not change the total cyclic AMP content of the kidneys of rats killed by microwave irradiation.

4 Acetazolamide produced a diuresis in the rat and a slight inhibition of the antidiuretic effect of Pitressin. Frusemide produced a diuresis and greatly reduced the antidiuretic response to Pitressin. Ethacrynic acid was ineffective as a diuretic in the rat and actually enhanced the antidiuretic response to Pitressin.

5 In investigating the possible influence of diuretics and PTH on the activity and state of phosphorylation of carbonic anhydrase it was found that: there was no correlation between the ability of diuretics to inhibit carbonic anhydrase activity and to inhibit carbonic anhydrase phosphorylation; neither PTH nor cyclic AMP (in the presence of adenosine triphosphate, Mg²⁺, K⁺ and incubation at 37°C) inhibited rat cortex homogenate carbonic anhydrase activity.

6 It seems unlikely that any of the tested diuretics exerts its pharmacological effect by means of changes in kidney cyclic AMP metabolism.

     

Use of a dimethylated oxirane to characterise microsomal mono-oxygenases in rat liver

A newly-synthesised dimethylated chlorocyclodiene epoxide is metabolised by NADPH-supplemented rat liver microsomes to produee either one or both of two major metabolites. M1 and M2. Microsomes from adult male rat liver produce both metabolites. By contrast, microsomes made from the livers of mature or immature female rats make only metabolite M1, as do hepatic microsomes from pre-pubertal male rats. Male rats castrated when 7 days old provide liver microsomes that make little of metabolite M2 upon killing at 3–5 months post partum. After induction with phenobarbital, microsomes from adult male and female rat liver make metabolites M1 and M2. together with a further unidentified metabolite. After induction with 3-methylcholanthrene, hepatic microsomes from mature female rats continue to make only metabolite M1 but to make it in larger quantities. These differences, which appear to result from the activity of three or more mono-oxygcnases. could possibly he exploited to monitor the mono-oxygenase status of rat liver microsomes.