N-thiocarboxyanhydrides, amino acid-derived enzyme-activated H2S donors, enhance sperm mitochondrial activity in presence and absence of oxidative stress.

BACKGROUND: Hydrogen sulfide (H2S) donors are crucial tools not only for understanding the role of H2S in cellular function but also as promising therapeutic agents for oxidative stress-related diseases. This study aimed to explore the effect of amino acid-derived N-thiocarboxyanhydrides (NTAs), which release physiological H2S levels in the presence of carbonic anhydrase, on porcine sperm function during short-term incubation with and without induced oxidative stress. For this purpose, we employed two H2S-releasing NTAs with release half-lives (t1/2) in the range of hours that derived from the amino acids glycine (Gly-NTA) or leucine (Leu-NTA). Because carbonic anhydrase is crucial for H2S release from NTAs, we first measured the activity of this enzyme in the porcine ejaculate. Then, we tested the effect of Gly- and Leu-NTAs at 10 and 1 nM on sperm mitochondrial activity, plasma membrane integrity, acrosomal status, motility, motile subpopulations, and redox balance during short-term incubation at 38 °C with and without a reactive oxygen species (ROS)-generating system. RESULTS: Our results show that carbonic anhydrase is found both in spermatozoa and seminal plasma, with activity notably higher in the latter. Both Gly- and Leu-NTAs did not exert any noxious effects, but they enhanced sperm mitochondrial activity in the presence and absence of oxidative stress. Moreover, NTAs (except for Leu-NTA 10 nM) tended to preserve the sperm redox balance against the injuries provoked by oxidative stress, which provide further support to the antioxidant effect of H2S on sperm function. Both compounds also increased progressive motility over short-term incubation, which may translate into prolonged sperm survival. CONCLUSIONS: The presence of carbonic anhydrase activity in mammalian spermatozoa makes NTAs promising molecules to investigate the role of H2S in sperm biology. For the first time, beneficial effects of NTAs on mitochondrial activity have been found in mammalian cells in the presence and absence of oxidative stress. NTAs are interesting compounds to investigate the role of H2S in sperm mitochondria-dependent events and to develop H2S-related therapeutic protocols against oxidative stress in assisted reproductive technologies.

Effect of Seed Treatment with Salicylic Acid on the Carbonic Anhydrase Activity, Photosynthesis Rate, Stomatal Conductance, and Pigments Content in Wheat Leaves at Zinc Excess.

The effect of seed treatment with salicylic acid (SA) on the carbonic anhydrase (CA) activity, photosynthesis rate, stomatal conductance, and pigment content in wheat leaves was studied at an optimal zinc content (2 µM) and zinc excess (1500 µM). It was shown for the first time that the CA activity and stomatal conductance increased upon seed treatment with SA at the optimal zinc content as compared with untreated plants, while the photosynthesis rate was not affected. When zinc was in excess in the root zone, seed treatment with SA decreased the CA activity to a greater extent, but the photosynthesis rate was higher than in untreated plants, apparently due to an increase in the contents of chlorophylls and carotenoids and stomatal conductivity. It was concluded that SA is involved in the protective and adaptive responses of wheat plants to excess environmental zinc along with other nonhormonal factors and hormones.

Autocrine boost of NMDAR current in hippocampal CA1 pyramidal neurons by a PMCA-dependent, perisynaptic, extracellular pH shift.

The plasma membrane Ca(2+)-ATPase (PMCA) is found near postsynaptic NMDARs. This transporter is a Ca(2+)-H(+) exchanger that raises cell surface pH. We tested whether the PMCA acts in an autocrine fashion to boost pH-sensitive, postsynaptic NMDAR currents. In mouse hippocampal slices, NMDAR EPSCs in a singly activated CA1 pyramidal neuron were reduced when buffering was augmented by exogenous carbonic anhydrase (XCAR). This effect was blocked by the enzyme inhibitor benzolamide and mimicked by the addition of HEPES buffer. Similar EPSC reduction occurred when PMCA activation was prevented by dialysis of BAPTA or the PMCA inhibitor carboxyeosin. Using HEPES, BAPTA, or carboxyeosin, the effect of XCAR was completely occluded. XCAR similarly curtailed NMDAR EPSCs of minimal amplitude, but had no effect on small AMPAR responses. These results indicate that a significant fraction of the postsynaptic NMDAR current is reliant on a perisynaptic extracellular alkaline shift generated by the PMCA.

pH buffering of single rat skeletal muscle fibers in the in vivo environment.

Homeostasis of intracellular pH (pHi) has a crucial role for the maintenance of cellular function. Several membrane transporters such as lactate/H(+) cotransporter (MCT), Na(+)/H(+) exchange transporter (NHE), and Na(+)/HCO3 (-) cotransporter (NBC) are thought to contribute to pHi regulation. However, the relative importance of each of these membrane transporters to the in vivo recovery from the low pHi condition is unknown. Using an in vivo bioimaging model, we pharmacologically inhibited each transporter separately and all transporters together and then evaluated the pHi recovery profiles following imposition of a discrete H(+) challenge loaded into single muscle fibers by microinjection. The intact spinotrapezius muscle of adult male Wistar rats (n = 72) was exteriorized and loaded with the fluorescent probe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (10 µM). A single muscle fiber was then loaded with low-pH solution [piperazine-N,N'-bis(2-ethanesulfonic acid) buffer, pH 6.5, ∼2.33 × 10(-3) µl] by microinjection over 3 s. The rats were divided into groups for the following treatments: 1) no inhibitor (CONT), 2) MCT inhibition (by α-Cyano-4-hydroxyciannamic acid; 4 mM), 3) NHE inhibition (by ethylisopropyl amiloride; 0.5 mM), 4) NBC inhibition (by DIDS; 1 mM), and 5) MCT, NHE, and NBC inhibition (All blockade). The fluorescence ratio (F500 nm/F445 nm) was determined from images captured during 1 min (60 images/min) and at 5, 10, 15, and 20 min after injection. The pHi at 1-2 s after injection significantly decreased from resting pHi (ΔpHi = -0.73 ± 0.03) in CONT. The recovery response profile was biphasic, with an initial rapid and close-to-exponential pHi increase (time constant, τ: 60.0 ± 7.9 s). This initial rapid profile was not affected by any pharmacological blockade but was significantly delayed by carbonic anhydrase inhibition. In contrast, the secondary, more gradual, return toward baseline that restored CONT pHi to 84.2% of baseline was unimpeded by MCT, NHE, and NBC blockade separately but abolished by All blockade (ΔpHi = -0.60 ± 0.07, 72.8% initial pHi, P < 0.05 vs. CONT). After injection of H(+) into, or superfusion onto, an adjacent fiber pHi of the surrounding fibers decreased progressively for the 20-min observation period (∼7.0, P < 0.05 vs. preinjection/superfusion). In conclusion, these results support that, after an imposed H(+) load, the MCT, NHE, and NBC transporters are not involved in the initial rapid phase of pHi recovery. In contrast, the gradual recovery phase was abolished by inhibiting all three membrane transporter systems simultaneously. The alteration of pHi in surrounding fibers suggest that H(+) uptake by neighboring fibers can help alleviate the pH consequences of myocyte H(+) exudation.

Apoptotic vesicles derived from human red blood cells promote bone regeneration via carbonic anhydrase 1.

Apoptotic vesicles (apoVs) are nanoscale vesicles derived from billions of apoptotic cells involved in the maintenance of the human body's homeostasis. Previous researches have shown that some apoVs, such as those derived from mesenchymal stem cells, contribute to bone formation. However, those apoVs cannot be extracted from patients in large quantities, and cell expansion is needed before apoV isolation, which limits their clinical translation. Mature RBCs, which have no nuclei or genetic material, are easy to obtain, showing high biological safety as a source of extracellular vesicles (EVs). Previous studies have demonstrated that RBC-derived EVs have multiple biological functions, but it is unknown whether RBCs produce apoVs and what effect these apoVs have on bone regeneration. In this study, we isolated and characterized RBC-derived apoVs (RBC-apoVs) from human venous blood and investigated their role in the osteogenesis of human bone mesenchymal stem cells (hBMSCs). We showed that RBCs could produce RBC-apoVs that expressed both general apoVs markers and RBC markers. RBC-apoVs significantly promoted osteogenesis of hBMSCs and enhanced bone regeneration in rat calvarial defects. Mechanistically, RBC-apoVs regulated osteogenesis by transferring carbonic anhydrase 1 (CA1) into hBMSCs and activating the P38 MAPK pathway. Our results indicated that RBC-apoVs could deliver functional molecules from RBCs to hBMSCs and promote bone regeneration, pointing to possible therapeutic use in bone tissue engineering.

Carbonic anhydrases in development: morphological observations and gene expression profiling in sea urchin embryos exposed to acetazolamide.

Carbonic anhydrases (CANs) are conserved metalloenzymes catalysing the reversible hydration of carbon dioxide into protons and bicarbonate, with important roles in cells physiology. Some CAN-coding genes were found in sea urchin genome, although only one involved in embryonic skeletogenesis was described in Paracentrotus lividus. Here, we investigated gene expression patterns of P. lividus embryos cultured in the presence of acetazolamide (AZ), a CAN inhibitor, to combine morphological defects with their molecular underpinning. CAN inhibition blocked skeletogenesis, affected the spatial/temporal expression of some biomineralization-related genes, inhibited embryos swimming. A comparative analysis on the expression of 127 genes in control and 3 h/24 h AZ-treated embryos, using NanoString technology, showed the differential expression of genes encoding for structural/regulatory proteins, with different embryonic roles: biomineralization, transcriptional regulation, signalling, development and defence response. The study of the differentially expressed genes and the signalling pathways affected, besides in silico analyses and a speculative 'interactomic model', leads to predicting the presence of various CAN isoforms, possibly involved in different physiological processes/activities in sea urchin embryo, and their potential target genes/proteins. Our findings provide new valuable molecular data for further studies in several biological fields: developmental biology (biomineralization, axes patterning), cell differentiation (neural development) and drug toxicology (AZ effects on embryos/tissues).

Piceatannol Protects against High Glucose-Induced Injury of Renal Tubular Epithelial Cells via Regulating Carbonic Anhydrase 2.

INTRODUCTION: We here evaluated the efficacy of piceatannol (PIC) in high glucose (HG)-induced injury of renal tubular epithelial cells HK-2. METHODS: After the establishment of an HG-induced cell injury model and the treatment with PIC at both high and low concentrations and/or acetazolamide (ACZ, the inhibitor of carbonic anhydrase 2 [CA2]), MTT and flow cytometry assays were carried out to confirm the viability and apoptosis of HK-2 cells. The levels of oxidative stress markers lactate dehydrogenase (LDH), malondialdehyde (MDA), and reactive oxygen species (ROS), the ratio of glutathione/oxidized glutathione (GSH/GSSG), and the CA2 activity were determined. Both quantitative reverse-transcription polymerase chain reaction and Western blot were used to calculate the expressions of CA2 (the predicted target gene of PIC via intersecting the data from bioinformatic analyses) and AKT pathway-related (phosphatase and tensin homolog [PTEN], phosphorylated [p]-AKT, AKT) and apoptosis-related proteins (Bcl-2 and cleaved caspase-3). RESULTS: HG suppressed cell viability and the levels of GSH/GSSG ratio, CA2, pThr308-AKT/AKT, pSer473-AKT/AKT, and Bcl-2, while promoting cell apoptosis, the levels of LDH, MDA, and ROS, and the expressions of PTEN and cleaved caspase-3. All effects of HG were reversed by PIC at a high concentration. CA2 was predicted and identified as the target of PIC. In HG-treated HK-2 cells, additionally, ACZ reversed the effects of PIC on the viability, apoptosis, and levels of both oxidative stress markers and AKT pathway- and apoptosis-related factors. CONCLUSION: PIC protects against HG-induced injury of HK-2 cells via regulating CA2.

Para-toluenesulfonamide, a novel potent carbonic anhydrase inhibitor, improves hypoxia-induced metastatic breast cancer cell viability and prevents resistance to αPD-1 therapy in triple-negative breast cancer.

Overexpression of the hypoxia-induced transmembrane enzyme carbonic anhydrase IX (CA9) has been associated with poor prognosis and chemoresistance in aggressive breast cancer. This study aimed to investigate the involvement of CA9 in the anti-tumor activity of para-toluenesulfonamide (PTS) and elucidate its mechanism of action against breast cancer both in vitro and in vivo. MCF-7 and MDA-MB-231 breast cancer cells were treated with PTS or subjected to hypoxic conditions using cobalt chloride (CoCl2), with acetazolamide serving as a positive control. Additionally, 4T1 breast cancer cell allograft mice were co-treated with PTS and α-programmed cell death 1 (αPD-1) monoclonal antibody for one month. The results demonstrated that PTS effectively reduced cell viability and reversed migration ability in MCF-7 and MDA-MB-231 cells under CoCl2-induced hypoxia. Furthermore, PTS upregulated the expression of apoptosis-related proteins and downregulated CA9, hypoxia-inducible factor-1α (HIF-1α), and vascular endothelial growth factor (VEGF) proteins, possibly through modulation of p38 MAPK and ERK1/2 phosphorylated proteins. In the animal model, PTS100 inhibited tumor growth and lung metastasis in mammary tumor allograft mice, exhibiting synergistic effects when combined with αPD-1 therapy. Collectively, our findings suggest that PTS inhibits breast cancer growth and metastasis through the p38 MAPK/ERK1/2 pathway. Moreover, PTS may have the potential to prevent the development of resistance to αPD-1 therapy in breast cancer.

Cloning, characterization and sulfonamide inhibition studies of an α-carbonic anhydrase from the living fossil sponge Astrosclera willeyana.

The α-carbonic anhydrase (CA, EC 4.2.1.1) Astrosclerin-3 previously isolated from the living fossil sponge Astrosclera willeyana (Jackson et al., Science 2007, 316, 1893), was cloned, kinetically characterized and investigated for its inhibition properties with sulfonamides and sulfamates. Astrosclerin-3 has a high catalytic activity for the CO(2) hydration reaction to bicarbonate and protons (k(cat) of 9.0×10(5) s(-1) and k(cat)/K(m) of 1.1×10(8) M(-1) × s(-1)), and is inhibited by various aromatic/heterocyclic sulfonamides and sulfamates with inhibition constants in the range of 2.9 nM-8.85 µM. Astrosclerin, and the human isoform CA II, display similar kinetic properties and affinities for sulfonamide inhibitors, despite more than 550 million years of independent evolution. Because Astrosclerin-3 is involved in biocalcification, the inhibitors characterized here may be used to gain insights into such processes in other metazoans.

Phenols from Origanum dictamnus L. and Thymus vulgaris L. and their activity against Malassezia globosa carbonic anhydrase.

Malassezia spp. are lipophilic fungi that are part of the normal flora of the human skin and are the etiological agents of dandruff and seborrheic dermatitis. ß-Carbonic Anhydrases (CAs; EC 4.2.1.1) expressed from the pathogenic fungi are an alternative/complementary drug target. Previous work by our groups demonstrated that flavonoids and depsides can effectively inhibit Malassezia globosa ß-CA (MgCA). In continuation of this study herein we report the inhibitory activity of a variety of phenols from Origanum dictamnus L. and Thymus vulgaris L. against ß-MgCA, among them I4-II7-di-carvacrol, a new natural product. Structure elucidation of the compounds was performed by 1 D, 2 D NMR and spectrometric analyses. Xanthomicrol and rosmarinic acid were active in the (sub)micromolar range (KIS 0.6 and 2.2 µM, respectively vs 40.0 µM of the standard inhibitor acetazolamide). Finally, the compounds were not cytotoxic, but showed in vitro no activity against Malassezia furfur.

Inhibition of carbonic anhydrases IX/XII by SLC-0111 boosts cisplatin effects in hampering head and neck squamous carcinoma cell growth and invasion.

BACKGROUND: Hypoxic tumor microenvironment (TME) contributes to the onset of many aspects of the cancer biology associated to the resistance to conventional therapies. Hypoxia is a common characteristic and negative prognostic factor in the head and neck squamous carcinomas (HNSCC) and is correlated with aggressive and invasive phenotype as well as with failure to chemo- and radio-therapies. The carbonic anhydrase isoenzymes IX and XII (CA IX/XII), regulators of extra and intracellular pH, are overexpressed in TME and are involved in adaptative changes occurring in cancer cells to survive at low O2. In this study, we aim to investigate in HNSCC cells and murine models the possibility to target CA IX/XII by the specific inhibitor SLC-0111 to potentiate the effects of cisplatin in hampering cell growth, migration and invasion. Furthermore, we analyzed the signal pathways cooperating in acquisition of a more aggressive phenotype including stemness, epithelial-mesenchymal transition and apoptotic markers. METHODS: The effects of cisplatin, CA IX/XII specific inhibitor SLC-0111, and the combinatorial treatment were tested on proliferation, migration, invasion of HNSCC cells grown in 2D and 3D models. Main signal pathways and the expression of stemness, mesenchymal and apoptotic markers were analyzed by western blotting. Molecular imaging using NIR-Annexin V and NIR-Prosense was performed in HNSCC xenografts to detect tumor growth and metastatic spread. RESULTS: HNSCC cells grown in 2D and 3D models under hypoxic conditions showed increased levels of CA IX/XII and greater resistance to cisplatin than cells grown under normoxic conditions. The addition of CA IX/XII inhibitor SLC-0111 to cisplatin sensitized HNSCC cells to the chemotherapeutic agent and caused a reduction of proliferation, migration and invasiveness. Furthermore, the combination therapy hampered activation of STAT3, AKT, ERK, and EMT program, whereas it induced apoptosis. In HNSCC xenografts the treatment with cisplatin plus SLC-0111 caused an inhibition of tumor growth and an induction of apoptosis as well as a reduction of metastatic spread at a higher extent than single agents. CONCLUSION: Our results highlight the ability of SLC-0111 to sensitize HNSCC to cisplatin by hindering hypoxia-induced signaling network that are shared among mechanisms involved in therapy resistance and metastasis.

Plasma-accessible carbonic anhydrase at the tissue of a teleost fish may greatly enhance oxygen delivery: in vitro evidence in rainbow trout, Oncorhynchus mykiss.

During a generalized acidosis in rainbow trout, catecholamines are released into the blood, activating red blood cell (RBC) Na(+)/H(+) exchange (ßNHE), thus protecting RBC intracellular pH (pH(i)) and subsequent O(2) binding at the gill. Because of the presence of a Root effect (a reduction in oxygen carrying capacity of the blood with a reduction in pH), the latter could otherwise be impaired. However, plasma-accessible carbonic anhydrase (CA) at the tissues (and absence at the gills) may result in selective short-circuiting of RBC ßNHE pH regulation. This would acidify the RBCs and greatly enhance O(2) delivery by exploitation of the combined Bohr-Root effect, a mechanism not previously proposed. As proof-of-principle, an in vitro closed system was developed to continuously monitor extracellular pH (pH(e)) and O(2) tension (P(O(2))) of rainbow trout blood. In this closed system, adding CA to acidified, adrenergically stimulated RBCs short-circuited ßNHE pH regulation, resulting in an increase in P(O(2)) by >30 mmHg, depending on the starting Hb-O(2) saturation and degree of initial acidification. Interestingly, in the absence of adrenergic stimulation, addition of CA still elevated P(O(2)), albeit to a lesser extent, a response that was absent during general NHE inhibition. If plasma-accessible CA-mediated short-circuiting is operational in vivo, the combined Bohr-Root effect system unique to teleost fishes could markedly enhance tissue O(2) delivery far in excess of that in vertebrates possessing a Bohr effect alone and may lead to insights about the early evolution of the Root effect.

Inhibition studies with anions and small molecules of two novel ß-carbonic anhydrases from the bacterial pathogen Salmonella enterica serovar Typhimurium.

Two new ß-carbonic anhydrases (CAs, EC 4.2.1.1) from the bacterial pathogen Salmonella enterica serovar Typhimurium, stCA 1 and stCA 2, were characterized kinetically. The two enzymes possess appreciable activity as catalysts for the hydration of CO(2) to bicarbonate, with k(cat) of 0.79×10(6) s(-1) and 1.0×10(6) s(-1), and k(cat)/K(m) of 5.2×10(7) M(-1) s(-1) and of 8.3×10(7) M(-1) s(-1), respectively. A large number of simple/complex inorganic anions as well as other small molecules (sulfamide, sulfamic acid, phenylboronic acid, phenylarsonic acid, dialkyldithiocarbamates) showed interesting inhibitory properties towards the two new enzymes, with several low micromolar inhibitors discovered. As many strains of S. enterica show extensive resistance to classical antibiotics, inhibition of the ß-CAs investigated here may be useful for developing lead compounds for novel types of antibacterials.

Bacillus thuringiensis Cry toxins bound specifically to various proteins via domain III, which had a galactose-binding domain-like fold.

Cry toxins have been reported to bind not only to receptors on insect cells but also to several unrelated proteins. In this study, we investigated the binding properties of Bacillus thuringiensis Cry toxins, focusing on domain III, a Cry toxin region with a structure that of the galactose-binding domain-like. Cry1Aa, Cry1Ac, and Cry8Ca specifically bound to several proteins unrelated to insect midgut cells. Cry1Aa binding to Cry toxin-binding proteins was inhibited by a monoclonal antibody, 2C2, indicating that Cry1Aa binds to these Cry toxin-binding proteins through domain III. Cry1Aa binding to Bombyx mori aminopeptidase N and other Cry toxin-binding proteins was inhibited by carbonic anhydrase, a Cry toxin-binding protein. The binding regions of carbonic anhydrase and Bombyx mori aminopeptidase N were narrowed to regions of less than 20 amino acids that did not have any similarity, suggesting that Cry toxin domain III has a binding pocket for multiple proteins.

Endogenous alkaline transients boost postsynaptic NMDA receptor responses in hippocampal CA1 pyramidal neurons.

In hippocampus, activation of the Schaffer collaterals generates an extracellular alkaline transient both in vitro and in vivo. This pH change may provide relief of the H+ block of NMDA receptors (NMDARs) and thereby increase excitability. To test this hypothesis, we augmented extracellular buffering in mouse hippocampal slices by adding 2 microM bovine type II carbonic anhydrase to the superfusate. With addition of enzyme, the alkaline transient elicited by a 10 pulse, 100 Hz stimulus train was reduced by 33%. At a holding potential (V(H)) of -30 mV, the enzyme decreased the half-time of decay and charge transfer of EPSCs by 32 and 39%, respectively, but had no effect at a V(H) of -80 mV. In current clamp, a 10 pulse, 100 Hz stimulus train gave rise to an NMDAR-dependent afterdepolarization (ADP). Exogenous enzyme curtailed the ADP half-width and voltage integral by 20 and 25%, respectively. Similar reduction of the ADP was noted with a brief 12 Hz stimulus train. The effect persisted in the presence of GABAergic antagonists or the L-type Ca2+ channel blocker methoxyverapamil hydrochloride but was absent in the presence of the carbonic anhydrase inhibitor benzolamide or when the exogenous enzyme was heat inactivated. The effects of the enzyme in voltage and current clamp were noted in 0 Mg2+ media but were abolished when (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine maleate was included in the patch pipette. These results provide strong evidence that endogenous alkaline transients are sufficiently large in the vicinity of the synapse to augment NMDAR responses.

Mechanism of Cd-induced inhibition of Na-glucose cotransporter in rabbit proximal tubule cells: roles of luminal pH and membrane-bound carbonic anhydrase.

BACKGROUND/AIMS: We have previously reported that a complex of cadmium-metallothionein (Cd-MT) directly affects the apical Na-glucose cotransporter on the luminal side in proximal tubules, suggesting that Cd-MT is more toxic than CdCl(2) in causing tubulopathy. To find the potential mechanisms, we evaluated the effect of luminal pH alteration and carbonic anhydrase (CA) inhibition on Cd-MT-induced reduction of glucose-dependent transmural voltage in rabbit S2 segments perfused in vitro. METHODS: Before and after the addition of Cd-MT (1 microg Cd/ml) to the lumen, the deflections of transmural voltage upon the elimination of glucose from the perfusate (DeltaVt(glu)) were measured as a parameter of activity of the Na-glucose cotransporter. RESULTS: During perfusion with a control solution of pH 7.4, the DeltaVt(glu) significantly decreased after addition of Cd-MT for 10 min. A reduction in pH to 6.8 significantly shortened the time needed to reduce the DeltaVt(glu) to <5 min, whereas an increase of pH to 7.7 significantly prolonged the time to >20 min. Furthermore, simultaneous addition of acetazolamide with control perfusate prevented the reduction. P-Fluorobenzyl-aminobenzolamide (pFB-ABZ), a membrane-impermeable CA inhibitor, added to the lumen also completely prevented the reduction in DeltaVt(glu). In rabbits with chronic Cd exposure, acetazolamide prevented the glucosuria. CONCLUSION: Cd-MT-induced inhibition of Na-glucose cotransporter activity in the S2 segment strongly depends on luminal pH, and that an increase in pH by inhibition of luminal membrane-bound CA is useful to prevent renal Cd toxicity.

Substituted carbamothioic amine-1-carbothioic thioanhydrides as novel trichomonicidal fungicides: Design, synthesis, and biology.

Sexually transmitted diseases like trichomoniasis along with opportunistic fungal infections like candidiasis are major global health burden in female reproductive health. In this context a novel non-nitroimidazole class of substituted carbamothioic amine-1-carbothioic thioanhydride series was designed, synthesized, evaluated for trichomonacidal and fungicidal activities, and was found to be more active than the standard drug Metronidazole (MTZ). Compounds were trichomonicidal in the MIC ranges of 4.77-294.1 µM and 32.46-735.20 µM against MTZ-susceptible and -resistant strains, respectively. Further, compounds inhibited the growth of at least two out of ten fungal strains tested at MIC of 7.50-240.38 µM. The most active compound (20) of this series was 3.8 and 9.5 fold more active than the MTZ against the two Trichomonas strains tested. Compound 20 also significantly inhibited the sulfhydryl groups present over Trichomonas vaginalis and was found to be more active than the MTZ in vivo. Further, a docking analysis carried out with cysteine proteases supported their thiol inhibiting ability and preliminary pharmacokinetic study has shown good distribution and systemic clearance.

Renal bicarbonate reabsorption in the rat. I. Effects of hypokalemia and carbonic anhydrase.

Free-flow micropuncture studies were carried out on superficial rat proximal and distal tubules to assess the participation of different nephron segments in bicarbonate transport. Particular emphasis was placed on the role of the distal tubule, and micro-calorimetric methods used to quantitate bicarbonate reabsorption. Experiments were carried out in control conditions, during dietary potassium withdrawal, and after acute intravenous infusions of carbonic anhydrase. We observed highly significant net bicarbonate reabsorption in normal acid-base conditions as evidenced by the maintenance of significant bicarbonate concentration gradients in the presence of vigorous fluid absorption. Distal bicarbonate reabsorption persisted in hypokalemic alkalosis and even steeper transepithelial concentration gradients of bicarbonate were maintained. Enhancement of net bicarbonate reabsorption followed the acute intravenous administration of carbonic anhydrase but was limited to the nephron segments between the late proximal and early distal tubule. The latter observation is consistent with a disequilibrium pH along the proximal straight tubule (S3 segment), the thick ascending limb of Henle, and/or the early distal tubule.

Luminal secretion of ammonia in the mouse proximal tubule perfused in vitro.

A major portion of the total ammonia (tNH3 = NH3 + NH+4) produced by the isolated perfused mouse proximal tubule is secreted into the luminal fluid. To assess the role of Na+-H+ exchange in net tNH3 secretion, rates of net tNH3 secretion and tNH3 production were measured in proximal tubule segments perfused with control pH 7.4 Krebs-Ringer bicarbonate (KRB) buffer or with modified KRB buffers containing 10 mM sodium and 0.1 mM amiloride. Net tNH3 secretion was inhibited by 90% in proximal tubule segments perfused with the pH 7.4 modified KRB buffer while tNH3 production remained unaffected. The inhibition of net tNH3 secretion by perfusion with the modified KRB buffer was only partially reversed by acidifying the modified KRB luminal perfusate from 7.4 to as low as 6.2. These data indicate that the Na+-H+ exchanger facilitates a major portion of net tNH3 secretion by the proximal tubule and that luminal acidification may play only a partial role in the mechanism by which the Na+-H+ exchanger mediates net tNH3 secretion.

Deficiency of carbonic anhydrase in the vasculature of rabbit kidneys.

The transit of 14CO2 and H14CO3- through the renal vasculature was studied in rabbit kidneys perfused without erythrocytes and in an in vivo preparation in which erythrocytes were present. In the absence of erythrocytes, the transit of 14CO2 from the renal artery to renal vein was much more rapid than that of H14CO3-. This suggests that (a) there is insufficient carbonic anhydrase (c.a.) in the vasculature between the renal artery and the exchange vessels of the kidney to ensure equilibration between CO2 and HCO3- and (b) CO2 can diffuse directly between arterial and venous vessels in the kidney. Following infusions of carbonic anhydrase, the renal venous outflow patterns of 14CO2 and H14CO3- became the same in the perfused kidneys. Although the initial recovery of 14CO2 remained greater than that of H14CO3- after infusions of acetazolamide (a c.a. inhibitor), arteriovenous diffusion of 14CO2 was diminished by this agent. This is attributed to inhibition of renal tubular c.a. The outflow patterns of H14CO3- and 14CO2 were nearly the same in the presence of erythrocytes, indicating that erythrocyte c.a. is sufficiently accessible to permit virtual equilibration of these radionuclides during the interval required for transit between the renal artery and exchange vessels. However, addition of carbonic anhydrase to the plasma seemed to accelerate transit of both 14CO2 and H14CO3- through the kidneys, and a small disequilibrium between CO2 and HCO3- may therefore normally be present in the renal interstitium and capillaries.