Repurposing FDA-approved sulphonamide carbonic anhydrase inhibitors for treatment of Neisseria gonorrhoeae.

Neisseria gonorrhoeae is a high-priority pathogen of concern due to the growing prevalence of resistance development against approved antibiotics. Herein, we report the anti-gonococcal activity of ethoxzolamide, the FDA-approved human carbonic anhydrase inhibitor. Ethoxzolamide displayed an MIC50, against a panel of N. gonorrhoeae isolates, of 0.125 µg/mL, 16-fold more potent than acetazolamide, although both molecules exhibited almost similar potency against the gonococcal carbonic anhydrase enzyme (NgCA) in vitro. Acetazolamide displayed an inhibition constant (Ki) versus NgCA of 74 nM, while Ethoxzolamide's Ki was estimated to 94 nM. Therefore, the increased anti-gonococcal potency of ethoxzolamide was attributed to its increased permeability in N. gonorrhoeae as compared to that of acetazolamide. Both drugs demonstrated bacteriostatic activity against N. gonorrhoeae, exhibited post-antibiotic effects up to 10 hours, and resistance was not observed against both. Taken together, these results indicate that acetazolamide and ethoxzolamide warrant further investigation for translation into effective anti-N. gonorrhoeae agents.

Myocardial and mitochondrial effects of the anhydrase carbonic inhibitor ethoxzolamide in ischemia-reperfusion.

We have previously demonstrated that inhibition of extracellularly oriented carbonic anhydrase (CA) isoforms protects the myocardium against ischemia-reperfusion injury. In this study, our aim was to assess the possible further contribution of CA intracellular isoforms examining the actions of the highly diffusible cell membrane permeant inhibitor of CA, ethoxzolamide (ETZ). Isolated rat hearts, after 20 min of stabilization, were assigned to the following groups: (1) Nonischemic control: 90 min of perfusion; (2) Ischemic control: 30 min of global ischemia and 60 min of reperfusion (R); and (3) ETZ: ETZ at a concentration of 100 µM was administered for 10 min before the onset of ischemia and then during the first 10 min of reperfusion. In additional groups, ETZ was administered in the presence of SB202190 (SB, a p38MAPK inhibitor) or chelerythrine (Chel, a protein kinase C [PKC] inhibitor). Infarct size, myocardial function, and the expression of phosphorylated forms of p38MAPK, PKCε, HSP27, and Drp1, and calcineurin Aß content were assessed. In isolated mitochondria, the Ca2+ response, Ca2+ retention capacity, and membrane potential were measured. ETZ decreased infarct size by 60%, improved postischemic recovery of myocardial contractile and diastolic relaxation increased P-p38MAPK, P-PKCε, P-HSP27, and P-Drp1 expression, decreased calcineurin content, and normalized calcium and membrane potential parameters measured in isolated mitochondria. These effects were significantly attenuated when ETZ was administered in the presence of SB or Chel. These data show that ETZ protects the myocardium and mitochondria against ischemia-reperfusion injury through p38MAPK- and PKCε-dependent pathways and reinforces the role of CA as a possible target in the management of acute cardiac ischemic diseases.

Staphylococcus aureus Genomes Harbor Only MpsAB-Like Bicarbonate Transporter but Not Carbonic Anhydrase as Dissolved Inorganic Carbon Supply System.

In recent years, it became apparent that not only autotrophic but also most other bacteria require CO2 or bicarbonate for growth. Two systems are available for the acquisition of dissolved inorganic carbon supply (DICS): the cytoplasmic localized carbonic anhydrase (CA) and the more recently described bicarbonate transporter MpsAB (membrane potential generating system). In the pathogenic species Staphylococcus aureus, there are contradictions in the literature regarding the presence of a CA or MpsAB. Here, we address these contradictions in detail. We could demonstrate by careful BLASTp analyses with 259 finished and 4,590 unfinished S. aureus genomes that S. aureus does not contain CA and that the bicarbonate transporter MpsAB is the only DICS system in this species. This finding is further supported by two further pieces of evidence: (i) mpsAB deletion mutants in four different S. aureus strains failed to grow under atmospheric air, which should not be the case if they possess CAs, since we have previously shown that both CA and MpsAB can substitute for each other, and (ii) S. aureus is completely resistant to CA inhibitors, whereas Staphylococcus carnosus, which has been shown to have only CA, was inhibited by ethoxyzolamide (EZA). Taken together, we demonstrate beyond doubt that the species S. aureus possesses only the bicarbonate transporter MpsAB as its sole DICS system. IMPORTANCE The discrepancies in the current literature and even in NCBI database, which listed some protein sequences annotated as Staphylococcus aureus carbonic anhydrase (CA), are misleading. One of the existing problems in publicly available sequence databases is the presence of incorrectly annotated genes, especially if they originated from unfinished genomes. Here, we demonstrate that some of these unfinished genomes are of poor quality and should be interpreted with caution. In the present study, we aimed to address these discrepancies and correct the current literature about S. aureus CA, considering the medical relevance of S. aureus. If left unchecked, these misleading studies and wrongly annotated genes might lead to a continual propagation of wrong annotation and, consequently, wrong interpretations and wasted time. In addition, we also show that bicarbonate transporter MpsAB-harboring bacteria are resistant to CA inhibitor, suggesting that pathogens possessing both MpsAB and CA are not treatable with CA inhibitors.

Inhibition of the ß-carbonic anhydrase from the protozoan pathogen Trichomonas vaginalis with sulphonamides.

Sulphonamides and their isosteres are classical inhibitors of the carbonic anhydrase (CAs, EC 4.2.1.1) metalloenzymes. The protozoan pathogen Trichomonas vaginalis encodes two such enzymes belonging to the ß-class, TvaCA1 and TvaCA2. Here we report the first sulphonamide inhibition study of TvaCA1, with a series of simple aromatic/heterocyclic primary sulphonamides as well as with clinically approved/investigational drugs for a range of pathologies (diuretics, antiglaucoma, antiepileptic, antiobesity, and antitumor drugs). TvaCA1 was effectively inhibited by acetazolamide and ethoxzolamide, with KIs of 391 and 283 nM, respectively, whereas many other simple or clinically used sulphonamides were micromolar inhibitors or did not efficiently inhibit the enzyme. Finding more effective TvaCA1 inhibitors may constitute an innovative approach for fighting trichomoniasis, a sexually transmitted infection, caused by T. vaginalis.

Novel pharmacological modulation of dystonic phenotypes caused by a gain-of-function mutation in the Na+ leak-current channel.

The Na leak-current channel (NALCN) regulates the resting membrane potential in excitable cells, thus determining the likelihood of depolarization in response to incoming signals. Gain-of-function (gf) mutations in this channel are associated with severe dystonic movement disorders in man. Currently, there are no known pharmacological antagonists or selective modulators of this important channel. A gain-of-function mutation in NALCN of C. elegans [known as unc-77(e625)] causes uncoordinated, hyperactive locomotion. We hypothesized that this hyperactive phenotype can be rescued with pharmacological modulators. Here, we summarize the results of targeted drug screening aimed at identification of drugs that corrected locomotion deficits in unc-77(e625) animals. To assay hyperactive locomotion, animals were acutely removed from food and characteristic foraging movements were quantified. Drug screening revealed that 2-aminoethoxydiphenyl borate (2-ABP), nifedipine, nimodipine, flunarizine and ethoxzolamide significantly decreased abnormal movements in unc-77(e625) animals. 2-APB also corrected egg release and coiling deficits in this strain. In addition, serotonin and dopamine both reduced hyperactive locomotion, consistent with regulatory interactions between these systems and the NALCN. 2-APB induced movement phenotypes in wild-type animals that faithfully mimicked those observed in NALCN knockout strains, which suggested that this drug may directly block the channel. Moreover, 2-APB and flunarizine showed significant structural similarities suggestive of overlap in their mode of action. Together, these studies have revealed new insights into regulation of NALCN function and led to the discovery of a potential pharmacological antagonist of the NALCN.

Carbonic anhydrase II/sodium-proton exchanger 1 metabolon complex in cardiomyopathy of ob-/- type 2 diabetic mice.

Heart failure is the leading cause of death among diabetic people. Cellular and molecular entities leading to diabetic cardiomyopathy are, however, poorly understood. Coupling of cardiac carbonic anhydrase II (CAII) and Na+/H+ exchanger 1 (NHE1) to form a transport metabolon was analyzed in obese type 2 diabetic mice (ob-/-) and control heterozygous littermates (ob+/-). Echocardiography showed elevated systolic interventricular septum thickness and systolic posterior wall thickness in ob-/- mice at 9 and 16 weeks. ob-/- mice showed increased left ventricular (LV) weight/tibia length ratio and increased cardiomyocyte cross sectional area as compared to controls, indicating cardiac hypertrophy. Immunoblot analysis showed increased CAII expression in LV samples of ob-/-vs. ob+/- mice, and augmented Ser703 phosphorylation on NHE1 in ob-/- hearts. Reciprocal co-immunoprecipitation analysis showed strong association of CAII and NHE1 in LV samples of ob-/- mice. NHE1-dependent rate of intracellular pH (pHi) normalization after transient acid loading of isolated cardiomyocytes was higher in ob-/- mice vs. ob+/-. NHE transport activity was also augmented in cultured H9C2 rat cardiomyoblasts treated with high glucose/high palmitate, and it was normalized after CA inhibition. We conclude that the NHE1/CAII metabolon complex is exacerbated in diabetic cardiomyopathy of ob-/- mice, which may lead to perturbation of pHi and [Na+] and [Ca2+] handling in these diseased hearts.

Anti-Helicobacter pylori activity of ethoxzolamide.

Ethoxzolamide (EZA), acetazolamide, and methazolamide are clinically used sulphonamide drugs designed to treat non-bacteria-related illnesses (e.g. glaucoma), but they also show antimicrobial activity against the gastric pathogen Helicobacter pylori. EZA showed the highest activity, and was effective against clinical isolates resistant to metronidazole, clarithromycin, and/or amoxicillin, suggesting that EZA kills H. pylori via mechanisms different from that of these antibiotics. The frequency of single-step spontaneous resistance acquisition by H. pylori was less than 5 × 10-9, showing that resistance to EZA does not develop easily. Resistance was associated with mutations in three genes, including the one that encodes undecaprenyl pyrophosphate synthase, a known target of sulphonamides. The data indicate that EZA impacts multiple targets in killing H. pylori. Our findings suggest that developing the approved anti-glaucoma drug EZA into a more effective anti-H. pylori agent may offer a faster and cost-effective route towards new antimicrobials with a novel mechanism of action.

Pharmacological evidence that DAPI inhibits NHE2 in Fundulus heteroclitus acclimated to freshwater.

Ionoregulation in the euryhaline killifish Fundulus heteroclitus has been intensively studied over the last two decades using a variety of techniques. However, there has been limited use of pharmacological inhibitors to identify proteins involved in ion transport for this species. In this study, we used a range of pharmacological inhibitors (EIPA, DAPI, ethoxzolamide, bumetanide, bafilomycin, phenamil, hydrochlorothiazide) to investigate the proteins involved in Na+ transport in freshwater (1 mM Na+) acclimated F. heteroclitus. Our results indicate that Na+ uptake under these conditions is sensitive to both EIPA (NHE-specific inhibitor) and DAPI (putative ASIC-specific inhibitor), but not to any of the other inhibitors. Results for EIPA are consistent with previous studies indicating F. heteroclitus relies solely on NHE2 for Na+ transport across the apical membrane of ionocytes. In contrast, results for DAPI are surprising given previous studies that have indicated the H+-ATPase is basolaterally located in F. heteroclitus and so cannot contribute to Na+ uptake via ASIC. The lack of bafilomycin sensitivity in the current study is consistent with a basolaterally located H+-ATPase. This suggests that DAPI is not an ASIC-specific inhibitor as has been previously assumed, and that it may also inhibit NHE2. Finally, we did not observe Na+ uptake to be sensitive to ethoxzolamide, suggesting that carbonic anhydrase may not be involved in generating the H+ needed to maintain NHE activity in freshwater as has been previously proposed.

Small Molecules That Sabotage Bacterial Virulence.

The continued rise of antibiotic-resistant bacterial infections has motivated alternative strategies for target discovery and treatment of infections. Antivirulence therapies function through inhibition of in vivo required virulence factors to disarm the pathogen instead of directly targeting viability or growth. This approach to treating bacteria-mediated diseases may have advantages over traditional antibiotics because it targets factors specific for pathogenesis, potentially reducing selection for resistance and limiting collateral damage to the resident microbiota. This review examines vulnerable molecular mechanisms used by bacteria to cause disease and the antivirulence compounds that sabotage these virulence pathways. By expanding the study of antimicrobial targets beyond those that are essential for growth, antivirulence strategies offer new and innovative opportunities to combat infectious diseases.

Carbonic anhydrase inhibitors reduce cardiac dysfunction after sustained coronary artery ligation in rats.

BACKGROUND: Two potent carbonic anhydrase (CA) inhibitors with widely differing membrane permeability, poorly diffusible benzolamide (BZ), and highly diffusible ethoxzolamide (ETZ) were assessed to determine whether they can reduce cardiac dysfunction in rats subjected to coronary artery ligation (CAL)-induced myocardial infarction. METHODS AND RESULTS: Rats with evidence of heart failure (HF) at 32 weeks following a permanent left anterior coronary artery occlusion were treated with placebo, BZ, or ETZ (4 mg kgday-1) for 4 weeks at which time left ventricular function and structure were evaluated. Lung weight/body weight (LW/BW) ratio increased in CAL rats by 17±1% vs. control, suggesting pulmonary edema. There was a trend for BZ and ETZ to ameliorate the increase in LW/BW by almost 50% (9±5% and 9±8%, respectively, versus CAL) (P=.16, NS). Echocardiographic assessment showed decreased left ventricular midwall shortening in HF rats, 21±1% vs. control 32±1%, which was improved by BZ to 29±1% and ETZ to 27±1%, and reduced endocardial shortening in HF rats 38±3% vs. control 62±1%, partially restored by BZ and ETZ to ~50%. Expression of the hypoxia-inducible membrane-associated CAIX isoform increased by ~60% in HF rat hearts, and this effect was blocked by ETZ. CONCLUSIONS: We conclude that CAL-induced myocardial interstitial fibrosis and associated decline in left ventricular function were diminished with BZ or ETZ treatment. The reductions in cardiac remodeling in HF with both ETZ and BZ CA inhibitors suggest that inhibition of a membrane-bound CA appears to be the critical site for this protection.

3-Amino-4-aminoximidofurazan derivatives: small molecules possessing antimicrobial and antibiofilm activity against Staphylococcus aureus and Pseudomonas aeruginosa.

AIM: The therapeutic treatment of microbial infections involving biofilm becomes quite challenging because of its increasing antibiotic resistance capacities. Towards this direction, in the present study we have evaluated the antibiofilm property of synthesized 3-amino-4-aminoximidofurazan compounds having polyamine skeleton. These derivatives were synthesized by incorporating furazan and biguanide moieties. METHODS AND RESULTS: Different 3-amino-4-aminoximidofurazan derivatives (PI1-4) were synthesized via protic acid catalysis and subsequently characterized by (1) H NMR and (13) C NMR spectra, recorded at 400 and 100 MHz respectively. We have tested the antimicrobial and antibiofilm activities of these synthetic derivatives (PI1-4) against both Staphylococcus aureus and Pseudomonas aeruginosa. The compounds so tested were also compared with standard antibiotics namely Tobramycin (Ps. aeruginosa) and Azithromycin (Staph. aureus) which were used as a positive control in all experimental sets. All these compounds (PI1-4) exhibited moderate to significant antimicrobial activities against both micro-organisms wherein compound PI3 showed maximum activity. Biofilm inhibition of both micro-organisms was then evaluated by crystal violet and safranin staining, estimation of biofilm total protein and microscopy methods using sub-MIC dose of these compounds. Results showed that all compounds executed anti biofilm activity against both Staph. aureus and Ps. aeruginosa wherein compound PI3 exhibited maximum activity. In relation with microbial biofilm inhibition, we have observed reduction in bacterial motility, proteolytic activity and secreted exo-polysaccharide (EPS) from both Staph. aureus and Ps. aeruginosa when they were grown in presence of these compounds. While addressing the issue of toxicity on host, we have observed that these molecules exhibited minimum level of R.B.C degradation. CONCLUSION: These findings establish the antibacterial and anti biofilm properties of 3-amino-4-aminoximidofurazan derivatives (PI1-4). SIGNIFICANCE AND IMPACT OF THE STUDY: Therefore, our current findings demonstrate that 3-amino-4-aminoximidofurazan derivatives (PI1-4) may hold promise to be effective biofilm and microbial inhibitors that may be clinically significant.

Catalytic activity of human carbonic anhydrase isoform IX is displayed both extra- and intracellularly.

Most carbonic anhydrases catalyse the reversible conversion of carbon dioxide to protons and bicarbonate, either as soluble cytosolic enzymes, in or at intracellular organelles, or at the extracellular face of the cell membrane as membrane-anchored proteins. Carbonic anhydrase isoform IX (CA IX), a membrane-bound enzyme with catalytic activity at the extracellular membrane surface, has come to prominence in recent years because of its association with hypoxic tissue, particularly tumours, often indicating poor prognosis. We have evaluated the catalytic activity of CA IX heterologously expressed in Xenopus laevis oocytes by measuring the amplitude and rate of cytosolic pH changes as well as pH changes at the outer membrane surface (pHs ) during addition and removal of 5% CO2 /25 mm HCO3-, and by mass spectrometry. Our results indicate both extracellular and intracellular catalytic activity of CA IX. Reduced rates of CO2 -dependent intracellular pH changes after knockdown of CA IX confirmed these findings in two breast cancer cell lines: MCF-7 and MDA-MB-231. Our results demonstrate a new function of CA IX that may be important in the search for therapeutic cancer drugs targeting CA IX.

The Carbonic Anhydrase Inhibitor Ethoxzolamide Inhibits the Mycobacterium tuberculosis PhoPR Regulon and Esx-1 Secretion and Attenuates Virulence.

Mycobacterium tuberculosis must sense and adapt to host environmental cues to establish and maintain an infection. The two-component regulatory system PhoPR plays a central role in sensing and responding to acidic pH within the macrophage and is required for M. tuberculosis intracellular replication and growth in vivo. Therefore, the isolation of compounds that inhibit PhoPR-dependent adaptation may identify new antivirulence therapies to treat tuberculosis. Here, we report that the carbonic anhydrase inhibitor ethoxzolamide inhibits the PhoPR regulon and reduces pathogen virulence. We show that treatment of M. tuberculosis with ethoxzolamide recapitulates phoPR mutant phenotypes, including downregulation of the core PhoPR regulon, altered accumulation of virulence-associated lipids, and inhibition of Esx-1 protein secretion. Quantitative single-cell imaging of a PhoPR-dependent fluorescent reporter strain demonstrates that ethoxzolamide inhibits PhoPR-regulated genes in infected macrophages and mouse lungs. Moreover, ethoxzolamide reduces M. tuberculosis growth in both macrophages and infected mice. Ethoxzolamide inhibits M. tuberculosis carbonic anhydrase activity, supporting a previously unrecognized link between carbonic anhydrase activity and PhoPR signaling. We propose that ethoxzolamide may be pursued as a new class of antivirulence therapy that functions by modulating expression of the PhoPR regulon and Esx-1-dependent virulence.

High glucose-induced mitochondrial respiration and reactive oxygen species in mouse cerebral pericytes is reversed by pharmacological inhibition of mitochondrial carbonic anhydrases: Implications for cerebral microvascular disease in diabetes.

Hyperglycemia-induced oxidative stress leads to diabetes-associated damage to the microvasculature of the brain. Pericytes in close proximity to endothelial cells in the brain microvessels are vital to the integrity of the blood-brain barrier and are especially susceptible to oxidative stress. According to our recently published results, streptozotocin-diabetic mouse brain exhibits oxidative stress and loose pericytes by twelve weeks of diabetes, and cerebral pericytes cultured in high glucose media suffer intracellular oxidative stress and apoptosis. Oxidative stress in diabetes is hypothesized to be caused by reactive oxygen species (ROS) produced during hyperglycemia-induced enhanced oxidative metabolism of glucose (respiration). To test this hypothesis, we investigated the effect of high glucose on respiration rate and ROS production in mouse cerebral pericytes. Previously, we showed that pharmacological inhibition of mitochondrial carbonic anhydrases protects the brain from oxidative stress and pericyte loss. The high glucose-induced intracellular oxidative stress and apoptosis of pericytes in culture were also reversed by inhibition of mitochondrial carbonic anhydrases. Therefore, we extended our current study to determine the effect of these inhibitors on high glucose-induced increases in pericyte respiration and ROS. We now report that both the respiration and ROS are significantly increased in pericytes challenged with high glucose. Furthermore, inhibition of mitochondrial carbonic anhydrases significantly slowed down both the rate of respiration and ROS production. These data provide new evidence that pharmacological inhibitors of mitochondrial carbonic anhydrases, already in clinical use, may prove beneficial in protecting the brain from oxidative stress caused by ROS produced as a consequence of hyperglycemia-induced enhanced respiration.

Ocean acidification alleviates low-temperature effects on growth and photosynthesis of the red alga Neosiphonia harveyi (Rhodophyta).

This study aimed to examine interactive effects between ocean acidification and temperature on the photosynthetic and growth performance of Neosiphonia harveyi. N. harveyi was cultivated at 10 and 17.5 °C at present (~380 µatm), expected future (~800 µatm), and high (~1500 µatm) pCO2. Chlorophyll a fluorescence, net photosynthesis, and growth were measured. The state of the carbon-concentrating mechanism (CCM) was examined by pH-drift experiments (with algae cultivated at 10 °C only) using ethoxyzolamide, an inhibitor of external and internal carbonic anhydrases (exCA and intCA, respectively). Furthermore, the inhibitory effect of acetazolamide (an inhibitor of exCA) and Tris (an inhibitor of the acidification of the diffusive boundary layer) on net photosynthesis was measured at both temperatures. Temperature affected photosynthesis (in terms of photosynthetic efficiency, light saturation point, and net photosynthesis) and growth at present pCO2, but these effects decreased with increasing pCO2. The relevance of the CCM decreased at 10 °C. A pCO2 effect on the CCM could only be shown if intCA and exCA were inhibited. The experiments demonstrate for the first time interactions between ocean acidification and temperature on the performance of a non-calcifying macroalga and show that the effects of low temperature on photosynthesis can be alleviated by increasing pCO2. The findings indicate that the carbon acquisition mediated by exCA and acidification of the diffusive boundary layer decrease at low temperatures but are not affected by the cultivation level of pCO2, whereas the activity of intCA is affected by pCO2. Ecologically, the findings suggest that ocean acidification might affect the biogeographical distribution of N. harveyi.

Poly-(cyclo)dextrins as ethoxzolamide carriers in ophthalmic solutions and in contact lenses.

Efficient ophthalmic therapy requires the development of strategies that can provide sufficiently high drug levels in the ocular structures for a prolonged time. This work focuses on the suitability of poly-(cyclo)dextrins as carriers able to solubilize the carbonic anhydrase inhibitor (CAI) ethoxzolamide (ETOX), which is so far used for oral treatment of glaucoma. Topical ocular treatment should notably enhance the efficiency/safety profile of the drug. Natural α-, ß- and γ-cyclodextrins and a maltodextrin were separately polymerized using citric acid as cross-linker agent under mild conditions. The resultant hydrophilic polymers exhibited larger capability to solubilize ETOX than the pristine (cyclo)dextrins. Moreover, they provided sustained drug diffusion in artificial lachrymal fluid. Interestingly the poly-(cyclo)dextrins solutions facilitate the loading of remarkably high doses of ETOX in poly(2-hydroxyethyl methacrylate)-based contact lenses. Exploiting ionic interactions between functional groups in the contact lenses and remnant free carboxylic acids in the citric acid linkers of poly-(cyclo)dextrins led to the retention of the drug-loaded poly-(cyclo)dextrins and, in turn, to sustained release for several weeks.

Emerging role of calcium-activated potassium channel in the regulation of cell viability following potassium ions challenge in HEK293 cells and pharmacological modulation.

Emerging evidences suggest that Ca(2+)activated-K(+)-(BK) channel is involved in the regulation of cell viability. The changes of the cell viability observed under hyperkalemia (15 mEq/L) or hypokalemia (0.55 mEq/L) conditions were investigated in HEK293 cells expressing the hslo subunit (hslo-HEK293) in the presence or absence of BK channel modulators. The BK channel openers(10(-11)-10(-3)M) were: acetazolamide(ACTZ), Dichlorphenamide(DCP), methazolamide(MTZ), bendroflumethiazide(BFT), ethoxzolamide(ETX), hydrochlorthiazide(HCT), quercetin(QUERC), resveratrol(RESV) and NS1619; and the BK channel blockers(2 x 10(-7)M-5 x 10(-3)M) were: tetraethylammonium(TEA), iberiotoxin(IbTx) and charybdotoxin(ChTX). Experiments on cell viability and channel currents were performed using cell counting kit-8 and patch-clamp techniques, respectively. Hslo whole-cell current was potentiated by BK channel openers with different potency and efficacy in hslo-HEK293. The efficacy ranking of the openers at -60 mV(Vm) was BFT> ACTZ >DCP ≥RESV≥ ETX> NS1619> MTZ≥ QUERC; HCT was not effective. Cell viability after 24 h of incubation under hyperkalemia was enhanced by 82+6% and 33+7% in hslo-HEK293 cells and HEK293 cells, respectively. IbTx, ChTX and TEA enhanced cell viability in hslo-HEK293. BK openers prevented the enhancement of the cell viability induced by hyperkalemia or IbTx in hslo-HEK293 showing an efficacy which was comparable with that observed as BK openers. BK channel modulators failed to affect cell currents and viability under hyperkalemia conditions in the absence of hslo subunit. In contrast, under hypokalemia cell viability was reduced by -22+4% and -23+6% in hslo-HEK293 and HEK293 cells, respectively; the BK channel modulators failed to affect this parameter in these cells. In conclusion, BK channel regulates cell viability under hyperkalemia but not hypokalemia conditions. BFT and ACTZ were the most potent drugs either in activating the BK current and in preventing the cell proliferation induced by hyperkalemia. These findings may have relevance in disorders associated with abnormal K(+) ion homeostasis including periodic paralysis and myotonia.

Inhibition of carbonic anhydrase prevents the Na(+)/H(+) exchanger 1-dependent slow force response to rat myocardial stretch.

Myocardial stretch is an established signal that leads to hypertrophy. Myocardial stretch induces a first immediate force increase followed by a slow force response (SFR), which is a consequence of an increased Ca(2+) transient that follows the NHE1 Na(+)/H(+) exchanger activation. Carbonic anhydrase II (CAII) binds to the extreme COOH terminus of NHE1 and regulates its transport activity. We aimed to test the role of CAII bound to NHE1 in the SFR. The SFR and changes in intracellular pH (pHi) were evaluated in rat papillary muscle bathed with CO2/HCO3(-) buffer and stretched from 92% to 98% of the muscle maximal force development length for 10 min in the presence of the CA inhibitor 6-ethoxzolamide (ETZ, 100 µM). SFR control was 120 ± 3% (n = 8) of the rapid initial phase and was fully blocked by ETZ (99 ± 4%, n = 6). The SFR corresponded to a maximal increase in pHi of 0.18 ± 0.02 pH units (n = 4), and pHi changes were blocked by ETZ (0.04 ± 0.04, n = 6), as monitored by epifluorescence. NHE1/CAII physical association was examined in the SFR by coimmunoprecipitation, using muscle lysates. CAII immunoprecipitated with an anti-NHE1 antibody and the CAII immunoprecipitated protein levels increased 58 ± 9% (n = 6) upon stretch of muscles, assessed by immunoblots. The p90(RSK) kinase inhibitor SL0101-1 (10 µM) blocked the SFR of heart muscles after stretch 102 ± 2% (n = 4) and reduced the binding of CAII to NHE1, suggesting that the stretch-induced phosphorylation of NHE1 increases its binding to CAII. CAII/NHE1 interaction constitutes a component of the SFR to heart muscle stretch, which potentiates NHE1-mediated H(+) transport in the myocardium.

Mechanisms of transepithelial ammonia excretion and luminal alkalinization in the gut of an intestinal air-breathing fish, Misgurnus anguilliacaudatus.

The weatherloach, Misgurnus angulliacaudatus, is an intestinal air-breathing, freshwater fish that has the unique ability to excrete ammonia through gut volatilization when branchial and cutaneous routes are compromised during high environmental ammonia or air exposure. We hypothesized that transepithelial gut NH(4)(+) transport is facilitated by an apical Na(+)/H(+) (NH(4)(+)) exchanger (NHE) and a basolateral Na(+)/K(+)(NH(4)(+))-ATPase, and that gut boundary layer alkalinization (NH(4)(+) → NH(3) + H(+)) is facilitated by apical HCO(3)(-) secretion through a Cl(-)/HCO(3)(-) anion exchanger. This was tested using a pharmacological approach with anterior (digestive) and posterior (respiratory) intestine preparations mounted in pH-stat-equipped Ussing chambers. The anterior intestine had a markedly higher conductance, increased short-circuit current, and greater net base (J(base)) and ammonia excretion rates (J(amm)) than the posterior intestine. In the anterior intestine, HCO(3)(-) accounted for 70% of J(base). In the presence of an imposed serosal-mucosal ammonia gradient, inhibitors of both NHE (EIPA, 0.1 mmol l(-1)) and Na(+)/K(+)-ATPase (ouabain, 0.1 mmol l(-1)) significantly inhibited J(amm) in the anterior intestine, although only EIPA had an effect in the posterior intestine. In addition, the anion exchange inhibitor DIDS significantly reduced J(base) in the anterior intestine although only at a high dose (1 mmol l(-1)). Carbonic anhydrase does not appear to be associated with gut alkalinization under these conditions as ethoxzolamide was without effect on J(base). Membrane fluidity of the posterior intestine was low, suggesting low permeability, which was also reflected in a lower mucosal-serosal J(amm) in the presence of an imposed gradient, in contrast to that in the anterior intestine. To conclude, although the posterior intestine is highly modified for gas exchange, it is the anterior intestine that is the likely site of ammonia excretion and alkalinization leading to ammonia volatilization in the gut.

Regulation of the cyanobacterial CO2-concentrating mechanism involves internal sensing of NADP+ and α-ketogutarate levels by transcription factor CcmR.

Inorganic carbon is the major macronutrient required by organisms utilizing oxygenic photosynthesis for autotrophic growth. Aquatic photoautotrophic organisms are dependent upon a CO(2) concentrating mechanism (CCM) to overcome the poor CO(2)-affinity of the major carbon-fixing enzyme, ribulose-bisphosphate carboxylase/oxygenase (Rubisco). The CCM involves the active transport of inorganic forms of carbon (C(i)) into the cell to increase the CO(2) concentration around the active site of Rubisco. It employs both bicarbonate transporters and redox-powered CO(2)-hydration enzymes coupled to membranous NDH-type electron transport complexes that collectively produce C(i) concentrations up to a 1000-fold greater in the cytoplasm compared to the external environment. The CCM is regulated: a high affinity CCM comprised of multiple components is induced under limiting external Ci concentrations. The LysR-type transcriptional regulator CcmR has been shown to repress its own expression along with structural genes encoding high affinity C(i) transporters distributed throughout the genome of Synechocystis sp. PCC 6803. While much has been learned about the structural genes of the CCM and the identity of the transcriptional regulators controlling their expression, little is known about the physiological signals that elicit the induction of the high affinity CCM. Here CcmR is studied to identify metabolites that modulate its transcriptional repressor activity. Using surface plasmon resonance (SPR) α-ketoglutarate (α-KG) and the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP(+)) have been identified as the co-repressors of CcmR. Additionally, ribulose-1,5-bisphosphate (RuBP) and 2-phosphoglycolate (2-PG) have been confirmed as co-activators of CmpR which controls the expression of the ABC-type bicarbonate transporter.