Carbonic Anhydrase Inhibitor Methazolamide Research Articles

Amyloid Beta Oligomers Activate Death Receptors and Mitochondria-Mediated Apoptotic Pathways in Cerebral Vascular Smooth Muscle Cells; Protective Effects of Carbonic Anhydrase Inhibitors.

Amyloid beta (Aβ) deposition within the brain vasculature is an early hallmark of Alzheimer's disease (AD), which triggers loss of brain vascular smooth muscle cells (BVSMCs) in cerebral arteries, via poorly understood mechanisms, altering cerebral blood flow, brain waste clearance, and promoting cognitive impairment. We have previously shown that, in brain endothelial cells (ECs), vasculotropic Aβ species induce apoptosis through death receptors (DRs) DR4 and DR5 and mitochondria-mediated mechanisms, while FDA-approved carbonic anhydrase inhibitors (CAIs) prevent mitochondria-mediated EC apoptosis in vitro and in vivo. In this study, we analyzed Aβ-induced extrinsic and intrinsic (DR- and mitochondria-mediated) apoptotic pathways in BVSMC, aiming to unveil new therapeutic targets to prevent BVSMC stress and death. We show that both apoptotic pathways are activated in BVSMCs by oligomeric Aβ42 and Aβ40-Q22 (AβQ22) and mitochondrial respiration is severely impaired. Importantly, the CAIs methazolamide (MTZ) and acetazolamide (ATZ) prevent the pro-apoptotic effects in BVSMCs, while reducing caspase 3 activation and Aβ deposition in the arterial walls of TgSwDI animals, a murine model of cerebral amyloid angiopathy (CAA). This study reveals new molecular targets and a promising therapeutic strategy against BVSMC dysfunction in AD, CAA, and ARIA (amyloid-related imaging abnormalities) complications of recently FDA-approved anti-Aβ antibodies.

FDA-approved carbonic anhydrase inhibitors reduce amyloid β pathology and improve cognition, by ameliorating cerebrovascular health and glial fitness.

Cerebrovascular pathology is an early and causal hallmark of Alzheimer's disease (AD), in need of effective therapies. Based on the success of our previous in vitro studies, we tested for the first time in a model of AD and cerebral amyloid angiopathy (CAA), the carbonic anhydrase inhibitors (CAIs) methazolamide and acetazolamide, Food and Drug Administration-approved against glaucoma and high-altitude sickness. Both CAIs reduced cerebral, vascular, and glial amyloid beta (Aβ) accumulation and caspase activation, diminished gliosis, and ameliorated cognition in TgSwDI mice. The CAIs also improved microvascular fitness and induced protective glial pro-clearance pathways, resulting in the reduction of Aβ deposition. Notably, we unveiled that the mitochondrial carbonic anhydrase-VB (CA-VB) is upregulated in TgSwDI brains, CAA and AD+CAA human subjects, and in endothelial cells upon Aβ treatment. Strikingly, CA-VB silencing specifically reduces Aβ-mediated endothelial apoptosis. This work substantiates the potential application of CAIs in clinical trials for AD and CAA.

Vascular dysfunction in CAA in the presence of cardiovascular risk factors: The role of the mitochondria and therapeutic approaches

AbstractBackgroundThe accumulation of amyloid‐β (Aβ) on the brain vasculature, known as Cerebral Amyloid Angiopathy (CAA), induces vascular dysfunction contributing to neurodegeneration. Cardiovascular (CV) risk factors, which are also mediators of vascular dysfunction and may increase the risk for developing Alzheimer’s Disease and dementia, may potentiate CAA‐mediated neurovascular pathology. Mitochondria are key cellular organelles that have a dual role as mediators of cell survival and death and have an important role in disease pathogenesis. However, the role of brain endothelial mitochondria in vascular amyloidosis complicated with CV risk factors is unknown. The carbonic anhydrase inhibitors (CAi) methazolamide and acetazolamide were able to reduce amyloid‐induced cell death and mitochondrial dysfunction. Our work focuses on understanding the mechanisms of vascular dysfunction induced by Aβ complicated with CV risk factors. We aim to understand the protective role of CAi in preventing the pathological changes associated to neurovascular dysfunction and neurodegeneration.MethodImmortalized human cerebral microvascular endothelial cells (ECs) were used to study the molecular changes that occur in the cerebral vasculature during amyloid challenge complicated with CV risk factors, such as hyperhomocysteinemia (HHC). We then tested CAi and their potential to prevent cell death and mitochondrial dysfunction in ECs challenged with Aβ in the presence or absence of CV risk factors such as HHC.ResultAβ and HHC increased EC barrier permeability and that the presence of HHC worsened amyloid‐induced barrier permeability. Aβ‐induced EC apoptosis was reduced by treatment with CAi. We further demonstrate that both Aβ and HHC decrease mitochondrial basal respiration. The presence of Aβ induces cytochrome‐c release, mitochondrial membrane potential loss, and ROS production which were reduced by treatment with CAi.ConclusionHHC and Aβ induce brain microvascular EC dysfunction and increase permeability of the EC barrier. Aβ induces cell death and mitochondrial dysfunction, which are both prevented by CAi. We also demonstrate that CV risk factors, specifically HHC, induce mitochondrial dysfunction on brain ECs which could further contribute to neurovascular dysfunction and neurodegeneration observed in CAA. Our studies suggest that CAi could serve as a novel therapeutic strategy towards vascular dysfunction during CAA complicated with CV risk factors.

Abstract 2013: A carbonic anhydrase inhibitor methazolamide potentiates efficacy of gemcitabine by modulating anti-proliferative activity and cancer stem cell markers in pancreatic carcinoma

Abstract Pancreatic cancer is the fourth cause for cancer related deaths globally. By 2020, incidence of new cases is expected to be 420,000 with equal number of associated mortalities. Overall 5-year survival has marginally improved over past despite of our growing understanding of the disease. Surgery remains standard of care for resectable tumors followed by adjuvant chemotherapy which resulted in median survival of 26 months. Newer therapies targeting stroma and immunotherapies hold promise for the future. Noticeably, a high unmet medical need warrant novel therapy to extend survival over a year in metastatic settings. Drug repositioning holds promise over the conventional drug discovery, as it reduces development time and cost. Using this approach, we report promising anticancer activity of Methazolamide (MZM) in pancreatic cancer. MZM, a synthetic derivative of sulphonamide and a carbonic anhydrase IX (CA) inhibitor is used for the treatment of ocular conditions for lowering intraocular pressure. Overexpression of CA is implicated in acidification of tumor microenvironment leading to hypoxia which in turn promotes growth and metastasis of the tumor. Therefore, modulation of extracellular tumor pH via inhibition of CA represents a promising course to anticancer therapy. Here, we report anti-proliferative activity of MZM in 12 pancreatic cell lines including patient derived (PDX) in 2D models and anchorage independent growth inhibition in 3D models. Combination treatment with GEM yielded significant growth inhibition than drug alone in all tested cell lines. Additive effects were observed for the combination in PDX-1986, PDX-546, Capan-2, Mia-Pa-Ca and PANC-1 cells. Further, in vivo efficacy of MZM in combination with GEM was performed using PDX-546 in nude mice. Combination group showed significant tumor growth inhibition (72%, p<0.05) as against drug alone, MZM at 60 mg/kg, p.o. (50%, p<0.05) and GEM at 50 mg/kg i.v. (61%, p<0.05). There was no significant body weight loss. For PK/PD studies, MZM levels were measured in tumors and PD effect was evaluated using various markers in tumor samples. Expression of target genes was measured by qRT-PCR. Significant reduction in expression of stem cell markers viz. RAC1, OCT4, CD34, CD14, Nanog and proliferation marker (Ki67) was observed in combination group as compared to drug alone. In addition, anti-angiogenic potential viz. would healing, tube formation and expression of important anti-angiogenic markers like HIF1α, VEGF were profoundly modulated in MZM and combination group. Thus, our study indicates that the combination of MZM, a CA inhibitor with GEM is a promising novel approach for the treatment of pancreatic cancer especially in metastatic settings. Citation Format: Kalpana Joshi, Jeevan D. Ghosalkar, Vinay R. Sonawane, Siddhika R. Raut, Geena Malhotra. A carbonic anhydrase inhibitor methazolamide potentiates efficacy of gemcitabine by modulating anti-proliferative activity and cancer stem cell markers in pancreatic carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2013.

Carbonic anhydrase inhibition selectively prevents amyloid β neurovascular mitochondrial toxicity.

SummaryMounting evidence suggests that mitochondrial dysfunction plays a causal role in the etiology and progression of Alzheimer's disease (AD). We recently showed that the carbonic anhydrase inhibitor (CAI) methazolamide (MTZ) prevents amyloid β (Aβ)‐mediated onset of apoptosis in the mouse brain. In this study, we used MTZ and, for the first time, the analog CAI acetazolamide (ATZ) in neuronal and cerebral vascular cells challenged with Aβ, to clarify their protective effects and mitochondrial molecular mechanism of action. The CAIs selectively inhibited mitochondrial dysfunction pathways induced by Aβ, without affecting metabolic function. ATZ was effective at concentrations 10 times lower than MTZ. Both MTZ and ATZ prevented mitochondrial membrane depolarization and H2O2 generation, with no effects on intracellular pH or ATP production. Importantly, the drugs did not primarily affect calcium homeostasis. This work suggests a new role for carbonic anhydrases (CAs) in the Aβ‐induced mitochondrial toxicity associated with AD and cerebral amyloid angiopathy (CAA), and paves the way to AD clinical trials for CAIs, FDA‐approved drugs with a well‐known profile of brain delivery.

The carbonic anhydrase inhibitor methazolamide prevents amyloid beta-induced mitochondrial dysfunction and caspase activation protecting neuronal and glial cells in vitro and in the mouse brain

Mitochondrial dysfunction has been recognized as an early event in Alzheimer's disease (AD) pathology, preceding and inducing neurodegeneration and memory loss. The presence of cytochrome c (CytC) released from the mitochondria into the cytoplasm is often detected after acute or chronic neurodegenerative insults, including AD. The carbonic anhydrase inhibitor (CAI) methazolamide (MTZ) was identified among a library of drugs as an inhibitor of CytC release and proved to be neuroprotective in Huntington's disease and stroke models. Here, using neuronal and glial cell cultures, in addition to an acute model of amyloid beta (Aβ) toxicity, which replicates by intra-hippocampal injection the consequences of interstitial and cellular accumulation of Aβ, we analyzed the effects of MTZ on neuronal and glial degeneration induced by the Alzheimer's amyloid. MTZ prevented DNA fragmentation, CytC release and activation of caspase 9 and caspase 3 induced by Aβ in neuronal and glial cells in culture through the inhibition of mitochondrial hydrogen peroxide production. Moreover, intraperitoneal administration of MTZ prevented neurodegeneration induced by intra-hippocampal Aβ injection in the mouse brain and was effective at reducing caspase 3 activation in neurons and microglia in the area surrounding the injection site. Our results, delineating the molecular mechanism of action of MTZ against Aβ-mediated mitochondrial dysfunction and caspase activation, and demonstrating its efficiency in a model of acute amyloid-mediated toxicity, provide the first combined in vitro and in vivo evidence supporting the potential of a new therapy employing FDA-approved CAIs in AD.

Carbonic anhydrase generates a pH gradient in Bombyx mori silk glands

Silk is a protein of interest to both biological and industrial sciences. The silkworm, Bombyx mori, forms this protein into strong threads starting from soluble silk proteins using a number of biochemical and physical cues to allow the transition from liquid to fibrous silk. A pH gradient has been measured along the gland, but the methodology employed was not able to precisely determine the pH at specific regions of interest in the silk gland. Furthermore, the physiological mechanisms responsible for the generation of this pH gradient are unknown.In this study, concentric ion selective microelectrodes were used to determine the luminal pH of B. mori silk glands. A gradient from pH 8.2 to 7.2 was measured in the posterior silk gland, with a pH 7 throughout the middle silk gland, and a gradient from pH 6.8 to 6.2 in the beginning of the anterior silk gland where silk processing into fibers occurs. The small diameter of the most anterior region of the anterior silk gland prevented microelectrode access in this region. Using a histochemical method, the presence of active carbonic anhydrase was identified in the funnel and anterior silk gland of fifth instar larvae. The observed pH gradient collapsed upon addition of the carbonic anhydrase inhibitor methazolamide, confirming an essential role for this enzyme in pH regulation in the B. mori silk gland. Plastic embedding of whole silk glands allowed clear visualization of the morphology, including the identification of four distinct epithelial cell types in the gland and allowed correlations between silk gland morphology and silk stages of assembly related to the pH gradient.B. mori silk glands have four different epithelial cell types, one of which produces carbonic anhydrase. Carbonic anhydrase is necessary for the mechanism that generates an intraluminal pH gradient, which likely regulates the assembly of silk proteins and then the formation of fibers from soluble silk proteins. These new insights into native silk formation may lead to a more efficient production of artificial or regenerated silkworm silk fibers.

HLA-B*5901 is Strongly Associated with Methazolamide-Induced Stevens–Johnson Syndrome/Toxic Epidermal Necrolysis

The carbonic anhydrase inhibitor methazolamide infrequently causes Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). An association between these diseases and the HLA-B59 serotype has been suggested in case reports. This study examined the disease-associated B*59 allele and investigated the association of these diseases with other HLA class I alleles. We performed high-resolution HLA-A, -B and -C genotyping in five patients with methazolamide-induced SJS/TEN using a PCR-sequencing-based typing method and analyzed the association between HLA-class I alleles and occurrence of methazolamide-induced SJS/TEN. B*5901 and Cw*0102 alleles were observed in all patients and A*2402 was observed in four patients. The B*5901 allele showed the strongest association with methazolamide-induced SJS/TEN (p < 0.001; odds ratio: 249.8; 95% CI: 13.4-4813.5), followed by Cw*0102 (p = 0.004; odds ratio: 22.1; 95% CI: 1.2-414.3), when compared with the general population as a control. The frequency of the patients carrying B*5901, Cw*0102 and A*2402 simultaneously was significantly higher than that in the general population (p < 0.001; odds ratio: 110.1; 95% CI: 11.7-1038.6). A strong association was observed between HLA-B*5901 and methazolamide-induced SJS/TEN in Korean patients. HLA-B*5901 may be a useful screening marker for predicting methazolamide-induced SJS/TEN in patients of Korean and Japanese ancestry.

Functional coupling of the downregulated in adenoma Cl−/base exchanger DRA and the apical Na+/H+exchangers NHE2 and NHE3

Non-nutrient-dependent salt absorption across the brush-border membrane of intestinal epithelial cells is primarily mediated by coupled apical Na(+)/H(+) (aNHE) and anion exchange transport, with the latter suspected of being mediated by DRA (downregulated in adenoma; SLC26A3) that is defective in congenital chloridorrhea. To investigate DRA in greater detail and determine whether DRA and NHE activities can be coupled, we measured (22)Na(+) and (36)Cl(-) uptake in Caco2BBE colon cells infected with the tet-off-inducible DRA transgene. Under basal conditions, DRA activity was low in normal and infected Caco2BBE cells in the presence of tetracycline, whereas NHE activities could be easily detected. When apical NHE activity was increased by transfection or serum-induced expression of the aNHE isoforms NHE2 and NHE3, increased (36)Cl(-) uptake was observed. Inhibition of DRA activity by niflumic acid was greater than that by DIDS as well as by the NHE inhibitor dimethylamiloride and the carbonic anhydrase inhibitor methazolamide. DRA activity was largely aNHE-dependent, whereas a component of DRA-independent aNHE uptake continued to be observed. Coupled aNHE and DRA activities were inhibited by increased cellular cAMP and calcium and were associated with synaptotagmin I-dependent, clathrin-mediated endocytosis. In summary, these data support the role of DRA in electroneutral NaCl absorption involving functional coupling of Cl(-)/base exchange and apical NHE.

CO2 chemosensing in rat oesophagus

Background:Acid in the oesophageal lumen is often sensed as heartburn. It was hypothesised that luminal CO2, a permeant gas, rather than H+, permeates through the epithelium, and is converted to...

Carbonic anhydrase inhibition prevents and reverts cardiomyocyte hypertrophy

Hypertrophic cardiomyocyte growth contributes substantially to the progression of heart failure. Activation of the plasma membrane Na+-H+ exchanger (NHE1) and Cl- -HCO3- exchanger (AE3) has emerged as a central point in the hypertrophic cascade. Both NHE1 and AE3 bind carbonic anhydrase (CA), which activates their transport flux, by providing H+ and HCO3-, their respective transport substrates. We examined the contribution of CA activity to the hypertrophic response of cultured neonatal and adult rodent cardiomyocytes. Phenylephrine (PE) increased cell size by 37 +/- 2% and increased expression of the hypertrophic marker, atrial natriuretic factor mRNA, twofold in cultured neonatal rat cardiomyocytes. Cell size was also increased in adult cardiomyocytes subjected to angiotensin II or PE treatment. These effects were associated with increased expression of cytosolic CAII protein and the membrane-anchored isoform, CAIV. The membrane-permeant CA inhibitor, 6-ethoxyzolamide (ETZ), both prevented and reversed PE-induced hypertrophy in a concentration-dependent manner in neonate cardiomyocytes (IC50=18 microm). ETZ and the related CA inhibitor methazolamide prevented hypertrophy in adult cardiomyocytes. In addition, ETZ inhibited transport activity of NHE1 and the AE isoform, AE3, with respective EC50 values of 1.2 +/- 0.3 microm and 2.7 +/- 0.3 microm. PE significantly increased neonatal cardiomyocyte Ca2+ transient frequency from 0.33 +/- 0.4 Hz to 0.77 +/- 0.04 Hz following 24 h treatment; these Ca2+ -handling abnormalities were completely prevented by ETZ (0.28 +/- 0.07 Hz). Our study demonstrates a novel role for CA in mediating the hypertrophic response of cardiac myocytes to PE and suggests that CA inhibition represents an effective therapeutic approach towards mitigation of the hypertrophic phenotype.

Prevention of acute mountain sickness by acetazolamide: as yet an unfinished story

since the 1970s, over 200 studies with acetazolamide have shown it safe and 60–80% effective in acute mountain sickness (AMS). Despite much investigation, our understanding of its action in AMS remains incomplete and more complicated than generally taught. This should come as no surprise

The carbonic anhydrase inhibitors methazolamide and acetazolamide have different effects on the hypoxic ventilatory response in the anaesthetized cat

We compared the effects of the carbonic anhydrase inhibitors methazolamide and acetazolamide (3 mg kg(-1), i.v.) on the steady-state hypoxic ventilatory response in 10 anaesthetized cats. In five additional animals, we studied the effect of 3 and 33 mg kg(-1) methazolamide. The steady-state hypoxic ventilatory response was described by the exponential function: *Vi= G exp(-D P(O2)) + A where *Vi is the inspired ventilation, G is hypoxic sensitivity, D is the shape factor and A is hyperoxic ventilation. In the first group of 10 animals, methazolamide did not change parameters G and D, while A increased from 0.86 +/- 0.33 to 1.30 +/- 0.40 l min(-1) (mean +/- s.d., P = 0.003). However, the subsequent administration of acetazolamide reduced G by 44% (control, 1.93 +/- 1.32; acetazolamide, 1.09 +/- 0.92 l min(-1), P = 0.003), while A did not show a further change. Acetazolamide tended to reduce D (control, 0.20 +/- 0.07; acetazolamide, 0.14 +/- 0.06 kPa(-1), P = 0.023). In the second group of five animals, neither low- nor high-dose methazolamide changed parameters G, D and A. The observation that even high-dose methazolamide, causing full inhibition of carbonic anhydrase in all body tissues, did not reduce the hypoxic ventilatory response is reminiscent of previous findings by others showing no change in magnitude of the hypoxic response of the in vitro carotid body by this agent. This suggests that normal carbonic anhydrase activity is not necessary for a normal hypoxic ventilatory response to occur. The mechanism by which acetazolamide reduces the hypoxic ventilatory response needs further study.

Carbonic Anhydrases and Mucosal Vanilloid Receptors Help Mediate the Hyperemic Response to Luminal CO2 in Rat Duodenum

The duodenal mucosa is exposed to PCO(2) >200 mm Hg due to the luminal mixture of gastric acid with secreted bicarbonate, which augments mucosal protective mechanisms. We examined the hyperemic response to elevated luminal PCO(2) in the duodenum of anesthetized rats luminally exposed to high CO(2) saline to help elucidate luminal acid-sensing mechanisms. Blood flow was measured by laser Doppler, and intracellular pH of epithelial cells by measured by ratio microimaging. The permeant carbonic anhydrase (CA) inhibitor methazolamide, relatively impermeant CA inhibitor benzolamide, vanilloid receptor antagonist capsazepine, or sodium-hydrogen exchanger 1 (NHE-1) inhibitor dimethyl amiloride were perfused with or without the high CO(2) solution. The high CO(2) solution increased duodenal blood flow, which was abolished by pretreatment with methazolamide or capsazepine or by dimethyl amiloride coperfusion. Sensory denervation with capsaicin also abolished the CO(2) effects. Benzolamide dose-dependently inhibited CO(2)-induced hyperemia and at 100 nmol/L inhibited CO(2)-induced intracellular acidification. The membrane-bound CA isoforms IV, IX, XII, and XIV and cytosolic CA II and the vanilloid receptor 1 (TRPV1) were expressed in duodenum and stomach. Dorsal root ganglion and nodose ganglion expressed all isoforms except for CA IX. The duodenal hyperemic response to luminal CO(2) is dependent on cytosolic and membrane-bound CA isoforms, NHE-1, and TRPV1. CO(2)-induced intracellular acidification was inhibited by selective extracellular CA inhibition, suggesting that CO(2) diffusion across the epithelial apical membrane is mediated by extracellular CA. NHE-1 activation preceding TRPV1 stimulation suggests that luminal CO(2) is sensed as H(+) in the subepithelium.

Effects of inhibition of epithelial ion transport and carbonic anhydrase on CO 2 ‐induced intracellular acidification in mouse duodenal villous cells

Epithelial ion transporters and carbonic anhydrase (CA) participate in the regulation of intracellular pH (pHi) in duodenal villous cells. Luminal acid or elevated luminal pCO2 (CO2 stress) acidifies duodenal epithelial cells. The Na/H exchange-1 (NHE-1) inhibitor dimethyl amiloride (DMA) or the Na:HCO3− cotransport (NBC) inhibitor DIDS enhances cellular acidification, whereas the CA inhibitors methazolamide (MTZ) or benzolamide (BNZ) abrogates acidification. We examined the effects of these inhibitors on pHi under CO2 stress in mouse duodenum. Mouse duodenal villous cells were exposed and loaded with a pH-sensitive fluorescence dye BCECF, and pHi was measured with in vivo fluorescent microscope. Duodenal mucosa was exposed to high CO2 solution (pH 6.4, pCO2 260 Torr) with or without DMA (0.1 mM), DIDS (1 mM), MTZ (1 mM), BNZ (1 μM) or novel cell-impermeant CA inhibitor sulfonamide compounds. CO2−stress rapidly acidified the cells followed by pHi recovery to the baseline after CO2 removal. DMA enhanced CO2-induced pHi decrease, whereas DIDS and MTZ reduced the acidification. BNZ and the cell-impermeant CA inhibitors had no effect. The divergent effects of DMA and DIDS on pHi suggest that NHE-1 is more important than is NBC for pHi regulation. Compared with complete inhibition of CO2−induced acidification by MTZ or BNZ in rat duodenum, MTZ was less effective, and the impermeant CA inhibitors had no effect in mouse duodenum, consistent with a marked species difference between rat and mouse for pHi regulation by CA and by plasma membrane acid-base transporters.

Stimulation of renal sulfate secretion by metabolic acidosis requires Na+/H+exchange induction and carbonic anhydrase

The acute effect of metabolic acidosis on SO(4)(2-) secretion by the marine teleost renal proximal tubule was examined. Metabolic acidosis was mimicked in primary cultures of winter flounder renal proximal tubule epithelium (fPTCs) mounted in Ussing chambers by reducing interstitial pH to 7.1 (normally 7.7). fPTCs with metabolic acidosis secreted SO(4)(2-) at a net rate that was 40% higher than in paired isohydric controls (pH 7.7 on interstitium). The stimulation was completely blocked by the carbonic anhydrase inhibitor methazolamide (100 microM). Although Na(+)/H(+) exchange (NHE) isoforms 1, 2, and 3 were identified in fPTCs by immunoblotting, administering EIPA (20 microM) to the interstitial and luminal bath solutions had no effect on net SO(4)(2-) secretion by fPTCs with a normal interstitial pH of 7.7. However, EIPA (20 microM) blocked most of the stimulation caused by acidosis when applied to the lumen but not interstitium, demonstrating that induction of brush-border NHE activity is important. In the intact flounder, serum pH dropped 0.4 pH units (pH 7.7 to 7.3, at 2-3 h) when environmental pH was lowered from 7.8 to approximately 4.3. Whereas serum [SO(4)(2-)] was not altered by acidosis, renal tubular SO(4)(2-) secretion rate was elevated 200%. Thus metabolic acidosis strongly stimulates renal sulfate excretion most likely by a direct effect on active renal proximal tubule SO(4)(2-) secretion. This stimulation appears to be dependent on inducible brush-border NHE activity.

Mechanism of augmented duodenal HCO3−secretion after elevation of luminal CO2

The proximal duodenum is exposed to extreme elevations of P(CO(2)) because of the continuous mixture of secreted HCO(3)(-) with gastric acid. These elevations (up to 80 kPa) are likely to place the mucosal cells under severe acid stress. Furthermore, we hypothesized that, unlike most other cells, the principal source of CO(2) for duodenal epithelial cells is from the lumen. We hence examined the effect of elevated luminal P(CO(2)) on duodenal HCO(3)(-) secretion (DBS) in the rat. DBS was measured by the pH-stat method. For CO(2) challenge, the duodenum was superfused with a high Pco(2) solution. Intracellular pH (pH(i)) of duodenal epithelial cells was measured by ratio microfluorometry. CO(2) challenge, but not isohydric solutions, strongly increased DBS to approximately two times basal for up to 1 h. Preperfusion of the membrane-permeant carbonic anhydrase inhibitor methazolamide, or continuous exposure with indomethacin, fully inhibited CO(2)-augmented DBS. Dimethyl amiloride (0.1 mM), an inhibitor of the basolateral sodium-hydrogen exchanger 1, also inhibited CO(2)-augumented DBS, although S-3226, a specific inhibitor of apical sodium-hydrogen exchanger 3, did not. DIDS, an inhibitor of basolateral sodium-HCO(3)(-) cotransporter, also inhibited CO(2)-augemented DBS, as did the anion channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid. CO(2) decreased epithelial cell pH(i), followed by an overshoot after removal of the CO(2) solution. We conclude that luminal CO(2) diffused in the duodenal epithelial cells and was converted to H(+) and HCO(3)(-) by carbonic anhydrase. H(+) initially exited the cell, followed by secretion of HCO(3)(-). Secretion was dependent on a functioning basolateral sodium/proton exchanger, a functioning basolateral HCO(3)(-) uptake mechanism, and submucosal prostaglandin generation and facilitated hydration of CO(2) into HCO(3)(-) and H(+).

Low-dose PGE2 mimics the duodenal secretory response to luminal acid in mice.

Luminal exposure to concentrated acid, the most accepted physiological stimulus for duodenal bicarbonate secretion (DBS), cannot be used with in vitro preparations due to potential tissue damage. We thus examined whether exposure to PGE(2), a well-characterized physiological duodenal secretagogue, could mimic the effects of acid perfusion. DBS was measured in C57/BL mice by pH-stat/back-titration and measurement of total dissolved CO(2) concentration ([CO(2)](t)). Anion transport inhibitor DIDS, anion channel inhibitor 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), carbonic anhydrase inhibitor methazolamide, and nonselective cyclooxygenase inhibitor indomethacin were used to inhibit separate components of HCO(3)(-) secretory pathway. Baseline DBS was not altered by exposure to methazolamide (0.1 mM) but was slightly reduced by DIDS (0.5 mM). DBS and [CO(2)](t) increased after acid and PGE(2) exposure. DIDS (0.5 mM) and NPPB (0.2 mM) abolished acid-induced DBS increase. Methazolamide (0.1 mM) and DIDS inhibited acid-induced [CO(2)](t) increase. DIDS, NPPB, or methazolamide had little effect on DBS in response to high concentration PGE(2) (100 microg/ml). Low concentration PGE(2) (1 microg/ml) increased DBS that was inhibited by DIDS, NPPB, and methazolamide. Pretreatment with indomethacin (5 mg/kg) inhibited DBS induced by acid exposure but not by PGE(2). High-dose PGE(2) substantially increases DBS by a mechanism that appears to be different than secretory response to luminal acid perfusion. Secretory response to low-dose PGE(2), at least in terms of inhibitor profile, closely resembles secretion in response to perfusion of physiological acid concentrations and may be a useful stimulus for in vitro study of DBS in isolated mouse duodenum.

Cortisol alters carbonic anhydrase-mediated renal sulfate secretion.

Active transepithelial sulfate secretion rate by winter flounder renal proximal tubule epithelium in primary culture (fPTC) is dependent on intracellular carbonic anhydrase (CA) and enhanced by cortisol. To further evaluate this relationship, a partial cDNA clone (327 bp) of carbonic anhydrase II (CAII) with high sequence similarity to CAII from numerous species including fish, chicken, and human was obtained from fPTCs. The majority of CA activity and CAII protein was present in the cytosol of fPTCs; however, significant amounts of both (in addition to SDS-resistant CA activity, i.e., CAIV-like isoform) were present in concentrated plasma membranes. CAII from concentrated membranes migrated differently than purified CAII on nondenaturing PAGE gels, suggesting that CAII associates with another membrane component. Treatment of fPTCs with the cell-soluble CA inhibitor methazolamide (100 microM) caused a 58% reduction in active transepithelial SO4(2-) secretion. fPTCs that were previously cultured under high-cortisol concentrations, when subjected to 5 days of low physiological levels of cortisol, had decreased CA activity (28%), CAII protein abundance (65%), and net active SO4(2-) secretion (28%), with no effect on epithelial differentiation. Methazolamide and low-cortisol treatment in combination inhibited net active SO4(2-) secretion 56%, which was not different than the effect of methazolamide treatment alone. These data indicate that cortisol directly increases renal CA activity, CAII protein abundance, and CA-dependent SO4(2-) secretion in the marine teleost renal proximal tubule.

Muscarinic receptor-induced acidification in sublingual mucous acinar cells: loss of pH recovery in Na+-H+ exchanger-1 deficient mice.

1. Intracellular pH (pHi) plays an important role in regulating fluid and electrolyte secretion by salivary gland acinar cells. The pH-sensitive, fluorescent dye 2', 7'-bis(carboxyethyl)-5(6)-carboxylfluorescein (BCECF) was used to characterize the mechanisms involved in regulating pHi during muscarinic stimulation in mouse sublingual mucous acinar cells. 2. In the presence of HCO3-, muscarinic stimulation caused a rapid decrease in pHi (0.24 +/- 0.02 pH units) followed by a slow recovery rate (0.042 +/- 0.002 pH units min-1) to the initial resting pHi in sublingual acinar cells. The muscarinic receptor-induced acidification in parotid acinar cells was of a similar magnitude (0. 25 +/- 0.02 pH units), but in contrast, the recovery rate was approximately 4-fold faster (0.181 +/- 0.005 pH units min-1). 3. The agonist-induced intracellular acidification was inhibited by the anion channel blocker niflumate, and was prevented in the absence of HCO3- by treatment with the carbonic anhydrase inhibitor methazolamide. These results indicate that the muscarinic-induced acidification is due to HCO3- loss, probably mediated by an anion conductive pathway. 4. The Na+-H+ exchange inhibitor 5-(N-ethyl-N-isopropyl)amiloride (EIPA) amplified the magnitude of the agonist-induced acidification and completely blocked the Na+-dependent pHi recovery. 5. To examine the molecular nature of the Na+-H+ exchange mechanism in sublingual acinar cells, pH regulation was investigated in mice lacking Na+-H+ exchanger isoforms 1 and 2 (NHE1 and NHE2, respectively). The magnitude and the rate of pHi recovery in response to an acid load in acinar cells isolated from mice lacking NHE2 were comparable to that observed in cells from wild-type animals. In contrast, targeted disruption of the Nhe1 gene completely abolished pHi recovery from an acid load. These results demonstrate that NHE1 is critical for regulating pHi during a muscarinic agonist-stimulated acid challenge and probably plays an important role in regulating fluid secretion in the sublingual exocrine gland. 6. In NHE1-deficient mice, sublingual acinar cells failed to recover from an acid load in the presence of bicarbonate. These results confirm that the major regulatory mechanism involved in pHi recovery from an acid load is not Na+-HCO3- cotransport, but amiloride-sensitive Na+-H+ exchange via isoform 1.