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PubMed1979

Volume expansion-induced alterations in proximal tubule chloride gradient in the rat.

To elucidate the mechanisms by which acute volume expansion (AVE) induces a decrease in proximal tubule transepithelial chloride gradient, male Sprague-Dawley rats were studied before and after AVE with Ringer lactate. In group 1, after AVE equivalent to 10% body wt, there were decreases in both tubule fluid to plasma inulin ratio ((TF/P)In) (from 2.28 +/- 0.10 to 1.57 +/- 0.05) and tubule fluid to ultrafiltrate chloride ratio ((TF/UF)Cl) (from 1.25 +/- 0.02 to 1.18 +/- 0.02). Group 2 was studied during carbonic anhydrase inhibition (CAI) produced by benzolamide before and during superimposed AVE (20% body wr). Both (TF/P)In (from 1.91 +/- 0.10 to 1.41 +/- 0.08) and (TF/UF)Cl (from 1.07 +/- 0.02 to 1.01 +/- 0.01) decreased. Group 3 was studied during maintained AVE (15% body wt) as a control for group 4, in which CAI was superimposed on maintained AVE. In group 3, (TF/P)In and (TF/UF)Cl did not change, but in group 4 CAI was associated with a decrease in (TF/P)In (from 1.55 +/- 0.05 to 1.21 +/- 0.05) and in (TF/UF)Cl (from 1.16 +/- 0.01 to 1.04 +/- 0.07). These data suggest that in the superficial proximal convoluted tubule of the rat, AVE-induced alterations in transepithelial chloride gradient are dependent on a mechanism(s) other than changes in carbonic anhydrase-mediated bicarbonate reabsorption.

Animals↗
PubMed1986

Can causality be determined from proximal tubular reabsorption and peritubular physical factors?

Many studies in the literature have drawn conclusions regarding the mechanism of change in absolute proximal tubular reabsorption (APR) based on steady-state measurements of proximal reabsorptive rates and the peritubular capillary. The proximal reabsorptive rate, APR, is the product of the effective reabsorptive pressure (ERP) and the peritubular capillary reabsorptive coefficient (LpAR) (APR = ERP . LpAR). The ERP is defined by the net hydrostatic and oncotic pressure gradient acting across the capillary wall from interstitium to peritubular capillary flow. The relationship APR = ERP . LpAR is predefined, and steady-state measurements do not permit determination of causality because primary changes in any variable obligate a proportional change in a second variable. As an example of the difficulties in interpretation of this type of analysis, we have examined the APR and factors contributing to ERP and LpAR before and after the administration of benzolamide, a carbonic anhydrase inhibitor, to saline-expanded Munich-Wistar rats. Alterations in peritubular capillary fluid uptake cannot always be interpreted as casual mechanisms for changes in absolute fluid reabsorption but may result from primary alterations in epithelial transport.

Absorption↗
PubMed1997

Ionic mechanisms of spontaneous GABAergic events in rat hippocampal slices exposed to 4-aminopyridine.

Ionic mechanisms of spontaneous GABAergic events in rat hippocampal slices exposed to 4-aminopyridine. J. Neurophysiol. 78: 2582-2591, 1997. Ion-selective (H+ and K+) microelectrode techniques as well as conventional extra- and intracellular recordings were used to study the ionic mechanisms of propagating spontaneous GABAergic events (SGEs) in rat hippocampal slices exposed to 4-aminopyridine (4-AP, 50-100 mu M). All experiments were made in the presence of antagonists of ionotropic glutamate receptors [10 mu M 6-nitro-7-sulphamoylbenzoquinoxaline-2,3-dione (NBQX) and 40 mu M -2-amino-5-phosphonopentanoic acid (AP5)]. The SGEs were composed of a negative-going change in field potential with a temporally coincident increase (0.7 +/- 0.3 mM; mean +/- SE) in extracellular K+ ([K+]o) and an alkaline transient (0.01-0.08 units) in extracellular pH (pHo) in stratum radiatum of the area CA1. Simultaneous intracellular recordings showed a triphasic hyperpolarization-depolarization-late hyperpolarization response in pyramidal cells. Application of pentobarbital sodium (PB, 100 mu M) decreased the interval between SGEs from a mean value of 35 to approximately 20 s and shortened the period of refractoriness of stimulus-evoked propagating events. This was accompanied by an increase in the amplitude of the field potential response of the [K+]o and the pHo shifts and of the depolarizing phase of the pyramidal-cell response. The SGEs were completely blocked by the gamma-aminobutyric acid-A (GABAA) receptor antagonist, picrotoxin (PiTX; 100 mu M). The amplitudes of the negative-going field potential and of the depolarizing phase of the pyramidal-cell response as well as the ionic shifts associated with SGEs were strongly suppressed in the nominal absence of CO2/HCO-3. There was a five-fold increase in the interevent interval, and propagating SGEs could not be evoked by stimuli given at intervals shorter than approximately 2-3 min. Exposure to inhibitors of carbonic anhydrase, benzolamide (BA; 10 micro M) or ethoxyzolamide (EZA; 50 mu M) fully blocked the alkaline pHo transients and turned them into acid shifts. The poorly membrane-permeant BA had no discernible effect on the other components of the SGEs, but application of EZA had effects reminiscent to those of CO2/HCO-3-free medium. Addition of the GABAA receptor-permeant weak-acid anion, formate (20 mM) reestablished the SGEs that were first suppressed by exposure to the CO2/HCO-3-free medium. No SGEs were seen in the presence of a similar concentration of the GABAA receptor-impermeant anion propionate. Unlike the alkaline transients associated with HCO-3-driven SGEs, those supported by formate were not blocked by BA. The present data suggest that an inward current carried by bicarbonate is necessary for the generation of SGEs and that the GABAA receptor-mediated excitatory coupling among GABAergic interneurons is essentially dependent on the availability of intracellular bicarbonate.

2-Amino-5-phosphonovalerate↗
PubMed1985

Role of carbonic anhydrase in bone: plasma acetazolamide concentrations associated with inhibition of bone loss.

Earlier reports from our laboratory have indicated that the carbonic anhydrase inhibitor acetazolamide blocks the hypercalcemic response to parathyroid hormone. In addition, we have reported that acetazolamide when administered by several routes partially prevents denervation-induced bone loss in a rat model of disuse osteoporosis. Continuous subcutaneous infusion required the least daily dose (8 mg/kg). The present study extends these earlier findings in several ways. It was found that in partially preventing denervation-induced bone loss: (1) incorporation of 1 M THAM [tris(hydroxymethyl)aminoethane] enhanced the potency of acetazolamide such that it was effective at daily doses of 0.6 mg/kg; (2) acetazolamide in the presence of 1 M THAM was effective at plasma concentrations as low as 50 ng/ml which are more than 500-fold less than peak plasma concentrations normally encountered in the human when acetazolamide is being used as a therapeutic agent; and (3) another inhibitor, benzolamide, was also effective by continuous subcutaneous infusion.

Acetazolamide↗
PubMed1982

Bicarbonate concentrations in the renal cortex.

Total CO2 (CO2+HCO3-) was measured in alkaline extracts of quickly frozen rat renal cortex. The mean concentration calculated for tissue water was 18.7 +/- SD 1.6 mEq/l (n = 10) when that measured in plasma water was 23.5 +/- 1.7. In other experiments (n = 6-10), the mean concentration in tissue water increased to 28.3 mEq/l after the animals were treated with benzolamide to inhibit carbonic anhydrase and to 38.1 mEq/l after they were infused with NaHCO3. On the other hand, the tissue concentration decreased to 14-15 mEq/l when they were hyperventilated or infused with HCl. Calculations based on published micropuncture data indicate that the usual intracellular concentration is 20-25 mEq/l or about twice that usually reported for skeletal muscle; and in contrast to muscle, the findings are suggestive of large variations in metabolic disorders.

Acidosis, Respiratory↗
PubMed1999

Temporal adjustment of the juxtaglomerular apparatus during sustained inhibition of proximal reabsorption.

Tubuloglomerular feedback (TGF) stabilizes nephron function by causing changes in single-nephron GFR (SNGFR) to compensate for changes in late proximal flow (VLP). TGF responds within seconds and reacts over a narrow range of VLP that surrounds normal VLP. To accommodate sustained increases in VLP, TGF must reset around the new flow. We studied TGF resetting by inhibiting proximal reabsorption with benzolamide (BNZ; administered repeatedly over a 24-hour period) in Wistar-Froemter rats. BNZ acutely activates TGF, thereby reducing SNGFR. Micropuncture was performed 6-10 hours after the fourth BNZ dose, when diuresis had subsided. BNZ caused glomerular hyperfiltration, which was prevented with inhibitors of macula densa nitric oxide synthase (NOS). Because of hyperfiltration, BNZ increased VLP and distal flow, but did not affect the basal TGF stimulus (early distal salt concentration). BNZ slightly blunted normalized maximum TGF response and the basal state of TGF activation. BNZ sensitized SNGFR to reduction by S-methyl-thiocitrulline (SMTC) and caused the maximum TGF response to be strengthened by SMTC. Sensitization to type I NOS (NOS-I) blockers correlated with increased macula densa NOS-I immunoreactivity. Tubular transport measurements confirmed that BNZ affected TGF within the juxtaglomerular apparatus. During reduced proximal reabsorption, TGF resets to accommodate increased flow and SNGFR through a mechanism involving macula densa NOS.

Absorption↗
PubMed1999

Does gill boundary layer carbonic anhydrase contribute to carbon dioxide excretion: a comparison between dogfish (Squalus acanthias) and rainbow trout (Oncorhynchus mykiss).

In vivo experiments were conducted on spiny dogfish (Squalus acanthias) and rainbow trout (Oncorhynchus mykiss) in sea water to determine the potential role of externally oriented or gill boundary layer carbonic anhydrase in carbon dioxide excretion. This was accomplished by assessing pH changes in expired water using a stopped-flow apparatus. In dogfish, expired water was in acid-base disequilibrium as indicated by a pronounced acidification (delta pH=-0.11+/-0.01; N=22; mean +/- s.e.m.) during the period of stopped flow; inspired water, however, was in acid-base equilibrium (delta pH=-0.002+/-0.01; N=22). The acid-base disequilibrium in expired water was abolished (delta pH=-0.005+/-0.01; N=6) by the addition of bovine carbonic anhydrase (5 mg l-1) to the external medium. Addition of the carbonic anhydrase inhibitor acetazolamide (1 mmol l-1) to the water significantly reduced the magnitude of the pH disequilibrium (from -0.133+/-0.03 to -0.063+/-0.02; N=4). However, after correcting for the increased buffering capacity of the water caused by acetazolamide, the acid-base disequilibrium during stopped flow was unaffected by this treatment (control delta [H+]=99.8+/-22.8 micromol l-1; acetazolamide delta [H+]=81.3+/-21.5 micromol l-1). In rainbow trout, expired water displayed an acid-base disequilibrium (delta pH=0.09+/-0.01; N=6) that also was abolished by the application of external carbonic anhydrase (delta pH=0.02+/-0.01). The origin of the expired water acid-base disequilibrium was investigated further in dogfish. Intravascular injection of acetazolamide (40 mg kg-1) to inhibit internal carbonic anhydrase activity non-specifically and thus CO2 excretion significantly diminished the extent of the expired water disequilibrium pH after 30 min (from -0.123+/-0.01 to -0.065+/-0.01; N=6). Selective inhibition of extracellular carbonic anhydrase activity using a low intravascular dose (1.3 mg kg-1) of the inhibitor benzolamide caused a significant reduction in the acid-base disequilibrium after 5 min (from -0.11+/-0.01 to -0.07+/-0. 01; N=14). These results demonstrate that the expired water acid-base disequilibrium originates, at least in part, from excretory CO2 and that extracellular carbonic anhydrase in dogfish may have a significant role in carbon dioxide excretion. However, externally oriented carbonic anhydrase (if present in dogfish) plays no role in catalysing the hydration of the excretory CO2 in water flowing over the gills and thus is unlikely to facilitate CO2 excretion.

Acetazolamide↗
PubMed2000

Interstitial carbonic anhydrase (CA) activity in brain is attributable to membrane-bound CA type IV.

We tested the hypothesis that extracellular membrane-bound carbonic anhydrase (CA) type IV is responsible for the regulation of interstitial pH (pH(o)) transients in brain. Rat hippocampal slices were incubated in phosphatidylinositol-specific phospholipase C (PI-PLC), which cleaves the link of CA IV to the external face of plasma membranes. Then evoked alkaline pH(o) shifts were studied in a recording chamber, using pH microelectrodes. Incubation fluid was saved for later analysis. The ability to buffer a rapid alkaline load was reduced markedly in PI-PLC-treated tissue as compared with adjacent, paired control slices. The effect of benzolamide (a poorly permeant CA inhibitor) on evoked pH(o) shifts was diminished greatly in the PI-PLC-treated tissue, consistent with the washout of interstitial CA. Treatment of the incubation fluid with SDS abolished nearly all of the CA activity in fluid from controls, whereas an SDS-insensitive component remained in the fluid from PI-PLC-treated slices. These data suggested that CA type II (which is blocked by SDS) leaked from injured glial cells in both slice preparations, whereas CA type IV (which is insensitive to SDS) was liberated selectively into the fluid from PI-PLC-treated tissue. Western blot analysis was consistent with this interpretation, demonstrating a predominance of CA IV in the incubation fluid from PI-PLC-treated tissue and variable amounts of CA II in fluid from PI-PLC-treated and control slices. These results demonstrate that interstitial CA activity brain is attributable principally to membrane-bound CA IV.

Animals↗
PubMed1988

Permeability of human cornea and sclera to sulfonamide carbonic anhydrase inhibitors.

Corneal penetration of sulfonamide carbonic anhydrase inhibitors for topical treatment of glaucoma has been tested in human eye bank and rabbit tissue. Paired corneas, with the epithelia intact or removed, and excised sclera were perfused in vitro. Corneal permeability (Kp) to methazolamide and ethoxzolamide was similar in both species, but for benzolamide and bromacetazolamide the Kp was greater in humans. Human corneas without epithelium had Kp the same as scleral Kp. Topical methazolamide (6 mmol/L) was studied in vivo in rabbits and in ten humans before cataract surgery. The mean (+/- SE) concentration in the rabbit aqueous was 3.2 +/- 1.4 mumol/L at eight minutes and 1.2 +/- 0.16 mumol/L at one hour. In humans, less than 0.2 mumol/L was detected at eight minutes; at one hour none was detected in three cases, and 0.4 +/- 0.08 mumol/L was detected in four cases. Lower permeability in humans than rabbits may result from a fourfold greater blinking rate, a twofold greater tear turnover, and a twofold lower corneal/conjunctival area.

Aged↗
PubMed1994

A physiological measure of carbonic anhydrase in Müller cells.

Carbonic anhydrase activity was characterized in freshly dissociated Müller cells of the salamander retina. Intracellular pH was monitored using ratio imaging of the indicator dye BCECF as extracellular PCO2 was varied. The extracellular solution was switched rapidly (141 ms rise time) from a HEPES buffered to a CO2-HCO3- buffered solution (both pH 7.4). Introduction of CO2-HCO3- produced a rapid cell acidification. Cell pH dropped from a steady-state pH of 7.02 in HEPES solution to pH 6.81 in CO2-HCO3-. Methazolamide, a carbonic anhydrase inhibitor, dramatically reduced the initial rate of acidification, demonstrating that the acidification was produced by the carbonic anhydrase-catalyzed hydration of CO2. The initial rate of acidification, 52.6 pH units per min (0.88 pH units per s), was reduced approximately 150-fold to 0.36 pH units per min by 10(-3) M methazolamide. Half-maximal inhibition occurred at a methazolamide concentration of 5.6.10(-7) M. The carbonic anhydrase inhibitor acetazolamide (10(-3) M) also greatly reduced the rate of cell acidification. The latency to the onset of carbonic anhydrase inhibition was 660 ms for methazolamide and 7.5 s for acetazolamide. The carbonic anhydrase inhibitor benzolamide (10(-4) M, 4 min exposure), which is poorly membrane permeant, had little effect on the rate of cell acidification, indicating that the site of carbonic anhydrase action was intracellular. The activity of Müller cell carbonic anhydrase may help to buffer extracellular CO2 variations in the retina.

Acetazolamide↗
PubMed1998

Inhibition and kinetic properties of membrane-bound carbonic anhydrases in rabbit skeletal muscles.

It was the aim of this study to investigate whether the carbonic anhydrases associated with the sarcoplasmic reticulum (SR) and sarcolemmal membranes differ in their kinetic and inhibitory properties. To this end, sarcolemmal and SR membrane vesicle fractions were prepared from rabbit white and red skeletal muscles, the white muscle sarcolemmal fraction (WSL), the red muscle sarcolemmal fraction (RSL), the white muscle SR fraction (WSR), and the red muscle SR fraction (RSR). WSL displayed a specific carbonic anhydrase activity of 22.1 U . ml/mg and RSL of 7.5 U . ml/mg, whereas the SR fractions showed a much lower activity of 0.5 U . ml/mg for WSR and of 2.4 U . ml/mg for RSR. In both SR fractions phase separation experiments with Triton X-114 demonstrated that the carbonic anhydrase activity is due to a membrane-bound enzyme and not due to a cytosolic isozyme. The kinetic properties of carbonic anhydrase from the four distinct membane fractions were evaluated by determination of the Michaelis constant, Km, and of the catalytic centre activity kcat. Km appears to be somewhat lower for SR than for SL. Inhibition constants of SR and SL carbonic anhydrases were determined applying six carbonic anhydrase inhibitors: chlorzolamide, ethoxzolamide, methazolamide, benzolamide, and acetazolamide, and also cyanate. The inhibition constants of the SR fractions were significantly different from those of the corresponding sarcolemmal fractions, indicating that the carbonic anhydrase measured in the SR fractions does not originate from contaminating sarcolemmal membrane vesicles, but appears to represent a distinct carbonic anhydrase associated with the SR membrane.

Animals↗
PubMed1998

Carbonic anhydrase II plays a major role in osteoclast differentiation and bone resorption by effecting the steady state intracellular pH and Ca2+.

Carbonic anhydrase II (CA II) expression in characteristic for the early stage of osteoclast differentiation. To study how CA II, which is crucial in proton generation in mature osteoclasts, influences the osteoclast differentiation process we performed rat bone marrow cultures. In this model, acetazolamide, a specific CA inhibitor, decreased the 1,25 (OH)2D3-induced formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cells, in a dose-dependent manner. We then performed intracellular pH (pHi) and Ca2+ (Cai2+) measurements for cultured osteoclasts and noticed that addition of acetazolamide caused a rapid, transient increase of both parameters. The increase in pHi was dependent neither on the culture substrate nor on the extracellular pH (pHe) but the increase could be diminished by DIDS or by bicarbonate removal. Membrane-impermeable CA inhibitors (benzolamide and pd5000) did not have this effect. Addition of CA II antisense oligonucleotides into the cultures reduced the pHi increase significantly. CA II inhibition was also found to neutralize the intracellular vesicles at extracellular pH (pHe) of 7.4, but at less extent at pHe 7.0. In mouse calvaria cultures, bone resorption was inhibited dose dependently by acetazolamide at pHe 7.4 while inhibition was smaller at pHe 7.0. We conclude that CA II is essential not only in bone resorption but also in osteoclast differentiation. In both processes, however, the crucial role of CA II is at least partially due to the effect on the osteoclast pHi regulation.

Acetazolamide↗
PubMed1990

Rat renal proximal tubular gluconeogenesis: possible involvement of nonmitochondrial carbonic anhydrase isozymes.

The carbonic anhydrase (CA) inhibitor ethoxzolamide decreases the rate of glucose synthesis from 10 mM pyruvate by tubules incubating in 25 mM HCO3- but not in 50 mM HCO3-: this is evidence that rat renal cortical mitochondrial CA (CA V) provides HCO3- for pyruvate carboxylation in renal tubular gluconeogenesis at physiological total CO2 (CO2 + HCO3-). In renal proximal tubules prepared from 48-h-starved rats and incubating in 10 mM pyruvate in 25 mM HCO3- buffered saline (Krebs-Henseleit buffer) the CA inhibitors acetazolamide (AZ) and benzolamide (BZ) decreased the rate of glucose synthesis. Maximal inhibition was reached with 125 microM AZ or with 450 microM BZ. The rate of glucose synthesis increased with increasing pyruvate concentration from 3.33 to 20 mM; including 600 microM BZ or 188 microM AZ results in glucose synthesis becoming independent of increasing pyruvate concentration. Doubling the physiological concentration of bicarbonate restored the dependence of glucose synthesis on pyruvate concentration and partly, but not completely, alleviated the inhibitory effect of AZ and BZ, leading to the conclusion that AZ and BZ influence gluconeogenesis by affecting enzymes in addition to CA V. Tubules were incubated with substrates which do not require pyruvate carboxylation for synthesis of oxaloacetate. When tubules were incubated in 10 mM malate the rate of glucose synthesis was unaffected by less than 100 microM AZ or 400 microM BZ and was decreased maximally by 40 and 20%, respectively, by 125 microM AZ, 450 microM BZ, and higher concentrations of these drugs. Increasing the malate concentration from 3.33 to 20 mM increased the rate of glucose synthesis; 600 microM BZ inhibited the rate of glucose synthesis only when the malate concentration was greater than 10 mM but 188 microM AZ decreased the rate of glucose synthesis at each concentration of malate. Results were similar when tubules were incubated in glutamine with CA inhibitors. The rate of glucose synthesis differed with the substrate metabolized and the substrate concentration except when 600 microM BZ was included.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMedDate not supplied

[3H]acetazolamide binding to carbonic anhydrase in normal and transformed cells.

The binding of [3H]acetazolamide (AZ), a carbonic anhydrase (CA) inhibitor, to soluble and particulate forms of CA was investigated. Sources for the assays were purified CA II, adult rat cortical, oligodendrocyte and neuronal enriched preparations; cultured murine glial cells, rat C-6 glioma, rat hepatoma and human glioblastoma cells. CA enzyme activity in the same preparations was also assayed by following change in pH during incubation. A gel permeation chromatographic method was developed to assess [3H]AZ binding to soluble CA, while glass fiber filter vacuum filtration was used for particulate CA binding. Saturable specific binding of [3H]AZ to rat cortical soluble and particulate CA preparations was demonstrated. Computer-assisted data analysis estimated the binding parameters of [3H]AZ to soluble rat cortical CA to be Bmax = 0.38 +/- 0.13 pmol/mg protein and Kd = 34.7 +/- 17.5 nM. The rat cortical particulate fraction Bmax was 2.05 +/- 0.28 pmol/mg protein with a Kd of 107.1 +/- 24.2 nM. Purified bovine CA-II bound 1.15 +/- 0.19 pmol [3H]AZ/mg protein with a Kd of 54.0 +/- 3.4 nM. The pH optima for [3H]AZ binding to soluble and particulate CA was between 6.5 and 7.5. Binding was linear with respect to protein up to 1.0 mg/mL. The particulate fraction bound 3-4 times more [3H]ligand per unit protein than the soluble fraction. Interestingly, no detectable CA enzyme activity or [3H]AZ binding was observed in the soluble or particulate fractions of human glioblastoma, rat C-6 glioma or rat hepatoma cells. Binding of [3H]AZ to other soluble enzymes or proteins was negligible. In competition binding experiments, a rank order of inhibition of [3H]AZ binding to rat cortical CA by established CA inhibitors was: dichlorphenamide greater than acetazolamide greater than or equal to benzolamide greater than methazolamide greater than hydrochlorothiazide greater than or equal to sulfanilamide. [3H]AZ binding was not affected by other classes of pharmacologic characterizing agents. The binding of [3H]AZ to the CA enzyme molecule is highly specific and sensitive and may prove useful in vitro or in situ as a probe for this enzyme.

Acetazolamide↗
PubMed1993

Refined structure of the aminobenzolamide complex of human carbonic anhydrase II at 1.9 A and sulphonamide modelling of bovine carbonic anhydrase III.

The binding of aminobenzolamide to human carbonic anhydrase (HCA II) has been investigated by X-ray crystallography. The three dimensional atomic structure of the enzyme inhibitor complex has been refined at 1.9 A resolution. The crystallographic R-factor is 17.8%. All inhibitor atoms are clearly possible to identify from the difference electron density map in the active site of the enzyme. The nitrogen of the sulphonamide group of the inhibitor is bound as a fourth ligand to the zinc ion, the other three are all histidyl residues. The binding conformation of the sulphonamide groups is similar to the previously described sulphonamide inhibitors. One of the oxygens of the outer sulphonamido group of the inhibitor forms a hydrogen bond to the amino group of Gln 92. The higher affinity of the benzolamide inhibitor compared with acetazolamide can be accounted for by the strong aromatic and hydrophobic interactions between the amino benzene ring of the inhibitor and the residues Phe 131 and Leu 198. In modelling studies of bovine carbonic anhydrase III (BCA III) it was evident that Phe 198 prevents an optimal interaction with sulphonamides.

Animals↗
PubMed1994

Depolarization-induced acid secretion in gliotic hippocampal slices.

Gliotic hippocampal slices were used to study glial acid secretion in a tissue largely devoid of neural elements. Rat hippocampal slices were prepared 10-28 days after sterotaxic injection of kainate. Cresyl Violet staining and immunohistochemistry for glial fibrillary acidic protein demonstrated a loss of neurons and a proliferation of reactive astrocytes in area CA3. Extracellular pH and K+ shifts were recorded in CA3 in response to K+ iontophoresis. Elevation of K+ evoked an extracellular acid shift that was two- to three-fold larger in gliotic versus unlesioned tissue. Ba2+ caused a slow extracellular acidification, and blocked both the depolarizing responses of the glial cells and the acid shifts evoked by K+. The K(+)-evoked acid shifts were abolished in Na(+)-free media, and diminished in HEPES-buffered solutions. Inhibition of extracellular carbonic anhydrase caused a reversible enhancement of the K(+)-evoked acid shifts, an effect that could be mimicked during H+ iontophoresis in agarose gels. Gliotic acid shifts were unaffected by amiloride or its analogs, stilbenes, zero Cl- media, zero or elevated glucose, lactate transport inhibitors, zero Ca2+ or Cd2+. Smaller acid shifts could be evoked in normal slices which were also enhanced by benzolamide, and blocked by Ba2+ and zero Na+ media. It is concluded that acid secretion by reactive astrocytes is Na+ and HCO3(-)-dependent and is triggered by depolarization. The similar pharmacological and ionic sensitivity of the acid shifts in non-gliotic tissue suggest that these properties are shared by normal astrocytes. These characteristics are consistent with the operation of an electrogenic Na(+)-HCO3- co-transporter. However, the enhancement of the acid shifts by inhibitors of extracellular carbonic anhydrase suggests that CO3(2-), rather than HCO3-, is the transported acid equivalent.

Acids↗
PubMed2005

Carbonic anhydrase inhibitors. Inhibition of the transmembrane isozyme XII with sulfonamides-a new target for the design of antitumor and antiglaucoma drugs?

The inhibition of a newly cloned human carbonic anhydrase (CA, EC 4.2.1.1), isozyme XII (hCA XII), has been investigated with a series of sulfonamides, including some clinically used derivatives (acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide, benzolamide, and sulpiride, or indisulam, a compound in clinical development as antitumor drug), as well as the sulfamate antiepileptic drug topiramate. Some simple amino-/hydrazine-/hydroxy-substituted aromatic/heterocyclic sulfonamides have also been included in the study. All types of activity have been detected, with several medium potency inhibitors (K(I)s in the range of 34-220 nM), whereas ethoxzolamide and several halogenated sulfanilamides showed stronger potency, with K(I)s in the range of 11-22 nM. The antiglaucoma sulfonamides used clinically, except dichlorophenamide, which is a moderate inhibitor (K(I) of 50 nM), as well as topiramate, indisulam, and sulpiride behave as very potent hCA XII inhibitors, with K(I)s in the range of 3.0-5.7 nM. Several subnanomolar inhibitors (K(I)s in the range of 0.30-0.85 nM) have also been detected. Compounds with excellent selectivity against hCA XII over hCA II have been found, showing selectivity ratios in the range of 177.7-566.7. Apparently, hCA XII is a target of the antiglaucoma sulfonamides, and potent hCA XII inhibitors may be developed/used for the management of hypoxic tumors, together with inhibitors of the other tumor-associated isozyme, CA IX.

Antineoplastic Agents↗
PubMed2002

Effects of antiepileptic drugs on extracellular pH regulation in the hippocampal CA1 region in vivo.

Intracellular and extracellular pH are known to influence neuronal activity and may play a role in seizure termination. In the pyramidal cell layer of the CA1 region of the hippocampus in the urethane anesthetized adult rat, there is an initial alkalinization in response to stimulus trains administered to the contralateral CA3 region. This is followed by an acidification that peaks after termination of the afterdischarge. Initial experiments demonstrated that the peak level of acidification correlated with the duration of the afterdischarge, but that the peak level of alkalinization did not. The effects of several antiepileptic drugs on the initial alkalinization were determined. Systemic administration of acetazolamide (50 mg/kg, n=4) and topiramate (45 mg/kg, n=7) and local administration of benzolamide (n=3), all of which inhibit carbonic anhydrase, decreased the initial alkalinization that occurs during the stimulus train. Diazepam (3 mg/kg, n=5) and phenobarbital (60 mg/kg, n=6), agonists at the GABA(A) receptor complex, increased the initial alkalinization, while sodium channel blockers phenytoin (80 mg/kg, n=5) and carbamazepine (50 mg/kg, n=5) had no significant effect. The data suggest that the alkalinization in CA1 in vivo is predominantly regulated through activity of the GABA(A) receptor, rather than through activation of glutamatergic receptors. The change in alkalinization does not appear to be related to the mechanism of the antiepileptic effect of the drugs that were tested.

Acetazolamide↗