Increased sarcolemma chloride conductance as one of the mechanisms of action of carbonic anhydrase inhibitors in muscle excitability disorders.

To get insight into the mechanism of action of carbonic anhydrase inhibitors (CAI) in neuromuscular disorders, we investigated effects of dichlorphenamide (DCP) and acetazolamide (ACTZ) on ClC-1 chloride channels and skeletal muscle excitability. We performed patch-clamp experiments to test drugs on chloride currents in HEK293T cells transfected with hClC-1. Using the two-intracellular microelectrode technique in current-clamp mode, we measured the effects of drugs on the resting chloride conductance and action potential properties of sarcolemma in rat and mouse skeletal muscle fibers. Using BCECF dye fluorometry, we measured the effects of ACTZ on intracellular pH in single rat muscle fibers. Similarly to ACTZ, DCP (100 µM) increased hClC-1 chloride currents in HEK cells, because of the negative shift of the open probability voltage dependence and the slowing of deactivation kinetics. Bendroflumethiazide (BFT, 100 µM), structurally related to DCP but lacking activity on carbonic anhydrase, had little effects on chloride currents. In isolated rat muscle fibers, 50-100 µM of ACTZ or DCP, but not BFT, induced a ~ 20% increase of the resting chloride conductance. ACTZ reduced action potential firing in mouse muscle fibers. ACTZ (100 µM) reduced intracellular pH to 6.8 in rat muscle fibers. These results suggest that carbonic anhydrase inhibitors can reduce muscle excitability by increasing ClC-1 channel activity, probably through intracellular acidification. Such a mechanism may contribute in part to the clinical effects of these drugs in myotonia and other muscle excitability disorders.

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.

Evaluation of the therapeutic potential of carbonic anhydrase inhibitors in two animal models of dystrophin deficient muscular dystrophy.

Duchenne Muscular Dystrophy is an inherited muscle degeneration disease for which there is still no efficient treatment. However, compounds active on the disease may already exist among approved drugs but are difficult to identify in the absence of cellular models. We used the Caenorhabditis elegans animal model to screen a collection of 1000 already approved compounds. Two of the most active hits obtained were methazolamide and dichlorphenamide, carbonic anhydrase inhibitors widely used in human therapy. In C. elegans, these drugs were shown to interact with CAH-4, a putative carbonic anhydrase. The therapeutic efficacy of these compounds was further validated in long-term experiments on mdx mice, the mouse model of Duchenne Muscular Dystrophy. Mice were treated for 120 days with food containing methazolamide or dichlorphenamide at two doses each. Musculus tibialis anterior and diaphragm muscles were histologically analyzed and isometric muscle force was measured in M. extensor digitorum longus. Both substances increased the tetanic muscle force in the treated M. extensor digitorum longus muscle group, dichlorphenamide increased the force significantly by 30%, but both drugs failed to increase resistance of muscle fibres to eccentric contractions. Histological analysis revealed a reduction of centrally nucleated fibers in M. tibialis anterior and diaphragm in the treated groups. These studies further demonstrated that a C. elegans-based screen coupled with a mouse model validation strategy can lead to the identification of potential pharmacological agents for rare diseases.

Acetazolamide prevents vacuolar myopathy in skeletal muscle of K(+) -depleted rats.

BACKGROUND AND PURPOSE: Acetazolamide and dichlorphenamide are carbonic anhydrase (CA) inhibitors effective in the clinical condition of hypokalemic periodic paralysis (hypoPP). Whether these drugs prevent vacuolar myopathy, which is a pathogenic factor in hypoPP, is unknown. The effects of these drugs on the efflux of lactate from skeletal muscle were also investigated. EXPERIMENTAL APPROACH: For 10 days, K(+)-depleted rats, a model of hypoPP, were administered 5.6 mg kg(-1) day(-1) of acetazolamide, dichlorphenamide or bendroflumethiazide (the last is not an inhibitor of CA). Histological analysis of vacuolar myopathy and in vitro lactate efflux measurements were performed in skeletal muscles from treated and untreated K(+)-depleted rats, and also from normokalemic rats. KEY RESULTS: About three times as many vacuoles were found in the type II fibres of tibialis anterioris muscle sections from K(+)-depleted rats as were found in the same muscle from normokalemic rats. In ex vivo experiments, a higher efflux of lactate on in vitro incubation was found in muscles of K(+)-depleted rats compared with that found in muscles from normokalemic rats. After treatment of K(+)-depleted rats with acetazolamide, the numbers of vacuoles in tibialis anterioris muscle decreased to near normal values. Incubation with acetazolamide in vitro inhibited efflux of lactate from muscles of K(+)-depleted rats. In contrast, bendroflumethiazide and dichlorphenamide failed to prevent vacuolar myopathy after treatment in vivo and failed to inhibit lactate efflux in vitro. CONCLUSIONS AND IMPLICATIONS: Acetazolamide prevents vacuolar myopathy in K(+)-depleted rats. This effect was associated with inhibition of lactate transport, rather than inhibition of CA.

Role of physiological HCO3-buffer on intracellular pH and histamine release in rat peritoneal mast cells.

The purpose of this study was to examine how intracellular pH (pHi) regulation and histamine release are affected by HCO3- in rat peritoneal mast cells. The pHi was measured using the pH-sensitive dye 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). We observed a pHi of 6.88+/-0.012 (n=24) in resting mast cells exposed to a HEPES buffer (pH 7.4), but a sustained drop of 0.21 pH units to 6.67+/-0.015 (n=23) when we exposed the mast cells to a HEPES/HCO3- buffer equilibrated at all time with 5% CO2 (pH 7.4). This fall in pHi is inhibited by the carbonic anhydrase inhibitor dichlorphenamide and is Na+-independent, indicating the involvement of Na+-independent Cl-/HCO3- exchange activity. Furthermore removal of external Cl- in the presence but not in the absence of HCO3- reversed the Cl-/HCO3- exchange and induced an alkaline load. The recovery from this alkaline load was dependent on external Cl- but independent of Na+. Both the alkalinization and the recovery were inhibited by the anion transport inhibitor 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). In addition, 36Cl- uptake measurements confirm the presence of a Cl-/HCO3- exchanger. Histamine release stimulated by antigen and compound 48/80 was substantially reduced in the presence of HEPES/ HCO3- buffer (pHo 7.4, pHi 6.66). Histamine release was increased, however, when pHi was clamped to 6.66 in HCO3--free media (pHo 6.9). We conclude that: (1) Na+-independent Cl-/HCO3- exchange determines steady-state pHi in rat peritoneal mast cells; and (2) the reduction in histamine release observed in the presence of HCO3- is not due to its effect on pHi per se, but rather on other changes in ion transport.

Carbonic anhydrase inhibitors are antiarthritic in the rat.

Adjuvant-induced arthritis in rats was attenuated by the therapeutic administration of carbonic anhydrase inhibitors. Female Lewis rats with established disease were treated daily (day 18 through day 50) with various carbonic anhydrase inhibitors; oedema and joint integrity (X-ray) were determined post-treatment. Acetazolamide, ethoxzolamide, methazolamide, and dichlorphenamide reduced paw oedema and attenuated the deterioration of the joints of rats with adjuvant arthritis. However, no carbonic anhydrase inhibitor tested possessed significant, acute, anti-inflammatory activity in the carrageenan-paw oedema test. The activity of carbonic anhydrase inhibitors in the chronic model of inflammation may be due to their reported inhibition of bone resorption.

Delayed protein digestion in the alligator following carbonic anhydrase inhibition.

Small alligators were injected with a carbonic anhydrase (CA) inhibitor (dichlorphenamide) and a day later they were fed large amounts of lean beef. The rate of protein digestion was determined and compared with that in controls fed the same amount. The controls began digestion at once, while in the CA-inhibited ones, digestion was blocked for over 10 hr. The inhibitor decreased gastric HCl production to one-third that in controls and delayed activation of pancreatic and intestinal proteases. After 15 hr, digestion began in the inhibited group and proceeded to completion. Once digestion began, further injections of the inhibitor could not block it. It seems that CA inhibition blocks digestion (for a time) by reducing HCl synthesis. NaHCO3 to neutralize the acid chyme in the intestine, was derived from that in the plasma.

Topical ocular hypotensive activity and ocular penetration of dichlorphenamide sodium in rabbits.

A single topical instillation (50 microliter) of the water soluble sodium salt of dichlorphenamide (10%) produced a pronounced and prolonged lowering of intraocular pressure (IOP), compared to suspensions of its free acid, in rabbits with alpha-chymotrypsin-induced ocular hypertension. In normal rabbits, instillation of dichlorphenamide sodium also produced a markedly elevated drug aqueous humor level relative to instillation of its free acid, indicating enhanced penetration into the eye. The IOP response after ocular instillation of dichlorphenamide sodium was equivalent to that produced by oral administration of 6 or 18 mg/kg dichlorphenamide sodium. Serum drug levels were lower and aqueous humor and iris-ciliary body levels were higher after instillation of dichlorphenamide sodium (10%) than after oral administration of 2 or 6 mg/kg. The data indicate that topically instilled dichlorphenamide sodium is capable of lowering IOP by a local action in the eye and provides a rationale for the potential utility of topical carbonic anhydrase inhibitors as a therapy for glaucoma in man.

Treatment of "permanent" muscle weakness in familial Hypokalemic Periodic Paralysis.

Three patients with Hypokalemic Periodic Paralysis (HOPP)-associated progressive interattack muscle weakness, who became unresponsive or worsened by acetazolamide, responded favorably to dichlorophenamide, a more potent carbonic anhydrase inhibitor. Dichlorophenamide in single-blind placebo-controlled trials, considerably improved functional strength in two of the patients and had a moderate but definite effect in the third. Muscle groups graded 4/5 (MRC scale)returned to normal; very weak (0-3/5) atrophic muscles, improved to a minor degree. In one patient with acetazolamide-resistant paralytic attacks, dichlorophenamide also diminished the frequency and severity of the acute attacks. Dichlorophenamide had, in the present study, less effect than acetazolamide in reducing serum HCO3(-) and elevating Cl-. Its effectiveness may be related to the degree of sensitivity of certain HOPP patients to alterations of Cl- and/or HCO3(-) serum levels or to a different action of the drug unrelated to carbonic anhydrase inhibition or acidosis. Dichlorophenamide should be considered as an alternate to acetazolamide in the treatment of patients with HOPP-associated interattack muscle weakness who have become unresponsive or worsened by acetazolamide.

Ocular hypotensive effects of carbonic anhydrase inhibitors in normotensive and glaucomatous Beagles.

Four carbonic anhydrase inhibitors (acetazolamide, dichlorphenamide, ethoxzolamide, and methazolamide) cause ocular hypotony in normotensive and glaucomatous Beagles. Four dosages of acetazolamide and methazolamide and three dosages of dichlorphenamide and ethoxzolamide were evaluated. The extent of ocular hypotony after these carbonic anhydrase inhibitors was usually greater in glaucomatous Beagles than it was in normotensive Beagles.

[The broncholytic effect of carboanhydrase inhibitors in subjects with healthy lungs (author's transl)]. / Die broncholytische Wirkung von Carboanhydrase-Hemmern bei Lungengesunden.

Carboanhydrase inhibitors are said to have a positive effect on respiratory insufficiency. We examined this, especially regarding the bronchial system. Actions and reactions of the human bronchial system can be followed by measurement of the anatomical dead space breath by breath. We have developed a new method which was tested by classical broncho-effective substances. Our experiments show in case of two men with healthy lungs that the three carboanhydrase inhibitors acetazolamide, methazolamide, and dichlorphenamide have a pronounced broncholytic effect. The effect is greater than that of fenoterol. In addition the bronchial peristalsis and the bronchial compliance are increased. In contrast to fenoterol carboanhydrase inhibitors have no cardiac side effect.