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

Abstract

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.

Figure 1

Haematoxylin-eosin staining in tibialis anterioris (TA) muscle sections from a K⁺-depleted rat and a normokalemic rat. (a) Haematoxylin-eosin staining revealed the abnormal presence of large subsarcolemmal vacuoles in the type II fibres of TA muscle from a K⁺-depleted rat (magnification × 10). (b) The vacuoles were found in the type II fibres as demonstrated by the ATPase (pH 4.3) staining, which was strong in the type I fibres (magnification × 20). (c) No vacuoles were observed in the type II fibres of muscle section from a normokalemic rat (magnification × 10).

Figure 2

Effects of the in vivo treatments of K⁺-depleted rats for 10 days with 5.6 mg kg⁻¹ day⁻¹ of acetazolamide, dichlorphenamide and bendroflumethiazide on vacuole formation in skeletal muscle. (a) Tibialis anterioris (TA) muscle sections from a K⁺-depleted rat treated with acetazolamide. No vacuoles were found in this muscle section following acetazolamide treatment suggesting that this drug prevented vacuole formation in the K⁺-depleted rats (magnification × 10). (b and c) In contrast, vacuoles were found in TA muscle sections from K⁺-depleted rats treated with bendroflumethiazide (b) or dichlorphenamide (c) indicating that these drugs were not effective in preventing the vacuolar myopathy in this animal model of hypokalemic periodic paralysis (magnification × 10).

Figure 3

NADH-tetrazolium reductase (NADH-TR) staining of flexor digitorum brevis muscle sections from K⁺-depleted rats treated for 10 days with 5.6 mg kg⁻¹ day⁻¹ of (a) acetazolamide and (b) dichlorphenamide. Tubular aggregates were observed in the type II fibres of muscles from K⁺-depleted rats, as demonstrated by the characteristic multifocal accumulation of materials in proximity of the sarcolemma stained by NADH-TR (magnification × 10).

Figure 4

In vitro effects of dichlorphenamide (DCP, 0.01–0.1 mM), bendroflumethiazide (BFT, 0.01–0.1 mM), acetazolamide (ACTZ, 0.01–0.1 mM) and cinnamate (Cinn., 10 mM) on lactate release from tibialis anterioris muscle of normokalemic rats. Lactate production and release were measured after incubating the muscles for the first hour in 0.5 mM K⁺ solution and during the second hour in normal Ringer solution containing 5 mM K⁺. The drugs under investigation were added in the normal Ringer solution during the second hour of the total incubation time. The data were compared with those obtained with the contralateral muscles of the same rats, used as controls, that were exposed to the same protocols but did not receive any drug treatment. ^*Data significantly different from that of the contralateral muscles of the same rats (P<0.05).