Increased membrane permeability to chloride in Duchenne muscular dystrophy fibroblasts and its relationship to muscle function.

Abstract

Previous studies have suggested an abnormality in Cl-metabolism in Duchenne muscular dystrophy (DMD) fibroblasts. In order to further characterize this abnormality, we have studied mCl-distribution and permeability in 11 DMD and 12 normal fibroblast lines. Under steady-state conditions Cl-efflux in fibroblasts is observed to be biphasic, revealing the presence of two major subcellular compartments. Each compartment contains approximately half of the cellular C1-. The faster of the two observed efflux components is significantly higher in DMD than in control fibroblasts (P < 0.001). To determine the results of a similar increase in Cl-permeability on skeletal muscle action potentials, we have simulated the effects of increased Cl-conductance on muscle by using a computer model. Effects on the simulated action potential include lower rates of membrane depolarization, lower overpotential, longer duration, and lower input resistance. These effects are similar to those actually observed in DMD muscle.Duchenne muscular dystrophy (DMD) is the most serious and rapidly progressive of the muscular dystrophies. Inherited as an X-linked recessive trait, the disease affects the skeletal muscle of boys in early childhood. Affected muscles show pseudohypertrophic changes secondary to the appearance of fat cells, macrophages, and fibroblasts between the necrotic muscle fibers (1). Ultrastructural changes in organelles within the fibers are suggestive of a compromised membrane. In the context of these pervasive cellular changes it has been difficult to identify the inherited biochemical abnormality. Recent investigations have, therefore, included studies of tissues such as erythrocytes, lymphocytes, and fibroblasts, which are not directly involved in the pathology of the disease (2-5).We have described three abnormalities in the lysosomes of DMD fibroblasts: (i) a 7-fold increase of cytoplasmic lamellar bodies (6, 7); (ii) decreased activity of dipeptidyl aminopeptidase I, a lysosomal proteolytic enzyme; and (iii) decreased structure-linked latency of this enzyme. Decreased structurelinked latency of lysosome-bound dipeptidyl aminopeptidase I is correlated with a decrease in the apparent K1/2 (the concentration having half-maximal effect) for Cl1 of this chloride-requiring enzyme. This difference in K1/2 is present only in intact lysosomes and disappears when the lysosomal membrane is disrupted (8). The requirement for an intact membrane and the correlation between latency and K112 for Cl-suggested that increased chloride permeability might be an important part of the etiology of DMD (9). In this study we 4732The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Purification and characterization of two Cl−‐activated aminopeptidases hydrolysing basic termini from human skeletal muscle

“…These enzymes may act primarily as dipeptidases, with a very restricted capacity for the degradation of naturally occurring oligopeptides. With the exception of the greater increase in activity in the presence of 0.15 M C1-and Br-anions, which we have observed with I and 11, the characteristics of these enzymes appear to correspond reasonably closely with those of the so-called aminopeptidase B previously described in other species and/or tissues 12, 7, 81. This paper describes the purification and characterization of two C1--activated aminopeptidases hydrolysing basic termini from human skeletal muscle, as part of a systematic investigation of the role of aminopeptidases in the protein turnover of normal and diseased muscle; it is of interest that Pato et al [9] have recently suggested that there is an increased permeability of the muscle membrane to C1-anions in muscular dystrophy. Table 5.…”

Altered chloride metabolism in cultured cystic fibrosis skin fibroblasts.

“…1; for each pair of cell cultures the number of experiments is given in parentheses. Data were analyzed assuming two parallel cellular compartments from which efflux occurred by first-order kinetics (7,9 this day-to-day variation, in any single experiment CF and N fibroblasts had comparable 3-O-MeGlc contents, corresponding to mean values of 4.96 and 5.20 jl/mg of protein. Therefore, because cell volume does not differ between CF and control fibroblasts, the lowered 36CL-capacity of CF cells corresponded to a lowered overall Cl-concentration.…”

“…Such kinetic analysis cannot identify the biological equivalents of compartments A and B without further work, nor can one assume that compartments identified by studies of 36CL-efflux correspond to the compartments (A' and B') found by analysis of 22Na+ efflux. Nevertheless, it is customary to assume that the more rapidly emptying compartment represents cytosol and that more slowly turning-over compartments reflect one or more subcellular organelle(s) or perhaps bound material (7,9,10). In this regard, it should be noted that the values derived in this analysis describe isotope efflux from parallel and independent compartments, and for simplicity we have tabulated the data in this way.…”

“…Chloride efflux was estimated under steady-state conditions as described by Pato et al (9) (10)(11)(12)(13)(14)(15) ,uCi/ml, 61-91 ptCi/mmol; New England Nuclear or Amersham) and 36C1-(10 tCi/ml, 72 pACi/mmol) during the loading period. 22Na' was determined in a y counter, and 36C1-was measured by liquid scintillation spectroscopy, with corrections for the contribution by 22Na+.…”

“…Explants were grown in 60-mm culture dishes (Falcon) using minimal essential medium with nonessential amino acids (GIBCO), 15% fetal calf serum (KC Biological, Lenexa, KS), 2 mM L-glutamine, penicillin (100 units/ml), and streptomycin (100 Ag/ml). After [6][7][8][9] days before an experiment and were fed every 2-3 days thereafter. Assays were performed using confluent cultures.…”