Increased endocytosis of acetylcholine receptors by dystrophic mouse myotubes in vitro

DEVELOPMENTAL

BIOLOGY

110, 362-368

(1985)

Increased Endocytosis of Acetylcholine Receptors Dystrophic Mouse Myotubes in Vifro G. Cossu, Istituto

F.

di Istologia

Received

May

EUSEBI,

M. I.

ed Embriologia

SENNI, genera&

22, 1984; accepted

in revised

AND

M.

MOLINARO

Universitci form

by

di Rma,

March

Italy

15, 1985

Multinucleated myotubes, grown in vitro from satellite cells of dystrophic mice (C57BL/&I/dydy) exhibit a reduced sensitivity to ACh. This reduction correlates with a reduced density of ‘%I-ol-bungarotoxin (‘=I-BTX) binding sites on the surface of dystrophic myotubes. Denervated adult muscle fibers from dystrophic mice respond to Ach similarly to denervated normal muscle fibers. Furthermore, cultured dystrophic myotubes, treated with a brain extract which induces AChR clusterization, still show an impaired response to ACh and reduced ‘%I-BTX binding. Thus AChR function appears altered in dystrophic muscle cells in culture while it appears normal in dystrophic adult muscle, regardless of whether the receptors are dispersed on the membrane or clustered at the junctional site. Metabolic studies on the reduced AChR level in dystrophic myotubes revealed a dramatically reduced half-life (2 vs 10 hr) while the rate of synthesis was unchanged. An increased rate of internalization of AChR was observed in dystrophic myotubes with a corresponding relative increase of the “hidden AChR pool,” which could be partially reduced by agents which disrupt the cytoskeleton. No structural alterations could be detected on the AChR molecule as its sedimentation coefficient and subunit composition appeared identical between normal and dystrophic myotubes. Thus the increased turnover of AChR in dystrophic myotubes either reflects subtle alterations of the molecule or a more generalized increase of endocytosis in this form of myopathy. Q 1985 Academic press, IX. INTRODUCTION

here the existence of a reduced half-life and increased internalization of this molecule by the dystrophic myotube membrane.

We reported previously the existence of a reduced acetylcholine (ACh) sensitivity and reduced expression of acetylcholine receptor (AChR) in dystrophic mouse myotubes in vitro (Cossu et al., 1984). This finding was unsuspected, since it is known that the sensitivity to the transmitter in the synaptic region of dystrophic muscle fibers in vivo is unchanged (Harris and Ribchester, 1979). Such apparently contradictory results might be explained by the existence of different forms of AChR in embryonic and adult muscle as suggested by several recent reports (Pumplin and Fambrough, 1982). Alternatively, the difference might be explained by the different membrane localization of the AChRs. The receptor present in myotubes is uniformly distributed on the surface of the cell and is free of lateral diffusion within the plane of the membrane whereas the receptor of adult muscle fibers is clustered at the synaptical junction where it interacts with the basal lamina outside (Anderson and Fambrough, 1983) and the cytoskeleton inside (Connoly, 1984) the membrane. In this paper we have investigated whether the altered features of dystrophic AChRs in vitro depend on their distribution on myotube surface or on alterations specific of an “embryonic” form of the molecule. We have also investigated the mechanism of the reduced steady-state level of the dystrophic AchR and report 0012-1606/85 Copyright All righis

$3.00

0 1985 by Academic Press. Inc. of reproduction in any form reserved.

MATERIALS

AND

METHODS

Cell cultures. Myogenic cells were isolated from the posterior legs of l- to 2-months-old dystrophic (C57 BL/GJ/dydy) or control (C57 BL/GJ) mice as detailed elsewhere (Cossu et al, 1980, 1983). Cultures were grown in minimum essential medium (GIBCO), supplemented with 10% horse serum, and 3% embryo extract. For biochemical studies, cytosine arabinoside (Ara C, Sigma Co.) at a final concentration of 2.5 pg/ml was added from Day 3 to Day 5 of culture. When indicated brain extract (Jesse11 et ah, 1979) was added to Day 5 cultures at a final concentration of 50 Ill/ml and cells were studied at Day 7. Electrophysiology. The ACh sensitivity of cells was determined by iontophoretic application of ACh and intracellular recording of the elicited depolarization. The ACh sensitivity is expressed in terms of the millivolts depolarization produced per nanocoulomb of iontophoretic charge (mV/nC). ACh pipettes filled with 1 M AChCl had resistance of 40-90 MQ. Iontophoretic current pulses 1 X lo-’ to 5 X lo-’ A and duration l500 msec were employed in measurements of sensitivity of cultured myotubes or of soleus fibers denervated for 5 days when the ACh sensitivity plateaus (Lomo, 1976). 362

COSSU

ET AL.

AChR

Turnover

Latency between onset of iontophoretic pulse and ACh response was less than 5 msec. Baking current, 3-8 nA, was used to avoid spontaneous ACh release. Determinations of ACh sensitivity of denervated fibers were carried out 3 mm from the Achilles tendon. For further details see Cossu et al. (1984). Assay for AChR. The number of specific lz51-bungarotoxin (lz51-BTX, Amersham, sp act 230 CVmmole) binding sites on the surface of myotubes was measured in triplicate cultures as described in detail before (Cossu et al., 1982); nonspecific binding was measured in sister cultures preincubated for 1 hr with 150 PLM D-tubocurarine (D-TC, Calbiochem) and was subtracted from the values obtained. In order to measure the loss of specifically bound lz51-BTX (Devreotes and Fambrough, 19’75; Patrick et al., 197’7) a number of cultures were incubated for 30 min in the presence of lz51-BTX. After the incubation, cultures were washed ~3 with PBS. Complete medium was added and the dishes were returned to the incubator. At different intervals, two dishes (+one pretreated with D-TC) were analyzed for the residual radioactivity in the cell fraction and for the radioactivity released into the medium. In order to discriminate between AChR-‘251-BTX complexes, lz51-BTX, and [1251]tyrosine, aliquots of the medium were run on a 10 X l-cm column of Sephadex G-50. The column was eluted in PBS and 0.5-ml fractions were collected. The complex is excluded from the column, the free toxin is eluted with a K,, of 0.6, and [1251]tyrosine with the V,. When this analysis was performed with the cell extracts, cells were homogenized in a Teflon Potter homogenizer with buffer II (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM EDTA, 2 mM PMSF, 5 mM benzamidine, 5 mM IAA, 1% Triton X-100), centrifuged in a Beckman microfuge, and the supernatant was applied to the same G-50 columns used for medium analysis and eluted in buffer II. To evaluate the fraction of AChR-lz51-BTX complex exposed on the cell surface at any period of the turnover experiment, we treated myotubes, prelabeled with lz51-BTX for 20 min at 37°C with 0.125% Sepharose-bound trypsin (Sigma Co.) for 3 min at 37°C. After trypsin treatment, trypsin inhibitor (0.125%, Sigma Co.) was added to the cell suspension which was centrifuged at 1,000 rpm for 10 min. Radioactivity was measured separately in the pellet and in the supernatant. Such a low concentration of insoluble trypsin does not result in cell breakage (Marino et ah, 1980); however, not all surface molecules are necessarily released by the treatment. In the case of AChR, about 80% of specifically bound toxin could be released by this treatment. We therefore assumed that this method could give us an acceptable quantification of the rate

in

Dystrophic

Myotubes

363

of AChR disappearance from the cell surface. To measure nonsurface ACh receptors, cultures were incubated for 1 hr at 37°C with 0.2 ~Munlabeled BTX to saturate cell surface receptors. Sister cultures also received cyclohexymide (10 pg/ml) for 3 hr before the extraction, in order to deplete the cells of AchR precursor pool. Control cultures were left untreated in order to measure the total amount of AChR. The cells of all the different cultures were then collected and extracted in buffer II. The extracts were centrifuged for 5 min in a Beckman microfuge and the supernatants were incubated with lz51-BTX for 30 min at 37°C. After the incubation the AChR-lz51-BTX complexes were separated on columns of Sephadex G-50 in buffer II as described above. The rate of AChR synthesis was measured as described by Patrick et al. (1977). Briefly 0.1 1iV cold BTX was added to each culture for 30 min at 37°C. The cells were washed three times with PBS and returned to the incubator in complete medium. Half of the cultures had also received 50 pug/ml of cycloheximide (Chx). At different intervals after the first incubation, two dishes (+two preincubated with 50 pug/ml of Chx) were assayed for newly exposed AChR as described above. The results are expressed as percentage of the total surface lz51-BTX binding sites measured in sister cultures at the beginning of the incubation. Sedimentation analysis. Cultures were incubated with lz51-BTX as described above, washed four times with PBS, and then extracted with buffer II. The extract was centrifuged in a Beckman microfuge and an aliquot of the supernatant was layered onto a 5-20% sucrose gradient in buffer II, and centrifuged for 16 hr at 38,000 rpm in a Beckman SW 41 rotor. Fractions (0.5 ml) were collected and assayed for radioactivity. pGalactosidase (11.3 S) was used as a sedimentation marker. Analysis of metabolically labeled AChR. For pulselabeling of cellular proteins, cultures were incubated in methionine-free medium supplemented with 2% FCS for 2 hr at 37°C with 50 &i/ml of [35S]methionine (NEN spec act 20 Ci/mmole). After the pulse, cells were washed X3 with PBS, scraped from the dish, and collected by centrifugation. Purification procedure was essentially as described by Gotti et aZ. (1982). The pellet was homogenized in a Teflon Potter homogenizer in buffer I (50 mM Tris-HCI, pH 7.5, 50 mM NaCl, 5 mM EDTA, 2 mM PMSF, 5 mM benzamidine, 5 mlM IAA) and centrifuged in a Beckman microfuge. The pellet was extracted in buffer II, centrifuged again and the supernatant was incubated with either 50 pi/ml of Sepharose 4-B or with 50 PI/ml of Sepharose 4B to which Naja naja toxin had been covalently bound. An aliquot of the supernatant was also processed in the presence of 150 ~LMD-TC to evaluate nonspecific binding

364

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to Naja naja-Sepharose. The incubation was for 30 min at 4’C with occasional mixing. The beads were washed five times in buffer III (50 mM Tris-HCl, pH 7.5, 1 M NaCl, 5 mM EDTA, 2 mM PMSF, 5 mM benzamidine, 5 mM IAA, 1% Triton X-100), until no radioactivity was found in the wash. The beads were then boiled in 50 ~1 of sample buffer (2% SDS, 1% ,8mercaptoethanol), 50 mM Tris-HCl, pH 7.4) for 2 min. The supernatant was separated on a 8% SDS-polyacrylamide gel electrophoresis (Laemmly, 1970). After the run, the gel was treated with Enhance (New England Nuclear) and processed for fluorography. Autoradiography of AChR. In order to localize AChR on myotube surface, normal and dystrophic myotubes at Day 5 of culture received 50 pi/ml of brain extract (Jesse11 et aZ., 1979) or PBS. At Day 7 of culture, cells were incubated with lz51-BTX as described above and processed for autoradiography as described (Cossu et al., 1980). Exposure time ranged between 4 and 7 days. RESULTS

ACh receptors were induced to form clusters on the surface of both normal and dystrophic myotubes by addition of a soluble fraction from mouse embryo brain (Jesse11 et al., 1979). Within 2 days of treatment, dystrophic myotubes developed AChR clusters although in lower number than normal myotubes. Furthermore both the particle density of the clusters and their distribution on the surface appeared similar between normal and dystrophic cells. We therefore investigated whether dystrophic myotubes, treated with brain extract, would regain a normal sensitivity to iontophoretically applied ACh and a normal BTX binding site density. Table 1 summarizes the results and shows that Ach sensitivity of dystrophic myotubes that were

EFFECT

TABLE 1 OF BRAIN EXTRACT ON ACETYLCHOLINE SENSITIVITY IN NORMAL AND DYSTROPHIC MYOTUBES

Cultures Normal myotubes (N = 16) Dystrophic myotubes (N = 19) Normal myotubes +BE(N=12) Dystrophic myotubes + BE (N = 15)

RP (mV)

ACh sensit mV/nC

‘%I-BTX bound fmol/mg protein

-48.4 + 2.1

17.8 f 2.9

42 I? 15

-45.3 f 1.9

5.3 + 1.7

-47.9 f 3.1

17.5 + 2.4

-43.1 + 2.3

4.1 k 1.5

ilk

5

78 T!z12 35*

7

Note. Brain extract (BE) treatment was as described under Materials and Methods. Binding data are the mean k SD of two experiments performed in triplicate cultures.

VOLUME 110,1985

treated with brain extract was not increased with respect to untreated dystrophic myotubes. In both cases sensitivity was strongly reduced as compared to both control and brain-extract-treated normal myotubes. Furthermore, even though brain extract increased lz51-BTX binding about two times in normal myotubes and about four times in dystrophic myotubes, the difference in the level of lz51-BTX binding between normal and dystrophic cells remains significant. Hind limbs were denervated in four normal and four dystrophic mice. Five days after denervation ACh sensitivity could be measured over the entire muscle fibers surface in the soleus thus indicating the appearance of extrajunctional ACh receptors (Lomo, 1976). ACh sensitivity was found to be slightly higher in dystrophic denervated soleus than in its normal denervated counterpart, the difference being statistically not significant (Table 2). Thus AChR, whether dispersed or clustered, is altered in dystrophic cultured muscle cells, while it is normal in dystrophic muscle fibers, regardless of its synaptic (Harris and Ribchester, 1979) or extrasynaptic localization. In order to investigate the possible mechanism responsible for the reduced level of AChR density on the surface of dystrophic myotubes, we studied the rate of synthesis of the receptor, measured as appearance of new lz51-BTX binding sites after presaturation with cold BTX. Figure 1 shows that the rate of AChR synthesis is the same for both normal and dystrophic myotubes, at least for the first 4 hr of measurement. The degradation of AChRs in normal and dystrophic myotubes was measured by analyzing the residual cellassociated lz51 radioactivity, at different intervals after an initial 1-hr incubation with radiolabeled toxin. Figure 2 shows that, in agreement with previous data from mouse cells (Patrick et al., 1977), the AChR in normal myotubes was degraded with a half-life of 910 hr. The rate of disappearance of the receptor was linear with time indicating a first order process. The degradation of the AChR was much faster in dystrophic myotubes, since about 70% of the bound radioactivity was lost within the first 2 hr of incubation. After this initial loss, which resulted in a half-life of 1.30-2 hr, the residual radioactivity decayed at a lower rate, comparable with that of normal myotubes. Two different mechanisms might explain this increased degradation of AChR in dystrophic myotubes: (1) increased endocytosis followed by lysosomal degradation; (2) proteolytic cleavage of the AChR polypeptides, followed by shedding of the extracellular domains. Although the first mechanism has been shown to occur in normal myotubes, it is known that several cellular components are lost through the membrane of the dystrophic muscle fibers. In order to discriminate be-

COSSU ET AL. TABLE

AChR

Turnover

in Dystrophic

365

Myotubes

2

EFFECTOF DENERVATIONON ACETYLCHOLINE RESPONSEINNORMAL AND DYSTROPHICSOLEUS MUSCLE ACh RP (mV) Normal soleus (N = 12) Dystrophic soleus (N = 10)

-56.2 -58.3

f 2.2 _+ 2.1

sensitivity mV/nC

176.5 f 59 237.9 f 84

tween these two possible mechanisms we have examined the release of macromolecular components into the culture medium at various periods after a initial incubation with 1251-BTX and we have found such accumulation to be negligible at least up to 6 hr of observation (data not shown). At the same time, cells were treated with trypsin for 3 min: such treatment resulted in detachment of cells from substrate. Radioactivity was measured separately in the pelleted cells and in the supernatant. Figure 3 shows the existence of a dramatic difference in the rate of internalization of the radioactivity from cell surface (measured as intracellular accumulation of trypsin-resistant AChR-‘251-BTX complex) between normal and dystrophic cells. At the end of the incubation period with ‘251-BTX, about 20% of radioactivity (receptor-toxin complexes) could not be solubilized from the membrane of both normal and dystrophyc

.P .r 40P .-c s zo-

\\ \\ \ +------L-

1

2

4

FIG. 1. Appearance of newly synthesized AChRs on the cell surface. Normal (0) and dystrophic (0) cultures, were incubated X30 min in the presence of 0.1 gM cold BTX, washed X3 with PBS, and returned to the incubator. At the indicated periods cultures were incubated with ‘%I-BTX as described under Materials and Methods. Data are expressed as percentage of total surface receptors (which were measured in untreated sister cultures) and represent the mean + SD of two separate experiments on duplicate cultures. The level of binding in sister cultures, treated with cycloheximide (50 pg/ml), is represented by (A).

6

8

hours FIG. 2. Loss of specifically bound ‘“I-BTX from the surface of myotubes. Normal (0) and dystrophic (0) cultures were incubated with iZI-BTX as described under Materials and Methods. After the incubation, cells were washed X3 with PBS and cell associated radioactivity was measured at the indicated periods. Data are the mean 2 SD of three separate experiments on triplicate cultures.

2

hours

-_ -_ --_ + 9

4 hours

6

FIG. 3. Trypsin-sensitivity of specifically bound ‘“I-BTX. Normal (0) and dystrophic (0) cultures were incubated with ‘%I-BTX as described under Materials and Methods, washed ~3 with PBS, and returned to the incubator. At each indicated period, cultures were treated with Sepharose-hound trypsin as described (Marino aL, 1980). After trypsinization, cells were collected by centrifugation and radioactivity was measured separately in the pellet and in the supernatant. Data are expressed as the percentage of total residual radioactivity which could not be solubilized by trypsin and represent the mean of two experiments on duplicate cultures.

et

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DEVELOPMENTAL

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myotubes. Within 6 hr after ‘=I-BTX removal, about 45% of total residual radioactivity had been internalized in normal myotubes, while more than 90% of radioactivity had become intracellular in dystrophic myotubes. The increased internalization of the AChR might result in an increased proportion of the nonsurface receptor pool. Table 3 shows that in fact the nonsurface binding sites of dystrophic cells represent a higher proportion of total binding sites as compared to normal cells. If, however, the precursor pool is depleted by a cycloheximide treatment, the nonsurface binding sites (hidden pool) of dystrophic cells become a significantly higher proportion of total binding sites. In order to dissect the endocytotic pathway of AChR, we treated both normal and dystrophic myotubes with cytochalasin B and with chloroquine. Cytochalasin B blocks endocytosis by disrupting the cytoskeleton (de Petris, 19’75), whereas chloroquine acts on lysosomal proteases, thus altering membrane traffic between lysosomes, Golgi, and the plasma membrane (GonzalesNoriega et al., 1930). Myotubes were treated with the drugs for 4 hr because the turnover of the AChR in dystrophic myotubes is rapid enough to detect specific effects before the occurrence of general cytotoxic effects. Table 4 shows that while chloroquine treatment resulted in a reduced level of AChR on the membrane of both normal and dystrophic cells, cytochalasin B exerted a differential effect on normal and dystrophic myotubes. It slightly inhibited (by 5%) the level of ‘%IBTX binding in normal myotubes but increased binding to 160% of control in dystrophic myotubes. We investigated whether the abnormal metabolism of the AChR in dystrophic myotubes could be explained by structural alterations of the receptor itself. Figure 4 shows that the migration of the AChR from dystrophic myotubes on a sucrose gradient is identical to that of AChR from normal myotubes. Myotubes from both normal and dystrophic mice

SUBCELLULAR

Cultures Normal myotubes Dystrophic myotubes

TABLE DISTRIBUTION

Total binding sites

62 26

3 OF ACh

VOLUME

110,1985

EFFECT

TABLE 4 OF CHLOROQUINE AND CYTOCHALASIN SPECIFIC 1251-BTX BINDING

Cultures Normal myotubes Dystrophic myotubes

Control

Chloroquine

Cytochalasin

22 (56)

39 (100) 10 (100)

B ON

6 (60)

B

37 (95) 16 (160)

Note. Cultures were treated with cytochalasin B (10 rg/ml) and with chloroquine (25 a) for 4 hr before AChR level measurement. Data are expressed as femtomoles ‘%I-BTX bound per milligrams of protein and, in parentheses, as percentage of control.

were metabolically labeled with [YS]methionine and newly synthesized AChR was purified by affinity chromatography on Naja naja-Sepharose. AchR subunits were resolved on 8% SDS-PAGE. Figure 5 shows that the relative proportions and mobility of the resolved polypeptides were similar between normal and dystrophic cells. Occasionally, reduced proportions of larger polypeptides were noted in the dystrophic-cell-derived AChR. These variable results are likely due to the occurrence of proteolytic degradation of the receptor subunits during purification. It should be noted that the identification of the subunits is based only on relative mobility on SDS-PAGE and specific-binding

RECEPTORS

Nonsurface binding sites

21 (33.8%) 16 (61.5%)

Nonsurfacenonprecursor binding sites

8 (12.9%) 7 (31.8%)

Note. Total, nonsurface and nonsurface-nonprecursor binding sites were measured as described under Materials and Methods. Numbers in parentheses indicate the percentage of total receptor represented by each class. Aspecific binding was measured by incubation of the extracts in the presence of 2 p&f BTX and was subtracted to the values obtained.

!i

10

fraction

1'5

2'0

number

FIG. 4. Sedimentation analysis of acetylcholine receptor. Normal (0) and dystrophic (0) cultures were incubated with 1251-BTX as described under Materials and Methods, washed X3 with PBS, extracted with buffer II (see Materials and Methods), and an aliquot of the extract was centrifuged at 38,000 rpm X 16 hr on a 5-20% sucrose gradient in buffer II. Fractions (0.5 ml) were collected from the bottom of the tube. The arrow represents the migration position of /3-galactosidase (11.3 s).

COSSU ET AL.

e

AChR

Turnover

+

-2oo-

-68

-

FIG. 5. SDS-polyacrylamide gel electrophoresis of AChR subunits. Cultures were labeled metabolically with [3SS]methionine; dystrophic (a, c, e) and normal (b, d, f) extracts were prepared as described under Materials and Methods and incubated with plain Sepharose 4-B (a, b), with Nuju nuju-Sepharose (c, d) or with Nuju najuSepharose in the presence of 150 PM D-TC (e. f). Bound polypeptides were separated on 8% SDS-PAGE. Molecular weight markers were from Pharmacia.

to Naja naja-Sepharose, and should be therefore considered tentative. However, since no differences could be detected at this level of resolution, we have not pursued more stringent criteria of analysis. DISCUSSION

The data presented in this paper show the existence of an altered metabolism of AChR in dystrophic myotubes in vitro, at variance with the situation in vivo, where no alterations of AChR were reported (Harris and Ribchester, 1979). These results are consistent with the idea that at least two different forms of AChR might exist in muscle cells: one embryonic and one adult form, only the first appearing altered in dystrophic muscle cells. AChRs are dispersed on the surface of aneurally cultured myotubes, while they are clustered at the synaptic site in adult fibers, where stabilization of the molecule occurs through interactions with extracellular as well as cytoskeletal components (Bloch and Geiger, 1980; Anderson and Fambrough, 1983; Connolly, 1984). This difference is unlikely to explain the alteration of AChR in dystrophic myotubes in vitro: in fact, if the situation is reversed (by brain-extract-induced clusterization of embryonic AChR or by denervation of adult muscle) embryonic AChRs still respond abnormally to ACh and bind reduced amounts of ‘%I-BTX while extrajunctional dispersed adult AChRs still appear

in

Llystrophic

Myotubes

367

identical to their normal counterparts (Tables 1 and 2). The reduced AChR concentration on the surface of dystrophic myotubes is due to increased disappearance of the receptor from the membrane of the muscle cells (Fig. 2). We do not presently understand whether this altered turnover of the AChR is selective or whether it might reflect a generalized increased degradation of dystrophic cell membrane proteins. However, other surface proteins such as acetylcholinesterase do not show any quantitative alteration in vitro, and therefore should not be reduced in concentration on the memthe total brane (Poiana et al., 1985); furthermore amount of sugar and amino acid incorporation into membrane proteins is identical between normal and dystrophic muscle cells even after 24-hr pulses, whereas an increased degradation should reduce the level of incorporation in dystrophic cells (Cossu and Boitani, unpublished) and, finally AChR is normal in the membrane of adult muscle fiber, which is known to be severely damaged, while is altered in the membrane are much of muscle cells in vitro, where alterations harder to demonstrate. Thus the AChR in dystrophic myotubes is synthesized, assembled, and inserted into the plasma membrane at a presumable normal rate (Fig. l), but then it is lost from the membrane at faster rate than its normal counterpart. The mechanism of such degradative process is not presently understood. The experiment reported in Fig. 3 indicates that internalization into the cell rather than shedding into the extracellular environment is the route of degradation of the AChR in dystrophic cells. The rate at which the AChR disappears from the surface (measured as resistance to trypsin treatment) is even faster than the overall rate of decay and therefore causes the transient accumulation of an intracellular (trypsin-inaccessible) macromolecular AChR-toxin complex. The existence of a “hidden” pool of AChR has been initially postulated by Devreotes and Fambrough (1975). These receptors may derive from the plasma membrane or from the precursor pool, do not reappear on the surface, and are probably degraded in the lysosomes. Interestingly, a short treatment with cytochalasin B, which interferes with endocytosis by disruption of the cytoskeleton, increased the level of ‘251-BTX binding up to 160% of the control, while it does not affect binding (95% of control) in normal cells. This differential effect might be explained by the different half-life of AChR in normal and dystrophic cells. The proportion of receptors which are internalized during a 4-hr period is certainly smaller in normal than in dystrophic cells and therefore it is reasonable that the latter cells should be more significantly affected by inhibition of endocytosis through disruption of the cytoskeleton. Inhibition of lysosomal

368

DEVELOPMENTAL BIOLOGY

proteases by chloroquine treatment did not cause any differential effect on the level of AChR in normal and dystrophic cells. Thus it is possible that increased endocytosis of AChR (blocked at least partially by disruption of the cytoskeleton), causes the accumulation of a surface-derived AChR into a prelysosomal pool. On the basis of the available data it is not possible to decide whether this increased hidden pool of AChR in dystrophic myotubes is a simple consequence of an increased rate of AChR internalization, or whether it reflects some basic abnormality of membrane traffic within the dystrophic cells. We favor the first possibility because of the selectivity of such alteration, although the rate of synthesis, assembly, intracellular processing, and turnover are presently known for very few proteins in muscle. In order to identify a possible structural alteration of the dystrophic AChR, which might explain its altered stability on the membrane, we have analyzed the sedimentation profile and the subunit composition of the molecule. No differences in sedimentation values, number of subunits, or subunit mobility in SDS-PAGE were observed. Since the total number of cells that one can obtain from primary cultures of normal and dystrophic muscle is limited, more detailed biochemical analysis have been difficult to pursue. For example it has been reported that tunycamicin-induced inhibition of glycosylation alters the metabolism of AChR (Prives and Olden, 1980). If an alteration in the glycosylation pattern of AChR exists, it would probably be much more subtle than the lack of all N-linked carbohydrates, and therefore might not be sufficient to change the migration in SDS-PAGE, but might, for example, alter the binding of AChR to immobilized lectins. However, preliminary experiments indicate that AChRs from both normal and dystrophic cells bind only to Con-A Sepharose with similar affinity, in agreement with previous data on a muscle cell line (Patrick et al, 1977). In conclusion, the increased degradation of AChR from membranes of dystrophic myotubes, but not of adult dystrophic muscles, supports the idea of molecular heterogeneity of the receptor. While this specific alteration is likely to be irrelevant to the pathogenetic mechanism of the disease, it may still be diagnostic of a more common defect affecting the structure and the functions of certain proteins in dystrophic muscle. This work was supported by grants from M.P.I. (60%) and National Groups “Differentiation” and “Biomembranes.” We thank Mr. M. Coletta for excellent technical assistance.

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