From the Departamento de Biología Celular y Molecular,
Facultad de Ciencias Biologicas, Pontificia Universidad Católica
de Chile, P. O. Box 114-D, Santiago, Chile
Collagen-tailed asymmetric
acetylcholinesterase (AChE) forms are believed to be anchored to
the synaptic basal lamina via electrostatic interactions involving
proteoglycans. However, it was recently found that in avian and rat
muscles, high ionic strength or polyanionic buffers could not detach
AChE from cell-surface clusters and that these buffers solubilized
intracellular non-junctional asymmetric AChE rather than synaptic forms
of the enzyme. In the present study, asymmetric AChE forms were
specifically solubilized by ionic buffers from synaptic basal
lamina-enriched fractions, largely devoid of intracellular material,
obtained from the electric organ of Torpedo californica and
the end plate regions of rat diaphragm muscle. Furthermore, foci of
AChE activity were seen to diminish in size, number, and staining
intensity when the rat synaptic basal lamina-enriched preparations were
treated with the extraction buffers. In the case of
Torpedo, almost all the AChE activity was removed from the
pure basal lamina sheets. We therefore conclude that a major portion of
extracellular collagen-tailed AChE is extractable from rat and
Torpedo synaptic basal lamina by high ionic strength and
heparin buffers, although some non-extractable AChE activity remains
associated with the junctional regions.
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INTRODUCTION |
The enzyme acetylcholinesterase
(AChE)1 plays a key role in
cholinergic neurotransmission (1). Its predominant form at the neuromuscular junction is the collagen-tailed asymmetric form, A12, which is located on the extracellular surface
positioned for the hydrolysis of acetylcholine. Most of this junctional
AChE is associated with the basal lamina (BL), located between the nerve ending and the muscle plasma membrane (2, 3), and can be removed
from the cell surface of muscle tissue (4, 5) and mouse myotubes (6) by
treatment with collagenase, indicating that the collagenic tail of the
enzyme is involved in its anchorage to the BL (7, 8). Although the
precise mechanisms by which asymmetric AChE forms are anchored to the
BL remain elusive (9), there is compelling evidence to suggest that
heparan sulfate proteoglycans (HSPGs) or related proteoglycans are
involved (7, 10). This evidence includes the recent finding that
A12 has two heparin-binding consensus sequences in its
collagenic tail (11). Asymmetric AChE forms have a high binding
affinity for BL components, particularly HSPGs (12) which are
themselves major constituents of basement membranes (13, 14). Heparin
and heparan sulfate have also been shown to release asymmetric AChE
activity from rat muscle end plate regions (15) and BL sheets purified
from the electric organ of Discopyge (16). The demonstration
that A12 could bind and be selectively eluted from
heparin-agarose columns, whereas non-collagenous forms and
A12 after collagenase treatment could not, proved the
direct interaction of A12 with heparin in vitro (11, 17). Direct interactions with heparin/heparan sulfate moieties
in vivo have also been demonstrated. Asymmetric AChE forms
were shown to bind the surface of HSPG-rich cells, such as mouse
myotubes and CHO-KI cells, in a saturable and
time-dependent manner, whereas pretreatment of these cells
with heparitinase almost abolished this binding. In addition, the
binding of A12 AChE was reduced by 80% in Chinese hamster
ovary clone 606, a mutant expressing undersulfated HSPGs (10).
Similarly, purified BL sheets from electric organ were found to release
only asymmetric AChE forms when treated with heparitinase but not
chondroitinase ABC (16). Finally, the assumption that HSPGs were likely
involved in the intracellular assembly, transport, and cell surface
deposition of asymmetric AChE is supported by the finding that a mutant
variant of rat PC12 neuronal cells lacking HSPGs expressed a
predominantly internal distribution of asymmetric AChE in contrast to
normal PC12 cells in which almost all asymmetric AChE was extracellular (18, 19).
Despite the overwhelming evidence supporting the notion that asymmetric
AChE forms are associated with the BL through electrostatic bonds, it
was recently suggested that the enzyme displaced by high ionic strength
and heparin buffers was in fact non-junctional and originated in
intracellular compartments (20, 21). In quail muscle fibers and myotube
cultures, these ionic buffers did not disaggregate previously formed
surface AChE clusters, as detected by immunofluorescence techniques;
however, the presence of heparin impeded their formation. Hence the
authors formulated the hypothesis that only the newly secreted pool of
asymmetric AChE was capable of binding heparin, as a transient event
prior to its permanent, covalent association with the BL (21).
Our aims were to study the solubilization properties of junctional AChE
first by quantifying the extraction capacity of high salt- and
heparin-containing buffers, and second by evaluating the effect of
these buffers on the AChE activity associated with BL-enriched
preparations derived from rat muscle end plate regions and
Torpedo californica electric organ. Our results strongly
suggest that both high salt and heparin buffers solubilize a major
portion of extracellular collagen-tailed AChE and demonstrate the
capacity of these buffers to dissolve AChE-rich regions from
neuromuscular junctions, as detected by histochemical staining.
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EXPERIMENTAL PROCEDURES |
Preparation of BL-enriched Fractions--
The tissues used were
frozen samples of T. californica electric organ, obtained
from Pacific Bio-Marine (Venice, CA), and end plate regions of rat
diaphragm muscle. The latter were obtained from anesthetized female
Sprague-Dawley rats (200-250 g) by removing the diaphragm muscles
together with the surrounding ribs and dissecting 2-mm strips of end
plate regions (1 mm on either side of the phrenic nerve) (2, 15). The
end plate strips were maintained in ice-cold saline solution (0.9%
NaCl). The tissues were then homogenized for 5 min using a
glass-to-glass homogenizer at full speed, in 1:10 (w/v) detergent
buffer: 10 mM Tris-HCl, pH 7.4, 1% Triton X-100, and
protease inhibitors (1 mM N-ethylmaleimide, 1 mM benzamidine, 0.1 mg/ml bacitracin, 100 mM
caproic acid, 5 mM EDTA) (22). The homogenates were
centrifuged at 15,000 rpm for 25 min in a fixed rotor using a Kubota
KR-20000T centrifuge. The supernatants were then removed and assayed
for activity, and the pellets were resuspended and rehomogenized as
above. This wash cycle was repeated a total of 5 times, removing the
majority of AChE globular forms and yielding a fibrous BL-enriched
preparation for both rat muscle end plate tissue and Torpedo
electric organ (23, 24). All procedures were carried out at 4 °C.
Aliquots were taken from the initial tissue homogenates for activity
assays and from the BL-enriched preparations for activity and
histochemical analyses.
Quantifying Residual Intracellular Material in the BL-enriched
Preparations--
Lactic dehydrogenase (LDH) was used as a
quantitative marker for intracellular contamination of the BL-enriched
preparations, before and after undergoing extractions. LDH activity was
measured using the Promega kit according to the manufacturer's
instructions, and the percentage of remnant activity was calculated
from the activity measured in the initial tissue homogenates (100%).
Sialyltransferase activity, a marker of the Golgi apparatus (25), was
used in some experiments to assess the presence of Golgi vesicles, and therefore intracellular contaminants, in our BL fractions.
Solubilization of Asymmetric AChE Activity from the BL--
The
BL-enriched preparations were divided into 3 groups, and duplicate
samples for each group were homogenized for 5 min in 1:10 (w/v) of one
of the following buffers: control buffer, 10 mM Tris-HCl,
pH 7.4, containing several protease inhibitors (as above); heparin
extraction buffer, 1 mg/ml heparin in control buffer; or high salt
extraction buffer, 1 M NaCl in control buffer. The
homogenates were then centrifuged at 15,000 rpm for 25 min and the
supernatants removed, and the extraction procedure was repeated. All
procedures were carried out at 4 °C. Aliquots were taken from the
extraction supernatants for activity assays and sedimentation analyses.
The post-extraction pellets were kept at 4 °C for subsequent
histochemical studies and collagenase digestion.
Perfusion of Asymmetric AChE Activity from Rat Diaphragm Muscle
Strips--
Adult rat diaphragm muscle was dissected and cut into thin
strips, pinned, and perfused for 1 h with high salt borate
extraction buffer, in the presence or absence of heparin, as described
by Rossi and Rotundo (20). After washing with phosphate-buffered saline, the muscle fibers were incubated with the histochemical reaction buffer of Karnovsky and Roots (26). In addition, the AChE activity released by these buffers was assayed in parallel experiments.
Sedimentation Analysis of AChE Forms--
The molecular forms of
AChE present in the tissue homogenates and BL-enriched fractions, and
those solubilized from the latter, were resolved by sedimentation
analysis on 5-20% linear sucrose gradients, as described previously
(6, 27). To release all the molecular forms present in the homogenate
samples, initial homogenates and BL preparations were homogenized in
detergent buffer containing 1 M NaCl for the purpose of
sedimentation analysis.
AChE Activity Assay--
AChE activity was measured by the
method of Ellman et al. (28). All incubations were carried
out at 37 °C for rat muscle samples and 25 °C for
Torpedo electric organ, in a 1-ml reaction mixture
containing 100 mM sodium phosphate buffer, pH 7.0, 0.3 mM dithionitrobenzoic acid, and 0.75 mM
acetylthiocholine iodide. 10 mM tetraisopropyl
pyrophosphoramide (iso-OMPA) was also included as a specific inhibitor
of butyrylcholinesterase activity. Absorbance was read at 412 nm in a
Shimadzu UV-150-02 double-beam spectrophotometer.
Collagenase Digestion of the BL Preparations following Ionic
Extractions--
Collagenase digestions were carried out as described
by Younkin et al. (29), to quantitatively determine the AChE
activity extracted from the BL preparations as a function of the total AChE present. In short, the BL pellets were incubated following either
control, high salt, or heparin extractions, at 25 °C for 4 h
with 0.1 mg/ml collagenase (Sigma, type V) in 1:10 (w/v) of a buffer
containing 20 mM Tris-HCl, pH 7.4, 5 mM
N-ethylmaleimide, 2 mM benzamidine, 10 mM CaCl2 but devoid of EDTA. The reaction was
stopped when the samples were centrifuged, and the supernatants were
finally assayed for AChE activity.
Preparation of Rat End Plate Muscle Honeycomb Ghosts--
Rat
muscle end plate honeycomb ghost preparations were obtained using a
method similar to that of Sanes and Hall (23), omitting the salt
extraction step. End plate regions were dissected as before from
diaphragm muscles and were maintained in ice-cold saline solution
(0.9% NaCl) before being thinly cut into transverse sections and
washed twice for 1 h with detergent buffer as follows: 10 mM Tris-HCl, pH 7.4, 1% Triton X-100, and protease
inhibitors. The end plate strips were then divided into three groups
and incubated overnight at 4 °C with 1:25 (w/v) of detergent buffer
alone, detergent buffer containing 1 M NaCl, or detergent
buffer plus 1 mg/ml heparin. The resultant honeycomb ghosts were then
analyzed for residual AChE activity by histochemical staining, and
solubilized AChE activity was also assayed in the preparation buffers.
Visualization of Surface End Plate AChE Activity by Histochemical
Staining--
AChE activity was detected by histochemical staining in
the BL-enriched fractions of both rat muscle end plates and
Torpedo electric organ, in the semi-intact perfused rat
muscle fibers and in the rat muscle end plate honeycomb ghost
preparations, before and after their particular extractions. For this,
the method of Karnovsky and Roots (26) was employed, using 2.2 mg/ml
acetylthiocholine iodide, 0.71 M malate buffer, pH 6.0, 0.1 M sodium citrate, 30 mM copper sulfate, 5 mM potassium ferricyanide, and 3% neutral formol. All
incubations were carried out at 4 °C, and all samples were processed
simultaneously to make their staining intensities comparable. In the
case of the BL-enriched preparations, samples weighed ~3 mg. For the
honeycomb ghosts, multiple section samples were stained for each group.
All samples were incubated for 20 min with iso-OMPA (10 mM), prior to the histochemical reaction, to inhibit
butyrylcholinesterase activity. Control samples were incubated with 10 mM iso-OMPA plus either 50 mM methanesulfonyl fluoride or 10 µM BW 284c51 dibromide to inhibit
cholinesterase activity or specifically AChE activity, respectively.
Samples were then viewed under the phase-contrast light microscope.
Purification of Asymmetric AChE from Torpedo Electric
Organ--
Affinity chromatography using an acridine-agarose column
was used to purify the collagen-tailed form of AChE, as described previously (30). Both specific activity (4,000 units/mg protein) and
staining intensities following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (a single band of 67 kDa) were used to verify purity. Sucrose sedimentation analysis was subsequently used to confirm
that the purified protein corresponded to the asymmetric A12 form of AChE.
Association of Torpedo AChE with Rat Myotubes--
Rat muscle
primary cultures were prepared from the hindlimb muscles of an
18-day-old rat embryo and were maintained as described by Koenig (31).
Approximately 3 × 105 cells were plated onto 35-mm
plastic tissue culture dishes coated with gelatin. The myoblasts
reached confluence 24 h after plating, and the onset of fusion and
large myotube formation began at 48 h. Most of the myotubes
presented spontaneous contractile activity around the 5th day in
culture. The medium was then removed, and the myotubes were washed
three times with Dulbecco's modified Eagle's medium containing 0.1 M NaCl and 0.5 mg/ml bovine serum albumin. The myotubes
were then incubated for 1 h at 4 °C with 50 milliunits of AChE
enzyme, purified from Torpedo electric organ, in the
presence or absence of 1 mg/ml heparin. The cells were then washed
twice with 2:5 Dulbecco's modified Eagle's medium/phosphate-buffered saline and incubated for 15 min at 4 °C with phosphate-buffered saline buffer containing 2 mg/ml bovine serum albumin and 2 mg/ml heparin. The cell surface-associated AChE activity solubilized by this
medium was then assayed. For the determination of total cell
surface-bound AChE, an additional incubation with the same medium
containing 1 M NaCl instead of heparin was performed.
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RESULTS |
Asymmetric AChE Is Associated with BL-enriched
Preparations--
Since it was recently reported that neither high
salt- nor heparin-containing buffers detached asymmetric
collagen-tailed forms of AChE from the BL of the vertebrate
neuromuscular junction (20, 21), we wanted to re-evaluate the
solubilization properties of the asymmetric forms present in
BL-enriched preparations, derived from both diaphragm muscle end plates
as well as Torpedo electric organ. Several
homogenization-centrifugation cycles in the presence of Triton X-100
detergent were used to remove most of the hydrophobic and soluble
proteins and yield BL-enriched preparations for both tissues. The AChE
forms present in these BL-rich fractions were compared with those
present in the corresponding initial homogenates by velocity
sedimentation analysis (Fig. 1).
Characteristic sedimentation profiles were obtained for both rat
diaphragm (Fig. 1A) and Torpedo electric organ
(Fig. 1B) homogenates, including asymmetric (A12 and A8) as well as globular forms (G1 and
G4 for rat and G2 for Torpedo). In
contrast, the asymmetric AChE forms were greatly enriched in the BL
preparations as seen by the absence of the peaks corresponding to the
globular forms. When the Torpedo BL preparations were
examined under the electron microscope, the typical cytoarchitecture of
Torpedo basement membranes was apparent and predominant
(Fig. 2). The presence of asymmetric AChE
associated with these BL sheets was an important confirmation that the
enzyme was in fact anchored to such extracellular structures.

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Fig. 1.
Molecular forms of AChE solubilized from the
end plate regions of rat diaphragm muscle and from Torpedo
electric organ. A corresponds to rat muscle end
plates, and B corresponds to the Torpedo electric
organ, in which represents the initial tissue homogenate and represents the BL-enriched fraction. For the purpose of sedimentation
analysis, fresh (rat) or frozen (Torpedo) tissue samples
were homogenized in a high salt detergent buffer (1:10, w/v) as
follows: 10 mM Tris-HCl, pH 7.4, 1 M NaCl, 1%
Triton X-100, and protease inhibitors. By having submitted the rest of the tissue to 5 homogenization-centrifugation cycles with detergent buffer (10 mM Tris-HCl, pH 7.4, 1% Triton X-100, protease
inhibitors) to remove the majority of globular forms, the resultant
BL-enriched fractions were also solubilized in high salt detergent
buffer for sucrose sedimentation analysis. The arrow
indicates migration of the catalase marker at 11.3 S.
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Fig. 2.
Basal lamina sheets are the major
constituents of the extracellular matrix material purified from
Torpedo electric organ. Typical electron micrograph of
basement membranes obtained after extensive detergent extraction of
electric organ tissue. The sample was fixed in Karnovsky's fixative,
post-fixed, stained with uranyl acetate, and finally prepared for
electron microscopy. (Magnification, ×70,000.)
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Remnant Cytoplasmic Material in the BL-enriched
Preparations--
To discard the possibility that intracellular
asymmetric AChE, destined to be exported to the BL, may be
contaminating our BL fractions (despite exhaustive washes with
detergent buffer), we analyzed such preparations for the presence of
two intracellular markers. First, LDH activity was used as an indicator
of remnant-soluble cytoplasmic material. After repeated Triton X-100
extractions, some residual LDH activity (~6% of the total) was
detectable in the final rat muscle end plate BL-enriched preparation
(Fig. 3). Furthermore, two subsequent
extraction steps with either high salt or heparin solubilized half of
this remnant activity (see inset of Fig. 3), indicating that
a residual intracellular material was probably trapped within or
retained electrostatically by the BL sheets. Similar results were
obtained for Torpedo electric organ preparations. These
results indicate that asymmetric AChE is not trapped in vesicles as a
soluble entity. Second, the absence of sialyltransferase activity, a
Golgi marker, in both the rat and Torpedo BL-enriched
preparations (data not shown), makes it unlikely that intracellular
asymmetric AChE remained associated, as a contaminant, to the BL
fractions used in these studies.

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Fig. 3.
A small portion of cytoplasmic material
remained associated with the extracellular matrix fractions. LDH
activity was measured in the initial rat muscle end plate homogenate
(Homog.), the five supernatant fractions of the preparative
wash cycle (SN1-5), and the final BL-enriched fraction
(BL). The latter was also analyzed following control,
heparin, and high salt extractions (inset). LDH activity was
determined using the Promega kit according to the manufacturer's
instructions.
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High Salt- and Heparin-containing Buffers Solubilized Extracellular
Asymmetric AChE from the BL Preparations of Rat Muscle End Plates and
Torpedo Electric Organ, and from Rat Muscle Honeycomb Ghost
Preparations, but Not from Perfused Rat Muscle Strips--
Having
identified the source of collagen-tailed AChE as predominantly BL, a
study of the solubilization properties of these forms was carried out
at short extraction times. First, samples of purified BL material were
submitted to two 5-min homogenization-centrifugation cycles with either
high salt- or heparin-containing buffers. Both AChE activity and the
molecular forms solubilized were then determined. As Fig.
4 shows, only asymmetric forms of AChE
were released by either high salt or heparin from both rat (Fig.
4A) and Torpedo (Fig. 4B) BL
preparations. Additional experiments were carried out using sectioned
rat muscle end plates treated with detergent so as to generate
honeycomb ghost preparations (23) with structurally integral junctional
regions. The AChE activity released by the high salt- or
heparin-containing buffers used for the ghost preparation was assayed;
heparin was able to solubilize 50% of that released by NaCl (data not
shown). Similarly, in rat muscle end plate BL, heparin extracted
approximately 40% of the activity solubilized by high salt, as shown
in Fig. 5A, whereas in
Torpedo electric organ, both buffers solubilized similar
quantities (80-90%) of asymmetric AChE (Fig. 5B). The
total fraction of asymmetric AChE solubilized by heparin was much
greater in Torpedo electric organ than in rat muscle end
plate regions (80% compared with 30%) (Table I), suggesting that a larger population
of heparin-extractable asymmetric AChE exists in the former. When high
salt and heparin-containing buffers were used to perfuse rat diaphragm
muscle strips for 1 h, no AChE activity was detectable in the
activity assays carried out on these buffers (data not shown).

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Fig. 4.
Solubilization of the molecular forms of AChE
from the rat and Torpedo BL-enriched fractions.
A corresponds to rat muscle end plates, and B
corresponds to Torpedo electric organ, in which represents the AChE forms solubilized by 1 M NaCl and represents those released by 1 mg/ml heparin from the BL-enriched fractions. The latter were homogenized for 5 min in a buffer containing 10 mM Tris-HCl, pH 7.4, protease inhibitors, and either 1 mg/ml heparin or 1 M NaCl. Following centrifugation, the
extraction procedure was repeated, and the molecular AChE forms
solubilized by the first extraction were analyzed on a 5-20% sucrose
gradient. Two distinct peaks were obtained for both heparin and NaCl,
corresponding to the asymmetric forms A12 and
A8. Absorbance values represent the proportion of AChE
activity extracted by each buffer with respect to the total extractable
activity, determined by collagenase digestion, as described for Table
I. The arrow indicates migration of the catalase marker at
11.3 S.
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Fig. 5.
AChE activity released from the rat and
Torpedo BL-enriched fractions by ionic extractions and
collagenase treatment. Rat (A) and Torpedo
(B) BL preparations were submitted to control and ionic
extractions, as described earlier, using Tris-HCl (10 mM),
heparin (1 mg/ml), or NaCl (1 M) buffers (white
bars). Following centrifugation, the BL pellets were treated with
collagenase (Sigma type V) for 4 h at 25 °C to solubilize the
remnant AChE activity resistant to extraction (gray bars).
Each value is the mean ± S.D. of three experiments carried out in
duplicate.
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Table I
Solubilization of asymmetric AChE from rat and Torpedo basal
lamina-enriched fractions
Extractions were carried out by homogenizing BL-enriched preparations
in Tris-HCl buffer (control) and either 1 M NaCl or 1 mg/ml
heparin. The samples were then centrifuged at 15,000 rpm for 25 min,
and the procedure was repeated. The activity in both supernatants was
then measured and summed. Each value is the mean ± S.D. of three
representative experiments done in duplicate, and the values given for
collagenase correspond to the AChE fraction remnant following the high
salt extractions. The number in parentheses corresponds to the
percentage of AChE activity solubilized by each procedure. The activity
released by collagenase treatment of the control samples was used to
calculate the total extractable activity (100%) associated with the BL
preparations.
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A Minor Portion of Junctional AChE Persists in the BL-enriched
Preparations following Extractions with High Salt- or
Heparin-containing Buffers--
A population of collagen-tailed AChE
associated with the BL persists for long periods after frog muscle
denervation (3). In rat diaphragm and gracilis muscles, most of the
asymmetric AChE activity is extracellular, of which only 15-25% is
considered to be a non-extractable fraction (29, 32). However, a recent report suggested that all junctional asymmetric AChE formed part of
this non-extractable pool, given that high salt and heparin buffers
seemed unable to disrupt or diminish the number of cell-surface AChE
clusters in quail myotube cells or end plate regions of avian muscles
(20, 21). Therefore in the present study, we sought to quantify the
extractable as well as the non-extractable fraction of BL-associated
AChE activity and ascertain whether a change in the quantity and
distribution in extracellular AChE-rich regions could be detected
following high salt or heparin extractions. First, collagenase
digestion after high salt extractions indicated that the
non-extractable pool of asymmetric AChE in rat end plate regions
accounted for 23% of the total BL-associated asymmetric AChE (Fig.
5A, see also Table I), in agreement with Younkin et al. (29). Moreover, in Torpedo electric organ, a minor
portion of junctional AChE remained after high salt or heparin
extractions, accounting for 9% of non-extractable enzyme (Fig.
5B).
Second, AChE activity was visualized by histochemical staining before
and after extractions on the BL-enriched fractions obtained from rat
and Torpedo tissues. In the former, the morphological appearance, size, and organization of the histochemically stained structures seen in Fig. 6 revealed the
presence of numerous isolated and well preserved motor end plates with
their synaptic gutters. These neuromuscular junctions, rich in AChE
activity, were also clearly visible in the rat muscle BL fractions
prior to high salt and heparin extractions (Fig.
7A). Following high salt
treatment, most of the activity in these end plates disappeared (Fig.
7B). Heparin extractions also produced a reduction in the
size and staining intensities of the end plates detected (Fig.
7C). Control experiments in the presence of inhibitors
specific for AChE did not show end plate hydrolytic activity (Fig.
7D). Histochemistry carried out on the BL sheets purified
from Torpedo electric organ demonstrated that these samples
contained isolated surfaces highly enriched in AChE activity (Fig.
8A). On the other hand, the
same preparations treated with either high salt (Fig. 8B) or
heparin (Fig. 8C) showed only minimal residual AChE activity
in both cases. Controls treated with AChE inhibitors showed no activity
(Fig. 8D). In the experiments using rat muscle end plate
honeycomb ghost preparations, histochemical staining of AChE revealed
an array of end plate regions in these muscle ghosts (Fig.
9A). However, when these
samples were prepared in the presence of high salt, most of the AChE
activity was removed from the end plate ghosts (Fig. 9B),
whereas less activity was removed when heparin was present. Almost no
AChE activity was visible in control samples incubated with 20 µM BW284c51 and 10 mM iso-OMPA (Fig.
9C). In contrast, when rat diaphragm muscle strips were
perfused with a mixture of high salt and heparin buffers, no variation
in AChE histochemical staining intensity was seen between the control and treated samples (data not shown). Together, these results clearly
show that most of the junctional AChE is readily detached by either
high salt or heparin from rat or Torpedo BL preparations as
well as muscle end plate ghosts, which retain the basic cytoskeletal architecture of the muscle, but not from semi-intact rat muscle strips.

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Fig. 6.
Visualization of isolated motor end plates in
the rat muscle BL-enriched preparation. Histochemical localization
and phase-contrast light microscopy were used to confirm the presence of end plate-associated AChE activity in the BL-enriched fraction of
rat diaphragm muscle. Low (×100) (A), medium (×300)
(B), and high field (×800) (C) views show
isolated motor end plates with their apparent postsynaptic folds.
Histochemical staining was carried out using the method of Karnovsky
and Roots (26), prior to which samples were incubated with 10 mM iso-OMPA for the specific inhibition of
butyrylcholinesterase activity.
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Fig. 7.
Residual activity of AChE seen in the rat
muscle end plate BL-enriched fractions following high salt and heparin
extractions. Histochemical localization of BL-associated AChE
activity (26) as viewed under the phase-contrast microscope is shown.
A, rat muscle BL-enriched fraction prior to heparin or high
salt treatment; B, following heparin extractions;
C, NaCl extractions. Samples weighed ~3 mg and were
processed simultaneously to make their staining intensities as
comparable and quantitative as possible. All samples used were
incubated with 10 mM iso-OMPA prior to the histochemical
reaction. D, no AChE activity was visible in control samples
incubated with 10 mM iso-OMPA and 50 mM
methanesulfonyl fluoride for the inhibition of all cholinesterase
activity. (Magnification, ×200.)
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Fig. 8.
A minor residual AChE activity is present in
the basal lamina sheets of Torpedo electric organ following
high salt and heparin extractions. Histochemical localization of
BL-associated AChE activity as viewed under the phase-contrast
microscope is shown. The histochemical reaction was carried out
according to the method of Karnovsky and Roots (26). A,
Torpedo basal lamina sheets prior to heparin or high salt
treatment; B, following heparin extractions; and
C, NaCl extractions. D, almost no AChE activity was visible in control samples incubated with 10 mM
iso-OMPA and 50 mM methanesulfonyl fluoride. Samples
weighed ~3 mg and were processed simultaneously to make their
staining intensities as comparable and quantitative as possible. All
samples were incubated with iso-OMPA (10 mM) prior to
histochemistry. (Magnification, ×600.)
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Fig. 9.
High salt treatment of honeycomb ghosts
derived from rat muscle end plate regions released an important portion
of junctional AChE activity. Honeycomb ghosts were prepared from
sectioned rat muscle end plates by incubating the sections with
detergent buffer overnight, either in the presence or absence of 1 M NaCl. Remnant end plate AChE activity was then stained
histochemically and viewed by phase-contrast light microscopy.
A, AChE activity was visible as large, densely stained end
plate regions in the ghost preparations (control).
B, honeycomb ghosts prepared in the presence of high salt,
to extract asymmetric AChE, contained only small dispersed foci of
histochemical activity, which displayed lower staining intensities than
their control counterparts. C, no AChE activity was detected
in honeycomb ghosts incubated with 20 µM BW284c51
dibromide and 10 mM iso-OMPA. (Magnification, ×400.)
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Association of Torpedo AChE to Rat Primary Culture
Myotubes--
Despite the fact that rat muscle and Torpedo
electric organ are phylogenetically distant, the anchorage mechanisms
of the collagen-tailed enzyme may not be fundamentally different in
both systems. To determine the extent to which AChE association in these systems was compatible, a study of the binding properties of
asymmetric AChE, purified from Torpedo, was carried out
using rat primary muscle cultures. Fig.
10 shows that Torpedo AChE
was capable of associating with rat myotubes in a reversible manner, as
demonstrated by its subsequent detachment from the cell surface using a
heparin-containing buffer. Of the total AChE activity associated (3.65 milliunits/ml), heparin was able to release approximately 70% (2.6 milliunits/ml). However, incubation with AChE in the presence of
heparin eliminated the capacity of the enzyme to bind to the myotube
cell surface. When fibroblasts were used instead of myotubes, no
association was observed with or without heparin co-incubation (Fig.
10). These results suggest that specific AChE-binding sites are present
on the cell surface of rat myotubes which are absent in fibroblasts.
Moreover, such sites are also recognized by Torpedo
asymmetric AChE, implying that the anchorage of the collagen-tailed
form of AChE involves either similar extracellular molecules or similar
interaction mechanisms in both species. Whether or not this association
occurs in clusters over the myotube surface is a matter that deserves
further study.

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Fig. 10.
Association of purified Torpedo
asymmetric AChE with rat myotubes. Fifty milliunits of
asymmetric AChE, purified from Torpedo electric organ, were
incubated for 15 min with 5-day-old rat myotubes in 35-mm culture
dishes, in the presence or absence of 2 mg/ml heparin. Fibroblasts
obtained from the same muscle primary cultures were used as controls
and submitted to the same treatments. Cell surface-associated AChE was
then released by incubating the cells with 2 mg/ml heparin buffer, and
the activity solubilized was determined for cells incubated with AChE
in the presence or absence of heparin (labeled as Heparin
co-incubation and Heparin extractable, respectively).
Total cell surface-associated AChE (Total extractable) was
the total amount of activity solubilized from the myotubes by 2 mg/ml
heparin followed by 1 M NaCl buffer. Asymmetric AChE
associated only with rat myotubes, in a manner that was inhibited by
heparin. No significant association of asymmetric AChE was detected in
fibroblasts. Values represent the average of three experiments done in
duplicate.
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DISCUSSION |
Prevailing opinion indicates that the collagen-tailed forms of
AChE are anchored to the synaptic BL through electrostatic interactions
with polyanionic components such as HSPGs (9, 10). This widely accepted
hypothesis was later reinforced by the identification of two
heparin-binding domains in the collagenous tail of the enzyme (11, 33).
The objective of the present study was to determine directly whether
the AChE molecules localized at the neuromuscular junction were
quantitatively removed by high salt and heparin extractions, in view of
the results of Rossi and Rotundo (20) in which perfusions with these
buffers were unable to detach clustered cell-surface AChE from quail
and rat muscle fibers. Furthermore, these authors suggested that the
ability of heparin to solubilize asymmetric AChE, as reported for other systems, stemmed from its apparent ability to release mainly
intracellular and non-junctional AChE molecules (20, 21). However, our
biochemical and histochemical results demonstrate that these
conventional ionic buffers do in fact specifically detach a major
portion of asymmetric AChE from purified synaptic BL devoid of
intracellular material and derived from two of the most studied
neuromuscular junction systems, rat muscle end plates and
Torpedo electric organ.
In view of this apparent discrepancy, we wanted first to confirm the
extracellular source of asymmetric AChE prior to its extraction.
Asymmetric AChE occupies less than 0.1% of the total surface area of
the BL at the rat neuromuscular synapse, yet its density increases
severalfold specifically at junctional regions (34). In contrast, the
electric organ of Torpedo is a tissue in which the synaptic
junctions are overdeveloped (35, 36). Therefore, the use of both these
tissues in the present study minimized the possibility that
extra-junctional asymmetric AChE was being solubilized during the
extractions. Furthermore, several end plates rich in junctional AChE
were observed in the rat and Torpedo BL-enriched
preparations prior to extraction. Treatment with high salt or heparin
buffers reduced both the number, size, and staining intensities of
these regions. The studies of Rossi and Rotundo (21) nevertheless
demonstrated that neither high salt- nor heparin-containing buffers
were able to reduce the number of immunoreactive AChE clusters on the
surface of quail myotubes or quail muscle fibers. Yet by incubating the
myotubes in the presence of heparin, newly synthesized asymmetric AChE
accumulated in the medium and not as cell-surface clusters. The authors
concluded that only a transient cell-surface interaction occurred in
avian cell cultures between newly formed asymmetric AChE and heparin or
HSPGs. However, in the present study, the use of purified BL, as seen
under the electron microscope, eliminated the possibility that newly
synthesized asymmetric AChE and its subsequent externalization to the
cell surface could account for the enzyme detached during the
extraction processes.
It should also be noted that in quail skeletal muscle only 40% of
total asymmetric AChE is BL-associated, with the remainder being
intracellular (37). Therefore, the subcellular localization of
collagen-tailed AChE in avian muscle differs from most of the other
systems studied, as virtually all findings agree that the majority of
asymmetric AChE is extracellular. For instance, studies performed in
rat diaphragm muscle in vivo showed that 22% of asymmetric AChE forms were intracellular in rat diaphragm end plate regions (29),
30% in rat gracilis muscle (32), and 20% in mouse C2 myotubes (38). Moreover, there is further evidence to support the
notion that AChE attachment in the avian system may be unusual. For
instance, chondroitinase ABC or AC was found to detach asymmetric AChE
from chicken muscle (39) but not from mouse C2 myotube cultures or from Discopyge electric organ, in which only
heparitinase released the enzyme (10, 16).
Altogether, the above evidence clearly indicates that differences in
species' extractability of asymmetric AChE, extracellular to
intracellular ratios and anchorage mechanisms, are likely to account
for some of the differences obtained in the present study and the work
of Rossi and Rotundo (20, 21). However, there is also a fundamental
difference in the sample preparation and extraction methods used,
subcellular fractionation versus intact muscle fibers and
homogenization versus perfusion. This difference could
explain the apparent controversy of different results obtained in the
same species (Ref. 20, Fig. 7). For this reason, we carefully repeated
the experiment of Rossi and Rotundo (20) in which rat muscle strips
were perfused and extracted for 1 h with buffers containing high
salt and heparin. In our experiment using rat diaphragm, we obtained
the same results as Rossi and Rotundo with rat gastrocnemius muscle
(Ref. 20, Fig. 7), whereby these high ionic buffers extracted no or
little AChE activity from the muscle fibers. This was visualized using
both the same histochemical method as Rossi and Rotundo (20, 21) and
also by activity assays. Considering that in both rat muscle honeycombs
and purified synaptic BL, we successfully solubilized asymmetric AChE
but were unable to do so in muscle strips, we believe that perfusion of a semi-intact muscle sample is not the most appropriate method for the
extraction of synaptic molecules such as AChE.
A non-extractable population of asymmetric AChE has been identified
previously, which can only be released by collagenase treatment (29,
32). This non-extractable pool of AChE, in rat diaphragm end plates,
accounts for 21% of the total asymmetric forms which in these regions
exist principally as extracellular enzyme (29). However, the
ultrastructure of this tissue may present a greater resistance to the
extraction procedure than that of Torpedo electric organ, as
in the latter there seems to be a larger pool of heparin-sensitive
collagen-tailed AChE. Although some asymmetric AChE seems to be
covalently associated with the synaptic BL, the majority of the
extracellular enzyme is extractable using high salt- and
heparin-containing buffers, indicating that electrostatic interactions
must be involved in its anchorage at the neuromuscular junction.
The cell surface AChE binding studies, carried out using
Torpedo enzyme and rodent muscle cultures, proved to be
effective heterologous systems for such studies. Indeed, Rotundo
et al. (40) recently carried out transplant experiments, in
which frog neuromuscular synapses were incubated with purified quail
asymmetric AChE, and binding was observed. These authors even suggest
the presence of specific collagen-tailed AChE-binding sites at the synaptic BL of the vertebrate neuromuscular junction. In our case, the
localization of the AChE bound to the myotube surface was not
investigated. However, bound AChE would most likely appear as clustered
foci of activity but would nonetheless present a "non-junctional"
distribution due to the absence of neuronal stimuli. Therefore, the
formation of AChE clusters in myotube cell cultures does not imply the
presence of "junctional" AChE (as in Torpedo electric
organ and muscle end plates), which is defined by the existence of the
neuromuscular junction and requires the presence of motor neurons. For
this reason, we suggest caution in the use of the term junctional when
referring to the clustering of AChE on the surface of cultured
myotubes. The fact that heparin could be used to release the asymmetric
AChE bound to the myotube surface or block its association in our
in vitro studies suggests first that HSPGs are involved in
the binding of the Torpedo enzyme to the rodent muscle
cells, and second that these proteoglycans may be universally
recognized in such anchorage mechanisms. These possibilities are
reinforced by the finding that HSPGs are indeed present in the
extracellular matrix of rodent myotubes (41, 42), and it would be
interesting to study the focalization of AChE exogenously added to
myotube cultures, both in the presence and absence of motor
neurons.
In conclusion, we have established that a major portion of junctional
collagen-tailed AChE is in fact associated with the BL through
electrostatic interactions. Extracellular asymmetric AChE foci can be
disrupted by high salt- or heparin-containing buffers. These results
are entirely consistent with previous work regarding the anchorage
mechanism(s) of collagen-tailed AChE to the BL (10, 16) and with the
current notion that HSPGs may interact with the heparin-binding domains
present in the collagenic tail of the enzyme (11). Further studies
using synthetic peptides, derived from the collagenic region, as well
as molecular modeling and docking, should help us to elucidate the
exact mechanisms involved in the anchorage of asymmetric AChE to the BL
at the neuromuscular junction.
We thank Dr. David J. Carey and Dr.
Enrique Brandan for helpful comments on the manuscript and to Dr. Ariel
Orellana for assistance in the sialyltransferase assays.