Tubular aggregates are from whole sarcoplasmic reticulum origin: alterations in calcium binding protein expression in mouse skeletal muscle during aging.

Tubular aggregates are observed in various muscle disorders and appear as densely packed tubules believed to arise from sarcoplasmic reticulum of striated muscle. They are found both in human skeletal muscle, especially from patients suffering from 'tubular aggregate myopathy' and in fast twitch skeletal muscle of the male inbred mouse during aging. In this work, we studied tubular aggregates present in inbred male mouse skeletal muscle using electron microscopy as well as histochemistry and Western blotting with the main markers of the sarcoplasmic reticulum. We show that mouse tubular aggregates include the proteins SERCA 1, sarcalumenin (longitudinal sarcoplasmic reticulum), calsequestrin (terminal cisternae) and RyR1 (junctional sarcoplasmic reticulum). We demonstrate also that 95 and 51 kDa triadin isoforms are present in mouse skeletal muscle and are both components of tubular aggregates. These results support the hypothesis that tubular aggregates form a tubular arrangement of a complete sarcoplasmic reticulum containing the junctional, cisternae and longitudinal components of sarcoplasmic reticulum implicated in calcium homeostasis. During mouse skeletal muscle aging, however, densitometry of Western blots reveals a persistent decrease in the expression of the calcium binding protein calreticulin as well as a continuous increase in calsequestrin-like protein expression which both appear unrelated to the tubular aggregate formation.

Expression of the thrombin receptor (PAR-1) during rat skeletal muscle differentiation.

The serine protease thrombin has been proposed to be involved in neuromuscular plasticity. Its specific receptor "protease activated receptor-1" (PAR-1), a G protein-coupled receptor, has been shown to be expressed in myoblasts but not after fusion (Suidan et al., 1996 J Biol Chem 271:29162-29169). In the present work we have investigated the expression of PAR-1 during rat skeletal muscle differentiation both in vitro and in vivo. Primary cultures of rat foetal skeletal muscle, characterized by their spontaneous contractile activity, were used for exploration of PAR-1 by RT-PCR, immunocytochemistry and Western blotting. Our results show that PAR-1 mRNA and protein are both present in myoblasts and myotubes. Incubation of myotubes loaded with fluo-3-AM in presence of thrombin (200 nM) or PAR-1 agonist peptide (SFLLRN, 500 microM), induced the intracellular release of calcium indicating the activation of PAR-1. Blockade of contractile activity by tetrodotoxin (TTX, 6 nM) did not modify either PAR-1 synthesis or its cellular localization. Investigation of PAR-1 on rat muscle cryostat sections at Day 18 of embryogenesis and postnatal Days 1, 5, and 10 indicated that this protein is first expressed in the cytoplasm and that it later localizes to the membrane. Moreover, its expression correlates with myosin heavy chain transitions occurring during post-natal period and is restricted to primary fibers. Taken together, these results suggest that PAR-1 expression is not related to contractile activity but to myogenic differentiation.

Thrombin receptor induction by injury-related factors in human skeletal muscle cells.

Thrombin is involved in tissue repair through its proteolytic activation of a specific thrombin receptor (PAR-1). Previous studies have shown that serine proteases and their inhibitors are involved in neuromuscular junction plasticity. We hypothesized that thrombin could also be involved during skeletal muscle inflammation. Thus we investigated the expression of PAR-1 in human myoblasts and myotubes in vitro and its regulation by injury-related factors. The functionality of this receptor was tested by measuring thrombin's ability to elicit Ca2+ signals. Western blot analysis and immunocytochemistry demonstrated the presence of PAR-1 in myoblasts but not in myotubes unless they were treated by tumor necrosis factor-alpha (10 ng/ml), interleukin-1beta (5 ng/ml), or transforming growth factor-beta(1) (10 ng/ml). The addition of 10 nM alpha-thrombin evoked a strong Ca2+ signal in myoblasts while a limited response in myotubes was observed. However, in the additional presence of injury-related factors, the amplitude of the Ca2+ response was significantly enhanced, representing 88, 65, 48% of their respective basal level, compared to 27% of that obtained in controls. Moreover, immunochemical studies on human skeletal muscle biopsies of patients suffering from inflammatory myopathies showed an overexpression of PAR-1. These results suggest that PAR-1 synthesis may be induced in response to muscle injury, thereby implicating thrombin signaling in certain muscle inflammatory diseases.

A 295-kDA intermediate filament-associated protein in radial glia and developing muscle cells in vivo and in vitro.

The RC2 antibody is frequently used to label mouse radial glial cells in all parts of the nervous system where neuronal migration occurs during embryonic and early postnatal life. The antigen recognized by this antibody still needs to be identified. We have characterized further its localization in vivo, its expression and subcellular localization in vitro, as well as its molecular nature. Histologic investigations of whole mouse embryos reveal an equally intense expression of RC2 immunostaining in radial glial cells in brain and spinal cord and in skeletal muscle. In glial cells cultures, the RC2 antibody recognizes an epitope located on the glial cytoskeleton and identified as an intermediate filament associated protein (IFAP) at the ultrastructural level. RC2 immunostaining in those cells is strongly dependent on the presence of a serum-derived activity. Serum-removal causes a decrease of the staining while adding serum back to the cells induces reexpression of RC2 immunoreactivity. By Western blotting, we find that in intermediate filament (IF) preparations obtained from cultured cerebellar glia, the RC2 antibody recognizes a 295-kDa protein whose expression is also dependent on the presence of serum in culture medium. In developing muscle cells, RC2 immunostaining is observed from the myoblast stage and disappears after complete myotube fusion. Both in vivo and in vitro, staining is first seen as a loose capping around myoblasts nuclei and progressively concentrates into Z-disks in association with the muscle IF protein desmin. The RC2 antibody also recognizes a 295-kDa protein band in muscle tissue protein extracts. Thus, the RC2 antibody recognizes a developmentally regulated cytoskeletal protein that is expressed, like other previously identified IFAPs, by cells of the glial and myogenic lineages and whose expression in vitro seems to be controlled by a signaling mechanism known to modulate astroglial morphology.

Protease nexin I expression is up-regulated in human skeletal muscle by injury-related factors.

Protease nexin I is a 43-50 kDa glycoprotein capable of inhibiting a number of serine proteases. In cultured differentiated human skeletal muscle (myotubes), we previously found that protease nexin I was localized in patches at their surface where it was active and able to inhibit thrombin. To understand the role of skeletal muscle protease nexin I after injury or in inflammatory conditions where thrombin might be extravasated by blood vessels, we examined the role of inflammatory factors on protease nexin I synthesis and secretion by myotubes in culture. By enzyme-linked immunosorbent assay (ELISA) and Western blotting, we found that this serine protease inhibitor is secreted by cultured human myotubes. Protease nexin I secretion is stimulated by tumor necrosis factor-alpha, transforming growth factor-beta and interleukin-1. Complex formation experiments with labeled thrombin reveal active protease nexin I bound to the surface of the treated cells. Secreted protease nexin I-thrombin complex was enhanced in the presence of transforming growth factor-beta and tumor necrosis factor-alpha. Protease nexin I mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis. Whatever the conditions, no significantly different levels were observed, indicating that the changes in cell and media protease nexin I concentration are elicited at the translational/posttranslational levels. Immunocytochemical studies on human skeletal muscle biopsies of patients suffering from inflammatory myopathies showed an overexpression of protease nexin I together with the above inflammatory factors. These findings suggest that skeletal muscle protease nexin I might play a role after injury or inflammatory pathologies.

Expression of matrix metalloproteinases 2 and 9 in regenerating skeletal muscle: a study in experimentally injured and mdx muscles.

Matrix metalloproteinases (MMPs) cooperatively degrade all components of the extracellular matrix (ECM). Remodeling of ECM during skeletal muscle degeneration and regeneration suggests a tight regulation of matrix-degrading activity during muscle regeneration. In this study, we investigated the expression of MMP-2 and MMP-9, in normal muscles and their regulation during regeneration process. We further investigated their secretion by C2C12 myogenic cell line. Two models of muscle degeneration-regeneration were used: (1) normal muscles in which necrosis was experimentally induced by cardiotoxin injection; (2) mdx muscles which exhibit recurrent signs of focal myofiber necrosis followed by successful regeneration. MMPs were studied by zymography; their free activity was quantified using 3H-labeled gelatin substrate and mRNA expression was followed by Northern hybridization. Muscle degeneration-regeneration was analyzed by conventional morphological methods and in situ hybridization was performed on muscle sections to identify the cells expressing these MMPs. Results show that MMP-2, but not MMP-9 expression, is constitutive in normal muscles. Upon injury, the active form of MMP-2 is transiently increased, whereas MMP-9 is induced within 24 h and remains present for several days. Quantitative assays of free gelatinolytic activity show a progressive and steady increase that culminates at 7 days postinjury and slowly returns to normal levels. In adult mdx mice, both pro and active forms of MMP-2 and MMP-9 are expressed. Northern blot results support these findings. Zymography of C2C12-conditioned medium shows that myogenic cells produce MMP-2. By in situ hybridization we localized MMP-9 mRNA in inflammatory cells and putative activated satellite cells in injured muscles. Our data allow the correlation of the differential expression of pro and/or active forms of MMP-2 and MMP-9 with different stages of the degeneration-regeneration process: MMP-9 expression is related to the inflammatory response and probably to the activation of satellite cells, whereas MMP-2 activation is concomitant with the regeneration of new myofibers.

Adenovirus-mediated transfer of the acid alpha-glucosidase gene into fibroblasts, myoblasts and myotubes from patients with glycogen storage disease type II leads to high level expression of enzyme and corrects glycogen accumulation.

Glycogen storage disease type II (GSD II) is an autosomal recessive disorder caused by defects in the lysosomal acid alpha-glucosidase (GAA) gene. We investigated the feasibility of using a recombinant adenovirus containing the human GAA gene under the control of the cytomegalovirus promoter (AdCMV-GAA) to correct the enzyme deficiency in different cultured cells from patients with the infantile form of GSD II. In GAA-deficient fibroblasts infected with AdCMV-GAA, transduction and transcription of the human transgene resulted in de novo synthesis of GAA protein. The GAA enzyme activity was corrected from the deficient level to 12 times the activity of normal cells. The transduced cells overexpressed the 110 kDa precursor form of GAA, which was secreted into the culture medium and was taken up by recipient cells. The recombinant GAA protein was correctly processed and was active on both an artificial substrate 4-methylumbelliferyl-alpha-D-glucopyranoside (4MUG) and glycogen. In GAA-deficient muscle cells, a significant increase in cellular enzyme level, approximately 20-fold higher than in normal cells, was also observed after viral treatment. The transduced muscle cells were also able to efficiently secrete the recombinant GAA. Moreover, transfer of the human transgene resulted in normalization of cellular glycogen content with clearance of glycogen from lysosomes, as assessed by electron microscopy, in differentiated myotubes. These results demonstrate phenotypic correction of cultured skeletal muscle from a patient with infantile-onset GSD II using a recombinant adenovirus. We conclude that adenovirus-mediated gene transfer might be a suitable model system for further in vivo studies on delivering GAA to GSD II muscle, not only by direct cell targeting but also by a combination of secretion and uptake mechanisms.

Developmental regulation of the serpin, protease nexin I, localization during activity-dependent polyneuronal synapse elimination in mouse skeletal muscle.

During vertebrate neuromuscular development, all muscle fibers are transiently innervated by more than one neuron. Among the numerous factors shown to potentially influence the passage from poly- to mononeuronal innervation, serine proteases and their inhibitors appear to play important roles. In this regard, protease nexin I (PNI), a potent inhibitor of the serine protease, thrombin, is highly localized to the neuromuscular junction (NMJ). In turn, thrombin is responsible for activity-dependent synapse elimination both in an in vitro model, and in vivo. In the present study, we used a monospecific anti-PNI polyclonal antibody to study the developmental kinetics of PNI expression in mouse leg skeletal muscle. By using immunoblotting, we detected PNI at embryonic day 16 (E16), as a 48-kDa band. This 48-kDa PNI band became prominent in leg muscle extracts at postnatal day 5 (P5) and remained so in extracts from adult muscle. In contrast, a higher molecular weight immunoreactive PNI band, which was sodium dodecyl sulfate- and beta-mercaptoethanol-resistant, was first detected at E16, increased at birth (P0), and then decreased at P15, i.e., after the wave of polyneuronal synapse elimination had occurred in these muscles. The results of an enzyme-linked immunosorbent assay, measuring active, complexed, and truncated PNI, correlated with Western blot data. We used immunocytochemistry to probe the localization of PNI at the NMJ and found that PNI was present in the cytoplasm of myotubes at E16, but neither then nor at birth did it colocalize with acetylcholine receptors. PNI became localized at NMJs by P5 and increased by P15, after which it remained stably concentrated there in the adult. Finally, we studied the gene expression of PNI mRNA, by using Northern blotting, and showed that PNI mRNA was present in skeletal muscle and remained stable throughout the time-course studies, suggesting that developmental regulation of muscle PNI occurs principally at the translational and/or post-translational levels. These results suggest that the localization of PNI, through a binding site or "receptor" may play an important role in differentiation and maintenance of synapse.

Mouse G2-GPI AChE is processed as a membrane-bound ectoenzyme in transfected mouse sarcoma cells but is not a homophilic adhesion molecule.

Acetylcholinesterase (AChE) is mainly involved in synaptic transmission by hydrolyzing acetylcholine in the synaptic cleft. It has been suggested that it could also be involved in other functions such as cell-cell adhesion. In this study, we have expressed mouse G2-GPI AChE at the membrane surface of S180 cells. We obtained a transfected cell line which permanently expresses high levels of AChE at the cell surface. However, transfected cells behave as single cells in culture. We performed cell aggregation and adhesion tests and found no significant aggregation or adhesion, which suggests that AChE is not a homophilic adhesion molecule.

Myoblast fusion promotes the appearance of active protease nexin I on human muscle cell surfaces.

Protease nexin I (PNI) is a 43- to 50-kDa glycoprotein capable of inhibiting a number of serine proteases and belongs to the serpin superfamily. PNI is identical to glia-derived nexin, a neurite outgrowth promoter by virtue of its thrombin-inhibiting activity. Of particular relevance to neuromuscular biology and pathology, PNI was the first serpin shown to be highly localized to the neuromuscular junction and it maps to precisely the same locus as autosomal recessive amyotrophic lateral sclerosis (ALSJ) at chromosome 2q33-35. In the present report, we now show that in cultures of human skeletal muscle, PNI protein is expressed only after myoblast fusion into multinuclear myotubes and is localized in patches on their surfaces. We performed complex formation experiments with labeled thrombin, another target protease for PNI, with intact human muscle cells in culture. We detected specific SDS-stable PNI/thrombin complexes in myotube extracts only, indicating that active PNI was bound to their surfaces. We studied the gene expression of PNI mRNA using a 300-bp cDNA synthesized from the published sequence of human PNI. Confirming the protein data, upregulation of PNI appears in myotubes using Northern blot analysis. The current results reinforce the hypothesis that the regulation of the balance of serine proteases and serpins, such as PNI, is involved in muscle differentiation. They also prompt us to explore PNI abnormalities in several neuromuscular diseases, including ALSJ.

Alpha-fetoprotein binding and uptake by primary cultures of human skeletal muscle.

alpha-Fetoprotein (AFP), a serum alpha-globulin mainly synthesized by the fetal liver and the yolk sac, is the major carrier of polyunsaturated fatty acids during embryo-fetal development. One property characteristic of fetal cells undergoing growth and differentiation is their ability to bind and internalize AFP. In the present work, we have studied the binding and endocytosis of AFP by human muscular cells developing in vitro. Primary cultures of human skeletal muscle, obtained from biopsies and examined at two stages of differentiation (myoblasts and myotubes), were incubated for different times, at 0 and 37 degrees C, with a colloidal-gold-conjugated human AFP probe and studied by light and electron microscopy, as well as by laser scanning confocal microscopy in the reflection mode. The results obtained show that (a) human myoblasts in primary culture bind and internalize the protein, probably through specific AFP receptors, (b) this property is strongly reduced or lost in well-differentiated myotubes, and (c) AFP is also bound, throughout culture development, to the extracellular matrix of fusing myoblasts and differentiated myotubes. The physiological significance of AFP uptake by human myoblasts undergoing growth and differentiation may be based on the ability of AFP to carry and deliver fatty acids to fetal cells.

Serine proteinase inhibitors in human skeletal muscle: expression of beta-amyloid protein precursor and alpha 1-antichymotrypsin in vivo and during myogenesis in vitro.

The balance of serine proteases and inhibitors in nerve and muscle is altered during programmed- and injury-induced remodeling. A serpin, alpha 1-antichymotrypsin (alpha 1-ACT), and Kunitz-inhibitor containing forms of the beta-amyloid precursor protein (beta APP) may be important components of this balance. In the present study, we analyzed their expression in primary cultures of human myogenic (satellite) cells that mimic myogenic differentiation using Western blotting and immunocytochemistry. In vitro results were compared to in vivo results from normal adult human skeletal muscle biopsies. Using an anti-alpha 1-ACT polyclonal antibody, we detected a 62 kDa immunoreactive band both in cultured human myogenic cells (mononucleated myoblasts as well as multi-nucleated myotubes) and in extracts of human muscle biopsies. With a polyclonal anti-beta APP antibody we found two bands (105 and 120 kDa) in myoblasts and myotubes in culture. However, the same antibody recognized only a single band at 92 kDa in biopsies. By immunocytochemistry, both alpha 1-ACT and beta APP were indistinctly present on localized to the surface of myoblasts in culture. In contrast, these inhibitors were dense on myotube surfaces, where they often formed distinct aggregates and frequently co-localized. In permeabilized muscle cells, alpha 1-ACT and beta APP appeared to be localized to the perikarya of both myoblasts and myotubes. Confirming previous results, both alpha 1-ACT and beta APP were present at the neuromuscular junction in human muscle sections. These developmental changes found during in vitro myogenesis for alpha 1-ACT and beta APP, both serine protease inhibitors, reinforce the hypothesis that regulation of the serine proteases and serine protease inhibitors plays an important role in neuromuscular differentiation.

Apolipoprotein E expression at neuromuscular junctions in mouse, rat and human skeletal muscle.

Apolipoprotein E (ApoE)-epsilon 4 allele has been associated with late onset familial Alzheimer's disease (AD). In both familial and sporadic AD brain, ApoE is localized to the vessel walls, senile amyloid plaques, and neurofibrillary tangles. ApoE is also an 'injury-response' macromolecule in peripheral nerves and was reported to increase in response to injury. We have demonstrated that Alzheimer beta-amyloid precursor protein and a serpin alpha 1-antichymotrypsin also found accumulated in senile plaques in AD brain, were also localized at neuromuscular junctions (NMJs). Using immunocytochemistry, our present results indicate that ApoE is found in normal mouse, rat and human skeletal muscle and concentrated at the NMJs as it is in the senile plaques in AD brain. Such experiments may shed light on the mechanism of synapse loss, as well as plaque formation in this neurodegenerative disease.

[Serine proteases and their inhibitors: their role in the differentiation in neuromuscular system]. / Sérine protéases et leurs inhibiteurs: leur rôle dans la différenciation du système neuromusculaire.

It is now well established that some serine proteases, such as plasminogen activators and thrombin, as well as their inhibitors, have roles in the development of both central and peripheral nervous systems. We have shown that muscle plasminogen activators, activated after denervation, were able to digest some components of the muscle basement membrane. We have also shown that several inhibitors of serine proteases were concentrated at the neuromuscular junction. These are protease nexin, I, also called glia-derived nexin, protease nexin II, a secreted form of the beta-amyloid precursor protein (APP), and alpha 1-antichymotrypsine (ACT). These results leads us to propose a model in which serine proteases would favor plasticity of motor nerve endings during neuromuscular development. On the contrary, the inhibitors of serine proteases would act to provide and secure maintenance of the synaptic contact. A dysequilibrium between serine proteases and their inhibitors might underlie one or more motor neuron diseases.

Developmental modulation of physicochemical variants of the tailed asymmetric (16S) acetylcholinesterase by neuromuscular activity and innervation in the mouse embryo.

We have studied the physicochemical properties of acetylcholinesterase (AChE) during embryonic development of normal and functionally impaired mouse skeletal muscle, focusing on the tailed asymmetric (16S) form of the enzyme. The muscle-specific 16S AChE exists in two different variants. One is associated with extracellular matrix and is high-salt soluble (HSS, also termed hydrophilic AChE), whereas the other form is anchored to cell membranes and is detergent extractable (DE, or hydrophobic AChE). Before innervation during normal embryonic development, both hydrophilic and hydrophobic 16S AChE exist in equal amounts. After muscle innervation, there was an increase (amounting three-fold on E18) in the levels of hydrophilic vs. hydrophobic 16S AChE. This alteration of the relative proportions of the two variants of 16S AChE did not occur in chronically inactive muscles either from the mouse mutant, muscular dysgenesis, or from tetrodotoxin-treated mouse embryos. Taken together with previous reports, the present results suggest that postsynaptic membrane depolarization-induced Ca2+ fluxes are important in modulating not only the synthesis of 16S AChE, but also the relative proportions of both physicochemical variants of this molecular form of AChE.

Phosphatidylinositol is involved in the attachment of tailed asymmetric acetylcholinesterase to neuronal membranes.

1. We analyzed the mode of attachment of 16 S tailed acetylcholinesterase (AChE; EC 3.1.1.7) to rat superior cervical ganglion (SCG) neuronal membranes. Using extractions by high-salt (HS) and nonionic detergent (Triton X-100), we found two pools of 16 S AChE. 2. The detergent-extracted (DE) 16 S AChE was tightly bound to membranes through detergent-sensitive, high-salt insensitive interactions and was distinct from high-salt-soluble 16 S AChE. The detergent-extracted (DE) 16 S AChE constituted a significant proportion of about one-third of the total 16 S AChE. 3. Treatment of the neuronal membranes by a phosphatidylinositol-specific phospholipase C (PIPLC) resulted in the release of some, but not all DE 16 S AChE, indicating that a significant amount of the neuronal DE 16 S AChE, about one-third, is anchored to membranes through a phosphatidylinositol containing residue. Thus, a covalent association of a glycolipid and catalytic or structural AChE polypeptidic chains occurs not only for dimeric AChE but also for the asymmetric species of AChE. 4. The complex polymorphism of AChE is due not only to different globular or asymmetric associations of catalytic and structural subunits but also to the alternative existence of a transmembrane domain or a glycolipid membrane anchor.

Cell modulation of hydrophobic tailed 16S acetylcholinesterase by intracellular calcium in rat superior cervical ganglion neurons.

In primary cell cultures of rat superior cervical ganglia (SCG) the tailed asymmetric 16S molecular form of acetylcholinesterase (AChE) possesses hydrophilic (high-salt soluble, HSS) and hydrophobic (detergent extracted, DE) variants. Hydrophobic tailed acetylcholinesterase is associated with membranes through a glycolipid anchor. In the presence of tunicamycin, an antibiotic which inhibits protein glycosylation, the cellular amount of the hydrophobic DE 16S AChE is increased. Exposure of the cells to the calcium ionophore A 23187 leads to a decrease in DE 16S AChE and a correlated increase in hydrophilic HSS 16S AChE. These results suggest the existence of an endogenous processing of tailed AChE, transforming the hydrophobic variant into an hydrophilic one controlled through glycosylation and intracellular calcium.

A dimeric form of acetylcholinesterase anchored through a glycolipid in mouse skeletal muscle.

A glycolipid anchorage for acetylcholinesterase (AChE) has been found in some tissues. In this paper, the possibility of such an anchorage has been explored in mammalian muscle membranes. We report that a phosphatidylinositol-specific phospholipase C (PIPLC) solubilizes AChE from microsomal membranes of mouse intercostal muscle. Among the several molecular forms of AChE, PIPLC specifically releases in a dose dependent manner one molecular form which migrates on linear sucrose gradients as a single peak of sedimentation coefficient 6.3 s. In other subcellular membrane fractions, including motor endplate enriched fraction, PIPLC fails to solubilize AChE. This type of membrane glycolipid mediated anchorage for AChE is then only detectable in a precise region of skeletal muscle.

Hydrophilic and hydrophobic attachment of both globular and asymmetric acetylcholinesterase to frog muscle basal lamina sheaths.

We prepared myofiber basal lamina sheaths (BLs) using the in vivo experimental procedure of Sanes et al. (J. Cell Biol.78, 176-198, 1978) on frog cutaneus pectoris muscle. On the 15 days post-operatively, acetylcholinesterase (AChE) is still found concentrated in native BLs and purified BLs preparations and both globular and asymmetric molecular forms coexist (Nicolet et al., J. Cell Biol., 107, 762-768, 1986). We describe here at least two distinct AChE pools, according to their differential solubility in non-ionic detergent and high-salt media. One is detergent-extracted (DE) and the other is detergent-insoluble, high-salt extracted (HSS). In the BLs preparation as well as in control motor end-plate rich regions (MEP-r) of muscle, both globular and asymmetric forms of AChE are found as DE and HSS variants. These observations suggest that all AChE forms are present in the extracellular muscle basal lamina and are bound through not only hydrophilic but also hydrophobic bonds, to probably distinct structural domains of the muscle basal lamina.

External and internal acetylcholinesterase in rat sympathetic neurones in vivo and in vitro.

The subcellular distribution of multiple molecular forms of acetylcholinesterase (AChE) in neurones of rat superior cervical ganglion (SCG) was determined both in vivo and in vitro by the use of selective lipid-soluble or -insoluble inhibitors. In vivo as well as in vitro, 10 S AChE is mainly outside the cell. In primary cultures of rat SCG neurones, both 4 S and 16 S AChE are mainly inside the cell. In near-term rat SCG, 4 S and 16 S are more external to the cell than in primary cultures. In adult rat SCG, 4 S AChE is equally distributed inside and outside and 16 S AChE is mainly outside the cell. Thus, specific AChE externalization probably occurs in neuronal cells as a developmentally regulated process.