Characterization and partial purification of a neuronal factor which increases acetylcholine receptor aggregation on cultured muscle cells

Medium conditioned by NG108-15 neuroblastoma × glioma hybrid cells contains a factor which increases the number of acetylcholine receptor (AChR) aggregates on cultured myotubes. Protease digestion indicates that the AChR aggregation factor is a protein, and the molecular weight is from 150,000 to 250,000 daltons as estimated by ultrafiltration and gel filtration. Preparative isoelectrofocusing indicates that the aggregation factor has a pI of about 4.7. The factor is found in the soluble cytoplasmic fraction but not in the plasma membrane fraction of NG108-15 cells. Aggregation activity is not detected in the cytoplasm of liver cells or in the cytoplasm of C6BU-1 glioma cells. A possible developmental role for the aggregation factor is suggested by its presence in embryonic rat brain but not in adult rat brain. AChR aggregation factors found in the cytoplasm or conditioned medium of NG108-15 cells or in the cytoplasmic fraction of fetal brain have similar molecular weights and isoelectric points.     

Nicotinic acetylcholine receptor from foetal human skeletal muscle

Nicotinic acetylcholine receptor protein has been purified from foetal and adult skeletal muscle by extraction in non-ionic detergent followed by purification on immobilised α-toxin. Purified foetal and adult receptors focused as single, sharp peaks whether directly labelled with ¹²⁵I or indirectly labelled with ¹²⁵I-α-bungarotoxin. Polyacrylamide gel electrophoresis of the purified foetal and adult receptors each showed four major protein bands with M_r 44,000, 51,000, 58,000 and 66,000; only that with M_r 44,000 was, in each case, labelled with the affinity reagent, 4-(N-maleimido) [³H]benzyltrimethylammonium. When the four major subunits, obtained by polyacrylamide gel electrophoresis, were labelled with ¹²⁵I-ConA, markedly different patterns of radioactivity were shown by the foetal and adult receptors, the band at 44,000 being less heavily labelled in the foetal case. Foetal and adult receptors behaved similarly with respect to inhibition by ConA of binding of ¹²⁵I-α-bungarotoxin; inhibition in both cases reaching a maximum of 70%. Foetal and adult receptors each showed single ¹²⁵I-α-bungarotoxin binding species in sucrose density gradient centrifugation with S_20w = 8.5Sand 9.5S, respectively, although the former peak was broader, possibly reflecting the relative instability of the purified foetal receptor. Our finding of marked differences in the glycosylation of foetal and adult human acetylcholine receptors suggest that, in otherwise very similar proteins, the carbohydrate moieties could determine the known differences in location and stability of the two receptor types.     

A maturational increase in rat neuromuscular junctional acetylcholine receptors despite disuse or denervation

Junctional acetylcholine receptors (AChR) of rat skeletal muscles were microassayed 4–7 days after denervation or total disuse. The normal growth-related increase in AChR number occurred despite denervation or muscle atrophy, but in disused muscles, this increase was less. Thus, at least for short periods, the developmental addition of junctional AChR is independent of muscle fiber size or innervation and partly independent of usage.     

An increase in intracellular free calcium ions by nicotinic acetylcholine receptors in a single cultured rat cortical astrocyte

Neuronal nicotinic acetylcholine receptors (nAChRs) are composed of an assembly between at least seven alpha (alpha2-alpha7, alpha9) and three beta (beta2-beta4) subunits in mammals. The addition of 50 mM KCl or 1 mM nicotine immediately increased the number of cells with high fluorescence intensity in rat cortical astrocytes on fluo-3 fluorescence measurement. Nicotine was effective at increasing the fluorescence intensity in astrocytes cultured for 2 days after replating, but not in those used 1 or 5 days after replating, without markedly affecting the cellular viability irrespective of the exposure period. Nicotine markedly increased the fluorescence intensity in a concentration-dependent manner at a concentration range of 10-100 microM in cultured astrocytes when analyzed on a responsive single cell. In these responsive single cells, the increase by nicotine was significantly prevented by the heteromeric alpha4/beta2 subtype antagonist dihydro-beta-erythroidine and the homomeric alpha7 subtype antagonist methyllycaconitine, as well as by nifedipine and EGTA but not thapsigargin. Methyllycaconitine failed to inhibit further the increase by nicotine in the presence of nifedipine, however, whereas the expression of mRNA was seen for all mammalian neuronal nAChR subunits in cultured rat cortical astrocytes as well as neurons. These results suggest that nicotine may increase intracellular free Ca2+ through the influx of extracellular Ca2+ across L-type voltage-gated Ca2+ channels rather than Ca2+ release from intracellular stores, in a manner related to the alpha4/beta2 and/or alpha7 nAChR channels functionally expressed in cultured rat cortical astrocytes.     

Specific effect of corticoids on acetylcholine receptor expression in rat skeletal muscle cell cultures.

The potential effect of different classes of steroids on the expression of acetylcholine receptors (AChR) was studied in different primary cultures of newborn-rat skeletal muscle cells. Comparison among three techniques for preparing newborn skeletal muscle cells showed that these systems were equivalent to study AChR expression. Only corticoids stimulated myogenesis as a twofold increase in AChR expression indicated. Among the corticoids, the glucocorticoids were the more potent, whereas the mineralocorticoid aldosterone had less marked effect. The sex hormones progesterone and testosterone partially blocked these effects, without inducing any significant effect when given alone. The steroids tested differed in efficacy in correlation with their different chemical structures. Among the glucocorticoids a clear structure-activity relationship could be established. These results emphasize the specificity of corticoid action on muscle cells and suggest an explanation for the effects induced by glucocorticoids used in treating human muscular or neuromuscular diseases.     

Relationship of a dystrophin-associated glycoprotein to junctional acetylcholine receptor clusters in rat skeletal muscle

The relationship of a member of the transmembrane dystrophin-associated glycoprotein (DAG) complex to acetylcholine receptors (AChRs) was investigated using immunofluorescence techniques at rat neuromuscular junctions (NMJs) viewed en face. These results were compared with those from a similar previous study of dystrophin and an autosomal homologue (utrophin or dystrophin-related protein, DRP) (Bewick et al. NeuroReport 1992; 3; 857–860). The region of highest 43 K DAG (43DAG) labelling projected beyond the AChRs by 0.3 μm, as does that for dystrophin. By contrast DRP labelling precisely co-localizes with the AChRs. These results suggest that at the NMJ, the region of high 43DAG concentration encompasses the area of highest intensity labelling for both DRP and dystrophin.     

Muscarinic acetylcholine receptors on cultured glia cells

Muscarinic acetylcholine receptors (76 fmol/mg protein) were detected on cultured glia cells (astroblasts) from embryonic chicken brain by specific [³H]quinuclidinylbenzilate (QNB) binding at physiological conditions. The QNB binding (K_d = 9.5 × 10⁻¹¹) to the intact cells seems to be cooperative (n_H= 1.98) as shown by graphical methods.     

Acetylcholine receptors of human skeletal muscle: a species difference detected by snake neurotoxins

The binding abilities of the nicotinic acetylcholine receptors (AChRs) of the skeletal muscles of man and other vertebrates to two typical curaremimetic toxins, erabutoxin b (Eb) and alpha-bungarotoxin (alpha-BT), were investigated. Fluorescent microscopy using rhodamine-labeled erabutoxin b (TMR-Eb) and FITC-labeled alpha-bungarotoxin (FITC-alpha-BT) revealed that AChRs of human and chimpanzee muscles were stained with FITC-alpha-BT, but not with TMR-Eb. In contrast, the AChRs of mouse muscle were stained with both fluorescent toxins. The stainings of human and chimpanzee AChRs with FITC-alpha-BT were inhibited by preincubation with unmodified alpha-BT, but not with either unmodified Eb or other short-chain neurotoxins. Binding experiments using 125I-labeled Eb ([125I]Eb) and 125I-labeled alpha-BT ([125I]alpha-BT) showed that the affinity of human AChRs for [125I]Eb was unusually low. Electrophysiological experiments showed that both acetylcholine potential and end-plate potential of human muscle were blocked by addition of alpha-BT, but not by Eb. On the contrary, acetylcholine potential of rat muscle was blocked by addition of Eb. All these results indicate that AChRs of human and chimpanzee muscles are different from those of other animals in having an exceptionally low affinity for Eb and other short-chain neurotoxins. The results suggest a heterogeneity among vertebrate AChRs concerning their reactivities to curaremimetic toxins.     

Qualitative and quantitative changes in acetylcholine receptor distribution at the neuromuscular junction following free muscle transfer

The qualitative and quantitative changes in acetylcholine receptor distribution were studied in the gracilis muscle of the Wistar rat following free neurovascular transfer. Even at 30 weeks after transfer, the morphology of the neuromuscular junction failed to return to the presurgical state. The number of acetylcholine receptors at the reinnervated neuromuscular junction also remained lower than the control. The persistent weakness following free neurovascular muscle transfer may be attributed to these qualitative and quantitative changes at the neuromuscular junction.     

The acetylcholine receptors of human muscles are not stained with rhodamine-labeled erabutoxin-b

The skeletal muscles from man and other vertebrates were incubated in rhodamine-labeled erabutoxin-b (TMR-Eb), and the distribution of the acetylcholine receptors (AChRs) at the neuromuscular junctions was examined under a fluorescence microscope. The AChRs of human muscles were not stained with TMR-Eb, whereas those of fish, frog, chicken, mouse, rat, cat and monkey muscles were stained under the same conditions. It was concluded that human AChRs have low or no binding ability for TMR-Eb.     

Botulinum toxin converts muscle acetylcholine receptors from adult to embryonic type

To assess the postsynaptic consequences of botulinum toxin injection into muscle we characterized the nicotinic acetylcholine receptor (nAChR) with the patch clamp technique, using adult mouse muscle after destruction of the nerve ending and after treatment with botulinum toxin (BoTX). In both, embryonic channels with a conductance of 30 and 34 pS could be identified, whereas on adult control muscle nAChR channels had a conductance of 48 pS. The mean open times were 1.2 ms for the channels in control, 2.5 ms in denervated, and 2.4 ms in BoTX-treated muscle. The dose–response curves of the maximal acetylcholine-elicited currents showed a K_m of 60 μmol/L for denervated, 70 μmol/L for BoTX-treated, and 100 μmol/L for control muscle. Destruction of the nerve ending and inhibition of acetylcholine release by BoTX has the same effect as far as the increase of sensitivity of the muscle to acetylcholine is concerned. In contrast to single-fiber EMG findings in patients treated for focal dystonia no distant changes could be found in the control muscle of the BoTX-treated animals. © 1994 John Wiley & Sons, Inc.     

Differential responses of L5 and rat primary muscle cells to factors in rat brain extract

Crude brain extract (100,000 g supernate from newborn or fetal rat brain homogenate) was studied for its effects on the number and distribution of acetylcholine receptors (AChRs) on myotubes of the L5 cloned myogenic cell line and compared to that of rat primary cultures. Gamma counting, light autoradiography and scanning electron microscopic autoradiography were used. We found that the L5 cells responded to the brain extract with an increase in the average AChR site density (2-5-fold) and with an increase in AChR clustering. Clustering was manifested by both an increase in the number of AChR clusters and in the ratio of receptor site density within clusters relative to that between clusters. The increase in average AChR site density was shown to be due to an increase in the rate of AChR insertion into the surface membrane with little change in the rate of receptor degradation. As also previously reported, the rat myotubes had a similar clustering response but only a very slight (approximately 1.2-fold) increase in average AChR site density. The surface area of myotubes was also increased slightly (approximately 1.2-1.3-fold) by the brain extract. Autoradiography viewed by scanning EM was found to be very useful in illustrating the shape and distribution of the receptor clusters. After the brain extract was fractionated on Sephadex G-200, the fractions with greatest clustering activity could be separated from those causing predominantly an increase in receptor site density. Increased receptor site density was primarily produced by the low molecular weight fractions (less than 12 kD), whereas the strongest (but not exclusive) effect on clustering was produced by the high molecular weight fractions (greater than 140 kD). Furthermore, the two cell types assayed had different sensitivity to the different factors. L5 cells responded to both the high and low molecular weight factors while rat primary cells are sensitive primarily to the high molecular weight factors.     

Influence of thyroid hormone on some electrophysiological properties of developing rat skeletal muscle cells in culture

The metabolic activity in the brains of adult spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY), and Wistar (W) rats was assessed before and after fasting using cytochrome oxidase (COX) histochemistry. Before fasting, metabolic activity in the paraventricular (PVH) and supraoptic (SON) nuclei of the hypothalamus in SHR was greater than in control rats. Fasting elicited a decrease in arterial pressure (AP) in SHR and WKY; in SHR the decrease in AP was accompanied by a decrease of metabolic activity in the PVH and SON. The findings of this study support the hypothesis that the PVH and SON are involved in the hypertension and in the increased levels of sympathetic nervous activity and vasopressin production known to occur in SHR. In addition, the PVH and possibly the SON may be involved in the suppression of sympathetic nervous system activity and the lowering of arterial pressure which are associated with fasting.     

Mechanism of action of lithium on acetylcholine receptor metabolism in skeletal muscle

Changes in the levels of cations within skeletal muscle are thought to mediate the neural regulation of turnover of extrajunctional acetylcholine receptors (AChRs). We have used lithium as a probe of these cation influences because of its resemblance to calcium and other ions. In the present experiments we studied the mechanism of action of lithium on AChR metabolism in cultured mammalian skeletal muscle. We measured the effects of lithium on AChR turnover (using [125I]alpha-bungarotoxin binding), and evaluated the resemblance of lithium and calcium in producing their effects on AChR metabolism. Our results provide insight into the mechanisms of action of lithium and the cellular processes controlling AChR metabolism in muscle. Lithium reduces the number of AChRs in skeletal muscle in vitro to a degree similar to that which we previously reported in vivo. Lithium appears to enter cells via both sodium and calcium channels. It then produces its effect on levels of AChRs primarily by selectively reducing AChR synthesis and insertion into the surface membrane. Lithium induces this change in AChR metabolism in a manner resembling neural and calcium-mediated effects on AChRs. Phosphoinositide pathways may be involved in the lithium-induced effects. Further analysis of the effects of lithium on AChR turnover should provide new information about the mechanisms underlying the cellular control of receptor metabolism.     

Reduced acetylcholine receptor density, morphological remodeling, and butyrylcholinesterase activity can sustain muscle function in acetylcholinesterase knockout mice

Nerve-evoked contractions were studied in vitro in phrenic nerve-hemidiaphragm preparations from strain 129X1 acetylcholinesterase knockout (AChE-/-) mice and their wild-type littermates (AChE+/+). The AChE-/- mice fail to express AChE but have normal levels of butyrylcholinesterase (BChE) and can survive into adulthood. Twitch tensions elicited in diaphragms of AChE-/- mice by single supramaximal stimuli had larger amplitudes and slower rise and decay times than did those in wild-type animals. In AChE-/- preparations, repetitive stimulation at frequencies of 20 and 50 Hz and at 200 and 400 Hz produced decremental muscle tensions; however, stimulation at 70 and 100 Hz resulted in little or no loss of tension during trains. Muscles from AChE+/+ mice maintained tension at all frequencies examined but exhibited tetanic fade after exposure to the selective AChE inhibitor 1,5-bis(4-allyldimethyl-ammoniumphenyl)pentane-3-one (BW 284C51). The ability of diaphragm muscles from AChE-/- mice to maintain tension at 70 and 100 Hz suggests a partial compensation for impairment of acetylcholine (ACh) hydrolysis. Three mechanisms--including a reliance on BChE activity for termination of ACh action, downregulation of nicotinic acetylcholine receptors (nAChRs), and morphological remodeling of the endplate region--were identified. Studies of neuromuscular transmission in this model system provide an excellent opportunity to evaluate the role of AChE without complications arising from use of inhibitors.     

Characterization of putative nicotinic acetylcholine receptors solubilized from rat brains

A membrane component of rat brains which binds [3H]acetylcholine in the presence of eserine and atropine was solubilized by various detergents and characterized. The affinity of this component for [3H]acetylcholine was increased 2-3-fold upon dissolution with non-ionic detergents. Pharmacological specificity of this component suggested its nicotinic cholinergic nature. The component cross-reacted with antisera raised against nicotinic acetylcholine receptors of Torpedo marmorata or Narke japonica. The component interacted with wheat germ agglutinin-conjugated Sepharose 4B, suggesting that it may be a glycoprotein, but did not bind appreciably to alpha-bungarotoxin-conjugated Sepharose 4B. Conversely, alpha-bungarotoxin binding component in the same preparation, which was bound to the toxin-conjugated gels, did not bind [3H]acetylcholine. These results demonstrate that nicotinic acetylcholine binding component and alpha-bungarotoxin binding component in brain membranes are distinct molecules and can be separated from each other.