Evidence for the presence of immunoreactive acetylcholine receptors on human thymus cells

Triton extracts from thymuses and thymomas from either normal subjects or MG patients specifically bind [125I] alpha-bungarotoxin (alpha Bgt). This binding is saturable (apparent Ks = 5.9 X 10(-9) M) and can be inhibited by cold alpha Bgt, acetylcholine or nicotinic agonists. These results demonstrate the presence on human thymus cells of acetylcholine receptor (ACh.R), 2.8% for normal human thymus in regard of ACh.R concentration in human muscle. Normal human thymic extracts were used as an antigen source in the radioimmunoassay for the detection of anti-ACh.R antibodies in positive MG patients' sera. 72% of these sera were also positive with the thymic antigen. The demonstration of ACh.R in human thymus brings further support to the hypothesis of intrathymic autosensitivity against ACh.R as a major factor in MG pathogenesis.     

Barbiturates block divalent cation action potentials in leech nociceptive cells

Phenobarbital (PNB), pentobarbital (PTB) and methohexital (MTX) decreased the maximum rate of depolarization Vmax and duration of divalent cation action potentials elicited in leech nociceptive neurons in Na+-free solutions containing the K+-channel blocker TEA, without significantly affecting resting membrane potential or conductance. The block of the divalent cation action potentials was reversible and dose-dependent, ED50 for inhibition of Vmax being 560 microM for MTX, 800 microM for PTB and 3000 microM for PNB. This order of potency correlated well with the ratio of unchanged/charged form of the drugs at physiological pH suggesting that in leech, as in other preparations, the non-ionized form was the active one. In Na+-containing Ringer, the 3 barbiturates depolarized and decreased membrane resistance in the lateral nociceptive cells, but not the medial nociceptive cells. These results provide additional information regarding the newly described pharmacological differences among closely related neurons. These membrane actions may be related to some of the excitatory properties described for other barbiturates in invertebrate and mammalian preparations.     

Expression of functional mouse muscle acetylcholine receptors in Chinese hamster ovary cells

CHO cells were transfected with cDNAs for all 4 subunits of the mouse muscle acetylcholine receptor (AChR) to obtain a stable cell line (CHO-AR) expressing the AChR on its surface. Immunoprecipitation experiments established that the AChR contained alpha- and beta-subunits assembled with gamma- and/or delta-subunits. In addition, one of the toxin-binding sites of the AChR was blocked by a myasthenic serum that specifically recognizes AChR containing the gamma-subunit. AChR from the CHO-AR cells had the same sedimentation rate, association rate constant for the binding of alpha-bungarotoxin (alpha BTX), and the same metabolic half-life as the AChR in myotubes of the mouse muscle cell line C2C12. Electrophysiological assay of CHO-AR cells by single-channel recording showed the presence of ACh-responsive ion channels with the characteristics of the embryonic AChR (gamma = 40 pS, tau = 5.6 msec). In some patches a smaller conductance channel was also seen that may represent partially assembled receptor. Fluorescence microscopy of fixed, permeabilized cells stained with rhodamine-alpha-BTX demonstrated both perinuclear and diffuse surface staining. The expression of fully assembled, functional mammalian muscle AChR in nonmuscle cells will allow detailed investigation of its properties and interactions with other cellular components.     

Metabolism of acetylcholine receptors on embryonic amphibian muscle

The functional properties of acetylcholine receptors are altered during the development of Xenopus myotomal muscle. A similar change also occurs in dissociated cell cultures of embryonic myotomal muscle. Metabolism of acetylcholine receptors was examined in order to ascertain whether the turnover rate changed at a time when the functional changes were detected. Incorporation of newly synthesized receptors occurred at a constant rate over a 24-hr period. The degradation of receptors followed first order kinetics with a half-life of 52 hr. Incorporation and degradation occurred at approximately equal rates such that 1 to 1.5% of the total receptor number was replaced every hour at equilibrium. Addition of neural tube cells to muscle cell culture, which altered the functional properties of channels, had no effect on either the incorporation or the degradation rate. We conclude that the changes in the functional properties of receptors occur independent of changes in the turnover rate of receptors.     

Desensitization of acetylcholine receptors in rat myotubes is enhanced by agents that elevate intracellular cAMP

Incubating skeletal muscle fibers with forskolin, an activator of adenylate cyclase, increases the rate at which nicotinic acetylcholine receptors (AChRs) desensitize when exposed to ACh. Several reports indicate that this is due to the phosphorylation of AChRs by cAMP-dependent protein kinase, but other studies suggest that forskolin interacts with AChRs directly and that second-messenger systems are not required. To help clarify this issue, we studied the effects of forskolin and several other drugs on AChR function in embryonic rat myotubes. AChR function was studied by recording ACh-induced membrane depolarizations and ACh-induced single-channel currents. Our results indicate that forskolin at low concentrations enhances AChR desensitization through the action of a second messenger, most likely cAMP. An analog of forskolin that is much less effective in activating adenylate cyclase (1,9-dideoxyforskolin) is also much less potent in enhancing desensitization. Forskolin at low concentrations does not alter single-channel conductance or mean channel open time. However, when used at concentrations above 20 microM, forskolin may also exert direct drug effects on AChRs.     

Mumps virus alters aggregation of acetylcholine receptors in cultured rat skeletal muscle cells

Cultured myoblasts, but not myotubes, from rat skeletal muscles were infected with the RW strain of mumps virus. Such myoblasts then fused to form myotubes containing viral antigen. The infected myotubes showed a significant decrease in the number of dorsal, linear acetylcholine receptor (AChR) aggregates as determined by FITC-conjugated alfa-bungarotoxin. Infected myotubes co-cultivated with spinal cord cells showed no increase in the number of dorsal, linear AChR aggregates, compared to normal, uninfected myotubes. In addition, an increased proliferation of the myoblasts, which remained uninfected in the infected cultures, was noted. This may indicate a release of a growth stimulating factor from the virus containing cells. This study shows that mumps virus infection can lead to an altered receptor organization in a morphologically preserved cell.     

Far-field potentials in the compound muscle action potential

It has long been believed that the compound muscle action potential (CMAP) in motor-nerve conduction studies (MCSs) records the action potential beneath the active electrode over the muscle belly. However, recent studies have revealed the contribution of the reference electrode to the CMAP, most prominent in the tibial nerve, followed by the ulnar nerve. This “reference electrode potential” is recorded when the conventional reference electrode distal to the muscle belly is connected to a proximal reference. It must be a far-field potential (FFP) considering its distribution, although the precise mechanism of its generation has not been clarified. The conventional theory of termination of the action potential at the muscle-tendon junction is insufficient. Regarding the ulnar CMAP, interosseous muscles mostly contribute to the FFPs. New understanding of CMAP based on the FFP theory may provide new insights into the interpretation of MCSs and related techniques, including motor unit number estimation.     

Mode of cembranoid action on embryonic muscle acetylcholine receptor

The mechanism of eupalmerin acetate (EUAC) actions on the embryonic muscle nicotinic acetylcholine receptor (nAChR) in BC3H-1 cells was studied by using whole-cell and single-channel patch-clamp current measurements. With whole-cell currents, EUAC did not act as an agonist on this receptor. Coapplication of 30 microM EUAC with 50 microM, 100 microM, or 500 microM carbamoylcholine (CCh) reversibly inhibited the current amplitude, whereas, with 20 microM CCh, current was increased above control values in the presence of EUAC. EUAC concentration curves (0.01-40 microM) obtained with 100 microM and 500 microM CCh displayed slope coefficients, n(H), significantly smaller than one, suggesting that EUAC bound to several sites with widely differing affinities on the receptor molecule. The apparent rate of receptor desensitization in the presence of EUAC and CCh was either slower than or equal to that obtained with CCh alone. The major finding from single-channel studies was that EUAC did not affect single-channel conductance or the ability of CCh to interact with the receptor. Instead, EUAC acted by increasing the channel closing rate constant. The results are not consistent with the competitive model for EUAC inhibition, with the sequential open-channel block model, or with inhibition by increased desensitization. The data are best accounted for by a model in which EUAC acts by closed-channel block at low concentrations, by positive modulation at intermediate concentrations, and by negative allosteric modulation of the open channel at high concentrations.     

Polymyositis: reduction of acetylcholine receptors in skeletal muscle

In polymyositis (PM), neither the precise target nor the mechanism of the attack against skeletal muscle have yet been defined. In this study, we report evidence of a pathologic process involving a membrane component of muscle, acetylcholine receptors (AChRs). Our results show that PM patients have significantly reduced AChRs at neuromuscular junctions, averaging 55% below control values (P less than 0.01). Incubation of cultured mammalian muscle cells with sera from PM patients significantly reduced (P less than 0.005) the number of surface AChRs and increased their rate of degradation in 7/8 cases (P less than 0.005). Similar effects were produced by purified IgG from PM patients. These results demonstrate the presence in PM patients of circulating IgG with functional effects on a component of the surface membrane of skeletal muscle. They suggest that PM and myasthenia gravis may have important features in common.     

Quantification of extrajunctional acetylcholine receptors in human muscle biopsies

Extrajunctional acetylcholine receptors were quantified in human muscle biopsies by in vitro autoradiography with 3H-alpha-bungarotoxin (alpha-Btx). Specific alpha-Btx binding was found in seven of eight biopsies with histological and/or histochemical signs of denervation. The highest binding (21 fmol/mg) was seen in the biopsy with the most pronounced signs of denervation. The specimens with co-existing signs of innervation had generally lower binding levels. Four of the control biopsies showed low binding (less than or equal to 1 fmol/mg) while no specific binding could be detected in the other six controls. The findings suggest that this method cannot only be used to demonstrate denervation in muscle biopsy specimens, but also to give a quantitative measure of the changes.     

Localization of acetylcholine receptors on isolated CNS neurons: cellular and subcellular differentiation

Significant progress has been made in determining regional expression of neurotransmitter receptors within the CNS, but little information is available at the neuronal level. In the current study, to begin characterizing cellular and subcellular aspects of receptor differentiation, we have localized ACh receptors on neurons isolated from the chicken CNS. Localization was determined autoradiographically using 2 cholinergic receptor probes: 3H-propylbenzilylcholine mustard (PrBCM) a muscarinic antagonist, and 125I-alpha-bungarotoxin (BTX), a putative nicotinic ligand in the avian CNS. To isolate neurons, we incubated embryonic chicken retinas (E13-E19) in Ca2+/Mg2+-free buffer containing 8 units/ml papain for 20 min and then gently agitated the tissue by trituration. Large numbers of dendrite-rich neurons, belonging to recognizable morphological subpopulations (e.g., multipolar neurons of various sizes, small bipolar and unipolar neurons), were collected on slides for autoradiography. Cell isolation had no effect on ligand binding levels, and a high ratio of specific to nonspecific binding allowed us to associate silver grains with receptor position. Muscarinic-receptor-positive cells comprised a recognizable subpopulation that had small rounded cell bodies (6-7 micron) and a single emergent arbor. The cells had an axial or elongated appearance. Muscarinic receptors were abundant over dendrites but absent from cell bodies. Segregation to dendrites was complete by E13, the age when synapses reportedly first begin to appear. Cells labeled with 125I-BTX were more heterogeneous in morphology. The most striking BTX-positive cells comprised neurons with large cell bodies (approximately 15 micron) and multiple processes. Dendrites were profusely labeled, but only sporadic labeling was seen on cell bodies, and often this was at sites crossed by labeled dendrites. Maximum labeling occurred in the distal, smallest-caliber ends of the dendritic arbors. All limbs of BTX-positive multipolar neurons expressed abundant receptors. Occasionally, a thin uniform-caliber process was seen branching from a primary dendrite, and such processes, which may have been axons, were never labeled. Very small neurons with bipolar morphology also showed minimal or no labeling on one process, despite dense labeling on the other. Photoreceptors and Mueller cells were never labeled with BTX or PrBCM. The data show that, within a discrete CNS region, specific subpopulations of neurons independently regulate expression of ACh receptors and that, even early in development, control mechanisms segregate receptors to physiologically appropriate regions of the cell surface.     

Mobility of extrajunctional acetylcholine receptors on denervated adult muscle fibers

We report the lateral mobility of extrajunctional acetylcholine receptors (AChR), marked with fluorescently labeled alpha-bungarotoxin, on rat flexor digitorum brevis single muscle fibers maintained in cell culture. Mobility is measured by a modification of the fluorescence photobleaching recovery technique. The denervated rat flexor digitorum brevis muscle fibers exhibit a nonuniform distribution of AChR on their sarcolemmas. Diffusely distributed AChR have an average diffusion coefficient of about 2.5 X 10(-11) cm2/sec, somewhat lower than that of diffusely distributed AChR on embryonic rat myotubes in culture. Extrajunctional AChR aggregated into small clusters (less than 20 microns) have an average diffusion coefficient of about 5 X 10(-12) cm2/sec. Both the clustered and nonclustered extrajunctional AChR have lateral mobilities several orders of magnitude greater than that of junctional AChR.     

Estimating the duration of intracellular action potentials in muscle fibres from single-fibre extracellular potentials

In situ recording of the intracellular action potential (IAP) of human muscle fibres is not yet possible, and consequently, knowledge concerning certain IAP characteristics is still limited. According to the core-conductor theory, close to a fibre, a single fibre action potential (SFAP) can be assumed to be proportional to the IAP second derivative. Thus, we might expect to be able to derive some characteristics of the IAP, such as the duration of its spike, from the SFAP waveform. However, SFAP properties not only depend on the IAP shape but also on the fibre-to-electrode (radial) distance and other physiological properties of the fibre. In this paper we, first, propose an SFAP parameter (the negative phase duration, NPD) appropriate for estimating the IAP spike duration and, second, show that this parameter is largely independent of changes in radial distance and muscle fibre propagation velocity. Estimation of the IAP spike duration from a direct measurement taken from the SFAP waveform provides a possible way to enhance the accuracy of SFAP models. Because IAP spike duration is known to be sensitive to the effects of fatigue and calcium accumulation, the proposed SFAP parameter, the NPD, has potential value in electrodiagnosis and as an indicator of IAP profile changes due to peripheral fatigue.     

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.     

Effect of desynchronized inputs on compound sensory and muscle action potentials

Stimulation of the second (S1) or third (S2) digit elicits a median sensory potential at the wrist. Similarly, a shock applied to the median (Sm) or ulnar (Su) nerve at the wrist evokes a sensory potential of the fourth digit and a muscle potential over the thenar eminence. Hence, a concomitant application of S1 and S2 or Sm and Su with varying interstimulus intervals simulates the effect of desynchronized inputs. In 10 hands, a shift in latency on the order of 1 msec between S1 and S2 or Sm and Su caused a major reduction in sensory potential by as much as 30-40% but little change in muscle action potential. A latency difference slightly less than one-half the total duration of unit discharge maximized the phase cancellation between the two components and consequently the loss of area under the waveform.