Effects of innervation on the distribution of acetylcholine receptors on cultured muscle cells.

1. Myotomal muscle cells from embryos of Xenopus laevis were cultured as a monolayer either alone or together with neural tube cells from the same embryos. 2. Spontaneous twitching and contractions evoked by electrical stimulation of neural perikarya were observed only in nerve-contacted muscle cells, and could be abolished by curare or alpha-bungarotoxin. 3. Within 2 days in culture muscle cells not contacted by nerve developed one or more discrete patches of acetylcholine (ACh) receptors as revealed by staining with fluorescent conjugates of alpha-bungarotoxin. Similar patches were also seen when staining was carried out after paraformaldehyde fixation, suggesting that they were not induced by the dyetoxin conjugate. 4. Radioautography after labelling with [125I]alpha-bungarotoxin revealed patches with grain densities approximately twenty-five-old greater than over the remainder of the cell. 5. Fluorescent stain on innervated cells was restricted to the path of nerve-muscle contact and sometimes extended for greater lengths than the largest patches seen on non-contacted muscle cells. 6. Similar long bands of stain associated with nerve--muscle contacts were observed when cultures were grown in high concentrations of curare and carbachol which prevented spontaneous twitching. They were also seen in cultures in which the addition of neural tube cells was delayed for 2-3 days. 7. It is concluded that innervation caused receptors to accumulate at sites of nerve-muscle contact and that this process can operate independently of muscle activity.     

The influence of basic fibroblast growth factor on acetylcholine receptors in cultured muscle cells.

Acetylcholine receptors (AChRs) in Xenopus muscle cells undergo changes in channel kinetics during development in culture and these changes are somehow related to innervation. Recently we showed that basic fibroblast growth factor (bFGF), when locally applied, can mimic the effect of nerve in inducing a postsynaptic-type development. In this study, we examined whether bFGF can influence the developmental changes of AChRs. Patch clamp method was employed to record single AChR channel currents from cultured Xenopus myotomal muscle cells and the kinetics of low-conductance AChR channels were analyzed. In cultures treated with 1 microgram/ml bFGF at an early stage (stage 23), the burst duration of low-conductance AChR channels showed a 1.5-fold decrease between the first and second day in culture, while it underwent a remarkable 3-fold decrease during the same period in the control. Histogram analyses showed that the low-conductance channels were composed of a fast and a slow component and that the decrease in burst duration was due to a shift in the population from the slow to the fast. bFGF treatment appeared to slow down this shift by retaining the slow channels for a longer period of time. This effect is probably not due to channel modulation as the burst duration of short channel in older cells (stage 40) was not affected by bFGF. These data suggest that bFGF may enhance the metabolic stability of intrinsically short-lived AChRs. This effect seems to parallel the stabilization of junctional AChRs at the innervated endplate. Thus, bFGF, or a related polypeptide growth factor, may mediate this and other innervation-induced changes in the postsynaptic membrane.     

Control of acetylcholine receptors in skeletal muscle.

An ACh receptor is the molecular entity that, in its native habitat, possesses the binding sites for ACh and all the other components required to generate the ion channels mediating the ACh response. Narrower definitions of an ACh receptor (as the binding site for ACh or the polypeptide chain that is folded to form the binding site) could lead to semantic arguments about receptor structure. Experimentally, ACh receptors are defined by their total function (when electrophysiological tests are used) or by ligand binding. There is no evidence that the ligand-binding portions of ACh receptors ever exist in vivo without the associated channel-forming mechanism and vice versa. Most data are consistent with the idea that detergent-solubilized glycoproteins retaining the ACh binding sites of the receptor also include the channel-forming components, although it appears that the mechanism is prone to denaturation or proteolytic damage. Studies of receptor-rich membranes and of solubilized receptor glycoprotein have not yet yielded a totally satisfactory image of receptor structure. Most evidence favors an ACh receptor composed of three or four different types of glycosylated polypeptide chains organized into a unit of aggregate molecular weight about 300,000--400,000 daltons. Plasma membranes are dynamic structures in two different ways. First, their constituent molecules are in rapid thermal motion and, when these molecules are not tethered to extramembranous structures or mired in large aggregates, they fairly rapidly change their position in the plane of the lipid bilayer. Second, all membrane components are continually being synthesized and degraded. Acetylcholine receptors participate in both aspects of this dynamism. In this review it is proposed that the number and the distribution of ACh receptors in skeletal muscle are controlled by modulation of receptor metabolism and modulation of associations between receptor molecules or between receptors and other, as yet unidentified, elements in neuromuscular junctions and at extrajunctional sites where receptors are clustered. The arrangements of receptors in skeletal muscle and the total number of receptors in skeletal muscle may be regulated by separate mechanisms. Clusters of ACh receptors apparently can form spontaneously in extrajunctional areas of denervated muscles and in tissue-cultured embryonic muscle. Such clusters may be positionally stable and the receptor molecules in them may be highly restricted in mobility. Nevertheless, these receptors have average lifetimes on the order of 20 h, just like the nonclustered, mobile extrajunctional receptors. Receptor clusters also form at sites of innervation. In the chick embryo the junctional receptor molecules remain short-lived. The metabolism of ACh receptors is highly regulated. The biosynthesis of receptors commences during myogenesis at about the time myogenic cells become competent to fuse. Later, biosynthesis is dramatically repressed by muscle activity and possibly by other factors...     

The distribution of acetylcholine receptors on muscle fibres of regenerating salamander limbs.

1. Muscle fibres developing during limb regeneration were examined for responsiveness to acetylcholine (ACh) applied iontophoretically along the membrane. 2. Fibres which were uninnervated as well as those with non-transmitting synapses had high over-all sensitivities, with only minor variations along their length. 3. Functionally innervated fibres in which depolarization did not yet elicit action potentials had high over-all sensitivities, even when the synaptic potentials ahd amplitudes of 40-50 mV. In these the membrane in the vicinity of synapses tended to have sensitivities above the background level. 4. Upon the appearance of action potentials, several weeks after fibre innervation, the responsiveness to ACh began to decline in synapse free regions of the membrane. In mature muscle the sensitivity to ACh is restricted to sites of synaptic contact.     

Collagen synthesis by cultured arterial smooth muscle cells during spontaneous phenotypic modulation

Quantitative and qualitative changes in collagen synthetic activity by rabbit arterial smooth muscle cells were monitored during spontaneous phenotypic modulation from days 2-15 of culture. The cultured smooth muscle cells transformed into a synthetic phenotype, reaching a maximum of 94.6% on day 4, and then gradually returned to a contractile phenotype accounting for 59.3% on day 15 of culture. The maximum collagen synthesis was found on day 7 when the cells were in early quiescent phase and showed a 91.7% synthetic phenotype. With an increasing proportion of cells in a contractile state, total collagen synthesis per cell decreased in parallel with the reduction in total protein synthesis. Synthesis of type I collagen was predominant, and the proportion of type I + III collagen was over 85% during the entire period of culture. Synthetic activity of type IV collagen, however, was relatively increased, and reached 3.8 +/- 0.4% at day 15 in comparison with 0.8 +/- 0.1% in the late logarithmic growth phase on day 4. This significant increment of type IV collagen in vitro seems to be correlated with the phenotypic modulation of cultured smooth muscle cells into a contractile phenotype.     

Role of calcium in the regulation of acetylcholine receptor synthesis in cultured muscle cells

The spleens of chloralose-urethane anaesthetized dogs were isolated, placed in a plethysmograph and perfused at constant arterial pressure from a cannulated femoral artery. Splenic venous pressure (Pv) was elevated by between 2.5 and 20 cm H₂O: it caused pressure-dependent increases in spleen volume of up to 98±12 ml/100 g spleen weight.The increase in spleen volume could be separated into an initial phase associated with passive distension of capacitance vessels (Phase-1) followed by a more continuous increase in spleen volume (Phase-2). We used techniques designed to distinguish these components of the volume response to Pv elevation, and found Phase-2 rates of increase in spleen volume of 5–10 ml min^–1 mm Hg^–1 100 g^–1 expressed in terms of Pv elevation and terminal, drained and trimmed spleen weight. Splenic venous haematocrit determination showed selective sequestration of RBCs during the Phase-2 volume response to Pv elevation which was related to the extent of the Pv elevation. Both the Phase-2 volume response and the haematocrit changes were reduced by splenic nerve stimulation or i.a. infusions of adrenaline but were unaffected by i.a. infusions of bradykinin.     

The acetylcholine receptors of Renshaw cells

Summary The pharmacological properties of Renshaw cells in the lumbar spinal cord of cats anaesthetised with pentobarbitone have been investigated by administering cholinomimetics, acetylcholine antagonists and cholinesterase inhibitors electrophoretically from multibarrelled glass micropipettes. The excitatory action of acetylcholine, nicotine and carbamylcholine was due predominantly to interaction with nicotinic receptors and could be blocked by dihydro--erythroidine. Acetyl--methylcholine and dl-muscarine interact with muscarinic receptors which could be blocked specifically by intravenously administered atropine. In the presence of dihydro--erythroidine and atropine, cholinomimetics may depress, and subsequently excite Renshaw cells. A third type of receptor, which may be a component of the membrane of many types of mammalian nerve cells, has been proposed to account for this depression. The enhancement of the acetylcholine excitation of Renshaw cells by physostigmine, edrophonium and neostigmine was complicated by a direct excitant action which was probably due to a combination with nicotinic receptors.     

Concurrent redistribution of antigen receptors and surface immunoglobulin on antigen-binding cells

Surface immunoglobulin can be demonstrated on almost all sheep erythrocyte rosette-forming cells in CBA and AKR mice, either by inhibition of antigen binding by anti-immunoglobulin or by immunofluorescence. When surface immunoglobulin on a rosette-forming cell is `capped' by anti-immunoglobulin, the bound red cells migrate with the immunoglobulin, demonstrating that the receptor-combining site and immunoglobulin antigenic determinants share the same mobile unit in the membrane. These observations strongly support the concept that the antigen receptor is surface immunoglobulin (at least for B cells), and illustrate the usefulness of the `capping' technique for studying the associations between membrane elements.     

Evidence that cultured airway smooth muscle cells contain bradykinin B2 and B3 receptors.

We examined bradykinin-induced 45Ca2+ efflux and prostaglandin synthesis in guinea pig tracheal smooth muscle cells maintained in tissue culture. We also studied the effects of a B1 receptor agonist and antagonist, a B2 receptor antagonist, and the cyclooxygenase inhibitor indomethacin. In cultured smooth muscle cells, bradykinin (0.1 nM to 10 microM) stimulated efflux of 45Ca2+ and induced the synthesis of prostaglandin E2 and the prostacyclin metabolite 6-keto-prostaglandin F1 alpha. DesArg9-bradykinin, a B1 receptor agonist, had no effect on 45Ca2+ efflux or prostaglandin synthesis, and no responses to bradykinin were unaffected by the B1 receptor antagonist desArg9-[Leu8]-bradykinin. Indomethacin (1 microM) abolished bradykinin-induced prostaglandin synthesis but was without effect on 45Ca2+ efflux. NPC 567 (DArg[Hyp3,DPhe7]-bradykinin), a B2 receptor antagonist, had no effect on bradykinin-induced 45Ca2+ efflux, but abolished prostaglandin synthesis. Unlike in membranes prepared freshly from guinea pig tracheal smooth muscle, the B2 receptor antagonist inhibited completely (Ki, 12 nM) binding of [3H]-bradykinin to membranes prepared from cultured tracheal smooth cells. These data suggest that tracheal smooth muscle cells, maintained in culture, express B2 receptors that mediate bradykinin-induced prostaglandin synthesis. The observation that bradykinin-induced efflux of calcium ions was unaffected by B1 or B2 antagonists provides further evidence that airway smooth muscle may contain a novel B3 receptor.     

Interaction of acetylcholine and cholecystokinin with dispersed smooth muscle cells.

Isolated gastric smooth muscle cells were prepared from the stomach of Bufo marinus by successive incubation in collagenase without added trypsin. Contraction was determined by image-splitting micrometry and expressed as the mean percentage decrease in cell length from control. Peak contractile response was attained within 30 s. Dose-response curves constructed from peak responses showed that the maximal responses to CCK-OP (37.2 +/- 3.8%), acetylcholine (35.3 +/- 2.5%), and Ca2+ (42.3 +/- 0.9%) were similar. The D50s for octapeptide of cholecystokinin (CCK-OP) and acetylcholine were around 10(-12) M and 10(-11) M, respectively. The response to a combination of submaximal concentrations of acetylcholine and CCK-OP exceeded the individual responses but did not exceed the maximal response to either agent alone. A low concentration of atropine (5 X 10(-10) M) inhibited specifically the maximal response to acetylcholine. A high concentration of atropine (5 X 10(-8) M) inhibited partially the maximal response to CCK-OP but had no effect on the maximal response to Ca2+. It was concluded that 1) dispersed gastric smooth muscle cells are highly sensitive to stimulation; 2) CCK-OP has a direct (myogenic) contractile effect on gastric smooth muscle; and 3) the effect of CCK-OP and acetylcholine are mediated by separate receptors.     

Fluorescent staining of acetylcholine receptors in vertebrate skeletal muscle

1. alpha-Bungarotoxin was labelled with fluorescent dyes and used as a stain for visualizing the distribution of acetylcholine receptors in vertebrate skeletal muscle fibres.2. Dye-toxin conjugates had the same pharmacological properties as native toxin, but their potencies were lower.3. Fluorescent staining was examined in teased muscle fibres. The stain was found to be confined to the neuromuscular junction and associated with the subsynaptic membrane.4. Staining intensity was reduced by curare and even more so by carbachol, but not by atropine or neostigmine. Pre-treatment of muscles with unlabelled alpha-bungarotoxin entirely prevented staining.5. The staining at amphibian neuromuscular junctions was characterized by a pattern of intense transverse bands occurring at intervals of approximately 0.5-1 mum, with fluorescence of lower intensity between them. Fluorescent staining was not detected on adjacent, extrasynaptic, muscle membrane. In side views the staining appeared as a fine line with small protuberances occurring at the same intervals as the intense bands seen face-on. These results indicate that acetylcholine receptors are associated with the entire subsynaptic membrane, including the membrane of the junctional folds and that their density changes abruptly at the border between synaptic and extrasynaptic muscle membrane.     

Agonist and blocking actions of choline and tetramethylammonium on human muscle acetylcholine receptors

Choline has been used widely as an agonist for the investigation of gain-of-function mutants of the nicotinic acetylcholine receptor. It is useful because it behaves like a partial agonist. The efficacy of choline is difficult to measure because choline blocks the channel at concentrations about four times lower than those that activate it. We have fitted activation mechanisms to single-channel activity elicited from HEK-expressed human recombinant muscle nicotinic receptors by choline and by tetramethylammonium (TMA). Channel block by the agonist was incorporated into the mechanisms that were fitted, and block was found not to be selective for the open state. The results also suggest that channel block is very fast and that the channel can shut almost as fast as normal when the blocker was bound. Single-channel data are compatible with a mechanism in which choline is actually a full agonist, its maximum response being limited only by channel block. However, they are also compatible with a mechanism incorporating a pre-opening conformation change ('flip') in which choline is a genuine partial agonist. The latter explanation is favoured by concentration jump experiments, and by the fact that only this mechanism fits the TMA data. We propose that choline, like TMA, is a partial agonist because it is very ineffective (approximately 600-fold less than acetylcholine) at eliciting the initial, pre-opening conformation change. Once flipping has occurred, all agonists, even choline, open the channel with similar efficiency.     

Nicotinic acetylcholine receptors of adrenal chromaffin cells

In the adrenal medulla, acetylcholine released by the sympathetic splanchnic nerves activates neuronal-type nicotinic acetylcholine receptors (nAChRs) on the membrane of chromaffin cells which liberate catecholamines into the bloodstream in preparation for the fight and flight reactions. On adrenal chromaffin cells the main class of nAChRs is a pentameric assembly of alpha3 and beta4 subunits that forms ion channels which produce membrane depolarization by increasing Na+, K+ and Ca2+ permeability. Homomeric alpha7 nicotinic receptors are expressed in a species-dependent manner and do not contribute to catecholamine secretion. Chromaffin cell nAChRs rapidly activate and desensitize with full recovery on washout. nAChR activity is subjected to various types of dynamic regulation. It is allosterically modulated by the endogenous neuropeptide substance P that stabilizes receptors in their desensitized state, thus depressing their responsiveness. The full-length peptide CGRP acts as a negative allosteric modulator by inhibiting responses without changing desensitization, whereas its N-terminal fragments act as positive allosteric modulators to transiently enhance nAChR function. nAChR expression increases when cells are chronically exposed to either selective antagonists or agonists such as nicotine, a protocol mimicking the condition of chronic heavy smokers. In this case, large upregulation of nAChRs occurs even though most of the extra nAChRs remain inside the cells, creating a mismatch between the increase in total nAChRs and increase in functional nAChRs on the cell surface. These findings highlight the plastic properties of cholinergic neurotransmission in the adrenal medulla to provide robust mechanisms for adapting catecholamine release to acute and chronic changes in sympathetic activity.     

Fibroblast growth factor, epidermal growth factor and insulin exert a neuronal-like influence on acetylcholine receptors in aneurally cultured human muscle

Fibroblast growth factor (FGF), epidermal growth factor (EGF) and insulin added in combination to the culture medium in which normal human muscle was cultured caused a 4.0-fold (P less than 0.005) increase of the total number of nicotinic acetylcholine receptors (AChRs) and a 4.5-fold (P less than 0.001) increase in AChR aggregation. Individually, only FGF caused a 3.0-fold increase (P less than 0.005) in AChR aggregation, without influencing the total number of AChRs. To the contrary, insulin alone caused a 2.0-fold increase (P less than 0.05) in the total number of AChRs without influencing AChR aggregation. These findings show that these three polypeptide growth factors exert a neuronal-like influence on cultured human muscle in regard to AChRs.