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L L Rubin

Publications and source records attributed to L L Rubin.

53 records · Page 3Linked to original sources
PubMed1988

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.

Animals↗
PubMed1987

Generation of subunit-specific antibody probes for Torpedo acetylcholinesterase: cross-species reactivity and use in cell-free translations.

The assembly of the collagen tailed A12 form of acetylcholinesterase (AChE) is regulated by muscle contraction. To begin to study this regulation, we derived antibody probes for the three subunits (100 kd, catalytic, and collagen tail) of AChE purified from Torpedo californica electric tissue. These included a polyclonal antiserum that recognizes all 3 subunits and 19 monoclonal antibodies; 16 of the monoclonals recognized the catalytic subunit, 2 recognized the tail subunit, and 1 recognized the 100 kd subunit on Western blots. We used immunohistochemical procedures to show that several of the anticatalytic and one of the antitail monoclonals cross-reacted with frog muscle AChE and Western blotting to show that several of the anticatalytic monoclonals cross-react with rat brain AChE. These antibodies were then used to immunoprecipitate AChE precursors from a cell-free translation system. There were generally three primary translation products, corresponding to the three enzyme subunits. Therefore, each subunit is probably derived from a separate mRNA. Occasionally there were two translation products corresponding to the catalytic subunit alone. The catalytic subunit was glycosylated following addition of canine microsomal membranes to the translation mix. The mRNA coding for this subunit appeared to be present in the poly(A)- RNA pool.

Acetylcholinesterase↗
PubMed1987

Regulation of nicotinic acetylcholine receptor phosphorylation in rat myotubes by forskolin and cAMP.

The nicotinic acetylcholine receptor (AcChoR) from rat myotubes prelabeled in culture with [32P]orthophosphate was isolated by acetylcholine affinity chromatography followed by immunoaffinity chromatography. Under basal conditions, the nicotinic AcChoR was shown to be phosphorylated in situ on the beta and delta subunits. Regulation of AcChoR phosphorylation by cAMP-dependent protein kinase was explored by the addition of forskolin or cAMP analogues to prelabeled cell cultures. Forskolin, an activator of adenylate cyclase, stimulated the phosphorylation of the delta subunit 20-fold over basal phosphorylation and induced phosphorylation of the alpha subunit. The effect of forskolin was dose dependent with a half-maximal response at 8 microM in the presence of 35 microM Ro 20-1724, a phosphodiesterase inhibitor. Stimulation of delta subunit phosphorylation was almost maximal within 5 min, whereas stimulation of alpha subunit phosphorylation was not maximal until 45 min after forskolin treatment. Stimulation of AcChoR phosphorylation by 8-benzylthioadenosine 3',5'-cyclic monophosphate was identical to that obtained by forskolin. Two-dimensional thermolytic phosphopeptide maps of the delta subunit revealed a single major phosphopeptide. These results correlate closely with the observed effects of forskolin on AcChoR desensitization in muscle and suggest that cAMP-dependent phosphorylation of the delta subunit increases the rate of AcChoR desensitization in rat myotubes.

Animals↗
PubMed1987

Acetylcholine receptor clustering and nuclear movement in muscle fibers in culture.

We have studied the formation of acetylcholine receptor (AChR) clusters and the behavior of myonuclei in rat and chick skeletal muscle cells grown in cell culture. These cells were treated with a factor derived from Torpedo electric extracellular matrix, which causes a large increase in their number of AChR clusters. We found that these clusters were located preferentially in membrane regions above myonuclei. This cluster-nucleus colocalization is explained by our finding that most of the nuclei near clusters remain relatively stationary, while most of those away from clusters are able to translocate throughout the myotube. In some cases, clusters clearly formed first, then nuclei migrated underneath and became immobilized. If clustered AChRs later dispersed, their associated nuclei resumed moving. These results suggest that AChR clustering initiates an extensive cytoskeletal rearrangement that causes the subcluster localization of organelles, potentially providing a stable source of newly synthesized AChRs for insertion into the cluster.

Animals↗
PubMed1985

Molecular forms of acetylcholinesterase in Xenopus muscle.

Xenopus adult muscle, whole Xenopus embryos, and cultured embryonic myocytes together contain five acetylcholinesterase forms which can be resolved by sucrose density gradient centrifugation. These are identified as the collagenase-sensitive asymmetric forms A12 and A8, and the globular forms G4, G2, and G1. Asymmetric forms rise in whole embryos during the period of neuromuscular synapse formation, but their rise is not prevented by tricaine methanesulfonate, which abolishes motor activity. Aneural myocyte cultures synthesize primarily asymmetric acetylcholinesterase, much of which is extracellular. Prior nerve contact is not required for its expression. The proportion of asymmetric forms is neither decreased by tetrodotoxin, nor enhanced by veratridine and aconitine. We conclude that muscle activity does not modulate the expression of asymmetric acetylcholinesterase in Xenopus.

Acetylcholinesterase↗
PubMed1985

Increases in muscle Ca2+ mediate changes in acetylcholinesterase and acetylcholine receptors caused by muscle contraction.

The synthesis of acetylcholinesterase (AcChoE; acetylcholine acetylhydrolase, EC 3.1.1.7) and of acetylcholine receptors (AcChoR) by cultured rat muscle fibers is influenced strongly by the level of muscle contractile activity. If fibers are grown in the presence of tetrodotoxin (TTX) to block spontaneous contraction, the total amount of AcChoE decreases markedly, as does the percentage of AcChoE assembled as the collagen-tailed presumed synaptic form of the enzyme. Under these conditions, however, the number of AcChoR increases. We demonstrate here that each effect of TTX can be prevented by treating the muscle cells with the calcium ionophore A23187. Thus, cells treated with A23187 and TTX have 30- to 40-fold higher levels of collagen-tailed AcChoE and lower levels of AcChoR by a factor of 4-5 than do cells grown in TTX alone. These results suggest that an increase in muscle cytoplasmic Ca2+ mediates the known effects of muscle contraction on these cholinergic macromolecules.

Acetylcholinesterase↗
PubMed1985

Cellular and secreted forms of acetylcholinesterase in mouse muscle cultures.

When grown in primary cell culture in the absence of neurons, muscle cells from a variety of species synthesize several forms of acetylcholinesterase (AChE), including the collagen-tailed A12 form. A12 AChE has been the subject of much study because it is thought to be a major functional enzyme form normally found in the basal lamina at the neuromuscular junction. In this paper, we show that muscle fibers derived from mouse embryos and neonates are also able to synthesize substantial percentages of their AChE as the A12 form when grown in vitro. This synthesis is modulated by a process associated with spontaneous muscle contractile activity since both total enzyme levels and the proportion of A12 AChE expressed on the cell surface are decreased when the cells are grown in the sodium channel blocker tetrodotoxin, which blocks muscle contraction. On the other hand, when the cells are treated with veratridine, which opens sodium channels, thereby mimicking one aspect of muscle contraction, their AChE levels are comparable to those of untreated cells. Although smaller in magnitude, these changes are similar to those seen in rat muscle cultures. A novel feature of mouse muscle cultures, not seen in those from rat and chick, is the presence of a secreted enzyme form that sediments in the same position as the cellular A12 form (when separated on sucrose density gradients containing high salt) and is also collagenase sensitive.

Acetylcholinesterase↗
PubMed1984

Transformation by Rous sarcoma virus prevents acetylcholine receptor clustering on cultured chicken muscle fibers.

Acetylcholine receptors aggregate in the membrane of cultured chicken myotubes; the process of receptor clustering can be stimulated by exogenous factors that we, among others, have begun to characterize. Chicken myoblasts transformed by temperature-sensitive mutants of Rous sarcoma virus, such as tsNY68, fuse to form multinucleated myotubes at 42 degrees C, the nonpermissive temperature for transformation. However, tsNY68-infected myotubes do not cluster acetylcholine receptors at 42 degrees C, even in the presence of active clustering agents. This phenomenon is not merely a result of viral infection, since myotubes infected with a transformation-deficient viral mutant, td107A, behave like noninfected myotubes with respect to receptor clustering; thus, the effects of tsNY68 on the clustering process must be mediated by the src gene product. These experiments may provide a method of identifying essential elements of acetylcholine receptor clusters.

Animals↗
PubMed1984

Components of Torpedo electric organ and muscle that cause aggregation of acetylcholine receptors on cultured muscle cells.

The synaptic portion of a muscle fiber's basal lamina sheath has molecules tightly bound to it that cause aggregation of acetylcholine receptors (AChRs) on regenerating myofibers. Since basal lamina and other extracellular matrix constituents are insoluble in isotonic saline and detergent solutions, insoluble detergent-extracted fractions of tissues receiving cholinergic input may provide an enriched source of the AChR-aggregating molecules for detailed characterization. Here we demonstrate that such an insoluble fraction from Torpedo electric organ, a tissue with a high concentration of cholinergic synapses, causes AChRs on cultured chick muscle cells to aggregate. We have partially characterized the insoluble fraction, examined the response of muscle cells to it, and devised ways of extracting the active components with a view toward purifying them and learning whether they are similar to those in the basal lamina at the neuromuscular junction. The insoluble fraction from the electric organ was rich in extracellular matrix constituents; it contained structures resembling basal lamina sheaths and had a high density of collagen fibrils. It caused a 3- to 20-fold increase in the number of AChR clusters on cultured myotubes without significantly affecting the number or size of the myotubes. The increase was first seen 2-4 h after the fraction was added to cultures and it was maximal by 24 h. The AChR-aggregating effect was dose dependent and was due, at least in part, to lateral migration of AChRs present in the muscle cell plasma membrane at the time the fraction was applied. Activity was destroyed by heat and by trypsin. The active component(s) was extracted from the insoluble fraction with high ionic strength or pH 5.5 buffers. The extracts increased the number of AChR clusters on cultured myotubes without affecting the number or degradation rate of surface AChRs. Antiserum against the solubilized material blocked its effect on AChR distribution and bound to the active component. Insoluble fractions of Torpedo muscle and liver did not cause AChR aggregation on cultured myotubes. However a low level of activity was detected in pH 5.5 extracts from the muscle fraction. The active component(s) in the muscle extract was immunoprecipitated by the antiserum against the material extracted from the electric organ insoluble fraction. This antiserum also bound to extracellular matrix in frog muscles, including the myofiber basal lamina sheath. Thus the insoluble fraction of Torpedo electric organ is rich in AChR-aggregating molecules that are also found in muscle and has components antigenically similar to those in myofiber basal lamina.

Animals↗
PubMed1980

Regulation of acetylcholinesterase appearance at neuromuscular junctions in vitro.

The appearance of acetylcholinesterase (AChE) at newly formed nerve-muscle synapses depends on synaptic transmission. Synapses form when cultures are grown in the presence of acetylcholine receptor antagonists, but AChE does not accumulate at these synapses. The important component of transmission seems to be muscle activity. Treatment with dibutyryl cyclic GMP mimics muscle activity, directly inducing synaptic AChE appearance.

Acetylcholinesterase↗
PubMed1978

Effect of concanavalin A on black widow spider venom activity at the neuromuscular junction: implications for mechanisms of venom action.

Concanavalin A (Con A) inhibits black widow spider venom-induced transmitter release at both tissue-cultured and adult neuromuscular junctions and also inhibits the venom-induced destruction of cultured neurites. This inhibitory action is partially or completely prevented by prior treatment with colchicine. Neither colchicine nor Con A interacts significantly with depolarization-induced transmitter release. These results are analogous to those obtained from experiments on lymphocyte surface receptor capping. They suggest that redistribution of neuronal membrane components may be a crucial step in spider venom action. This membrane redistribution appears to be modulated in neurons, as in other cell types, by microtubule-microfilament array. How such a redistribution causes increased transmitter release cannot as yet be specified. Changes in the ionic permeability of sodium and potassium were examined as likely mechanisms. Increased sodium influx (and seondary release of calcium from intracellular stores) cannot be the basis for spider venom action. Increased potassium efflux remains a possibility, but is not consistent with all of the data. Other possible mechanisms are also suggested.

Animals↗
PubMed1978

Double mode of action of black widow spider venom on frog neuromuscular junction.

Black widow spider venom (BWSV) contains a toxin, alpha-latrotoxin, which is capable of stimulating vesicle release, resulting eventually in depletion of vesicles and block of neuromuscular transmission at the frog neuromuscular junction. Since it has been shown that alpha-latrotoxin very markedly increases the cation conductance of artificial lipid bilayers, it was postulated that BWSV stimulates release by opening channels permeable to Ca2+ and, in the case of Ca2+-free Ringer's, to Na+ which would release Ca2+ from intracellular stores. To test this hypothesis we chose as a sodium substitute, glucosamine, which is impermeable to the venom-induced channels in the lipid bilayers and to the postsynaptic membrane of the frog neuromuscular junction. Electron microscopical analysis showed that up to 75 min perfusion in Na+ and Ca2+-free medium did not alter the ultrastructure of the nerve terminals. However when BWSV was applied in this medium a significant depletion was noticeable within 15 min and after 60 min the terminals were depleted of vesicles whereas the mitochondria were unchanged in number and structure. If BWSV is applied for 60 min in glucosamine Ringer's containing 1.8 mM Ca2+, most of the nerve terminals still have synaptic vesicles scattered in the cytoplasm or clustered around amorphous structures and the mitochondria are swollen. Application of large doses of BWSV in low Ca2+ Ringer's leads to damage of the mitochondria and to very pronounced swelling of the nerve endings, whereas this is not observed if the dose of venom is applied in Na+-free and Ca2+-free Ringer's. Electrophysiological recording showed that neuromuscular transmission is already blocked after 15 min treatment with BWSV in glucosamine-Ringer's. From these results we conclude that BWSV increases the conductance of the nerve terminal membrane to cations such as Na+ and Ca2+ and stimulates release by a mechanism which may not involve its ionophore property.

Animals↗
PubMed1978

Accumulation of acetylcholine receptors and acetylcholinesterase at newly formed nerve-muscle synapses.

Nerve-muscle synapses form and the postsynaptic membrane begins to differentiate rapidly in vitro. New clusters of AChRs have been located at transmitting synapses within 3 hours after a competent neurite contacted a receptive myotube. Receptors within new subneural clusters are not identical with receptors at adult end-plates: they are metabolized rapidly and their mean channel open time is prolonged. In this sense synapse formation must be considered a drawn out affair. AChE also accumulates at synapses soon after functional contacts are established. The cleft enzyme is apparently regulated differently than receptors in the postsynaptic membrane. Muscle activity is necessary for the early accumulation of AChE, but not for the clustering of receptors. Further studies in vitro should be useful for analysis of the role of muscle activity, of the local neural influence on the postsynaptic membrane, and of the mechanism of the muscle response.

Acetylcholine↗
PubMed1976

Black widow spider venom: effect of purified toxin on lipid bilayer membranes.

A purified toxin (the B5 fraction) from black widow spider venom added to the solution on one side of a lipid bilayer membrane interacts irreversibly with the membrane to produce a continuous, linear rise of membrane conductance with time. Conductances greater than 10(-4) reciprocal ohm per square centimeter can eventually be attained without any loss of membrane stability. Membranes treated with toxin are ideally selective for alkali cations over anions and are substantially permeable to calcium ion. These effects of the toxin result from the formation of permanent channels in the membrane of uniform conductance, 3.6 X 10(-10) reciprocal ohm (in 0.1 molar potassium chloride), that remain open almost all the time. Both the divalent cation permeability and the smaller conductances at low pH of toxin-treated membranes suggest that there is negative charge (possibly from carboxyl groups) associated with the channels. We discuss the possible relation of the action of this toxin on lipid bilayer membranes to its ability to stimulate massive transmitter release at the neuromuscular junction and to produce profound morphological changes on tissue cultured neurons.

Black Widow Spider↗