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B G Wallace

Publications and source records attributed to B G Wallace.

17 recordsLinked to original sources
PubMed1992

Mechanism of agrin-induced acetylcholine receptor aggregation.

Agrin induces the formation of specializations on chick myotubes in culture at which several components of the postsynaptic apparatus accumulate, including acetylcholine receptors (AChRs). Agrin also induces AChR phosphorylation. Several lines of evidence suggest that agrin-induced phosphorylation of tyrosine residues in the beta subunit of the AChR is an early step in receptor aggregation: agrin-induced phosphorylation and aggregation have the same dose dependence; treatments that prevent aggregation block phosphorylation; phosphorylation begins before any detectable change in receptor distribution, reaches a maximum hours before aggregation is complete, and declines slowly together with the disappearance of aggregates after agrin is withdrawn; agrin slows the rate at which receptors are solubilized from intact myotubes by detergent extraction; and the change in receptor extractability parallels the change in phosphorylation. A model for agrin-induced AChR aggregation is presented in which phosphorylation of AChRs by an agrin-activated protein tyrosine kinase causes receptors to become attached to the cytoskeleton, which reduces their mobility and detergent extractability, and leads to the accumulation of receptors in the vicinity of the activated kinase, forming an aggregate.

Agrin↗
PubMed1991

The mechanism of agrin-induced acetylcholine receptor aggregation.

Agrin, a protein isolated from the synapse-rich electric organ of Torpedo californica, induces the formation of specializations on myotubes in culture which resemble the post-synaptic apparatus at the vertebrate skeletal neuromuscular junction. For example, the specializations contain aggregates of acetylcholine receptors and acetylcholinesterase. This report summarizes the evidence that the formation of the post-synaptic apparatus at developing and regenerating neuromuscular junctions is triggered by the release of agrin from motor axon terminals and describes results of recent experiments which suggest that agrin-induced tyrosine phosphorylation of the beta subunit of the acetylcholine receptor may play a role in receptor aggregation.

Agrin↗
PubMed1991

Agrin induces phosphorylation of the nicotinic acetylcholine receptor.

Agrin causes acetylcholine receptors (AChRs) on chick myotubes in culture to aggregate, forming specializations that resemble the postsynaptic apparatus at the vertebrate skeletal neuromuscular junction. Here we report that treating chick myotubes with agrin caused an increase in phosphorylation of the AChR beta, gamma, and delta subunits. H-7, a potent inhibitor of several protein serine kinases, blocked agrin-induced phosphorylation of the gamma and delta subunits, but did not prevent either agrin-induced AChR aggregation or phosphorylation of the beta subunit. Experiments with anti-phosphotyrosine antibodies demonstrated that agrin caused an increase in tyrosine phosphorylation of the beta subunit that began within 30 min of adding agrin to the myotube cultures, reached a plateau by 3 hr, and was blocked by treatments known to block agrin-induced AChR aggregation. Anti-phosphotyrosine antibodies labeled agrin-induced specializations as they do the postsynaptic apparatus. These results suggest that agrin-induced tyrosine phosphorylation of the beta subunit may play a role in regulating AChR distribution.

Agrin↗
PubMed1990

Inhibition of agrin-induced acetylcholine-receptor aggregation by heparin, heparan sulfate, and other polyanions.

Heparin and heparan sulfate have been shown to block nerve-induced acetylcholine-receptor (AChR) aggregation at developing neuromuscular junctions. We found that heparin, heparan sulfate, and a wide variety of other polyanions also inhibited agrin-induced AChR aggregation. The more highly charged the polyanion, the more potent it was as an inhibitor. Inhibition of agrin-induced AChR aggregation was due, at least in part, to the formation of a complex between the polyanion and agrin that was inactive. These findings are consistent with the hypothesis that nerve-induced aggregation of AChRs is mediated by the release of agrin, or a closely related protein, from axon terminals and suggest that a polyanion, such as a sulfated proteoglycan, may be involved in the interaction of agrin with its receptor on the myotube surface.

Agrin↗
PubMed1989

Agrin-induced specializations contain cytoplasmic, membrane, and extracellular matrix-associated components of the postsynaptic apparatus.

The aims of the studies reported here were to determine the extent to which the specializations induced by agrin on cultured chick myotubes resemble the postsynaptic apparatus and examine how these specializations form. We found that agrin induces the formation of specializations at which at least 6 components of the postsynaptic apparatus are concentrated: one cytoplasmic component [a 43 kDa acetylcholine receptor (AChR)-associated protein], 3 membrane components [AChRs and globular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE)], and 2 extracellular matrix-associated proteins (A12 asymmetric AChE and a heparan sulfate proteoglycan). The accumulation of AChE and BuChE into agrin-induced aggregates occurred in the absence of any change in the amount, rate of synthesis, accumulation, and release, or molecular forms of either enzyme. Thus, agrin affects primarily the distribution of these components of the postsynaptic apparatus and not their metabolism. Agrin-induced formation of AChR aggregates was not prevented by inhibition of protein synthesis, consistent with our previous results that agrin-induced accumulation of AChRs occurs by lateral migration. The accumulation of components of the extracellular matrix would seem less likely to occur by lateral migration and so might require release of newly synthesized proteins; indeed, formation of aggregates of heparan sulfate proteoglycan was prevented by inhibitors of protein synthesis. Thus, different components of the postsynaptic apparatus accumulate in agrin-induced specializations by different mechanisms.

Acetylcholinesterase↗
PubMed1988

Regulation of agrin-induced acetylcholine receptor aggregation by Ca++ and phorbol ester.

Agrin, a protein extracted from the electric organ of Torpedo californica, induces the formation of specializations on cultured chick myotubes that resemble the postsynaptic apparatus at the neuromuscular junction. The aim of the studies reported here was to characterize the effects of agrin on the distribution of acetylcholine receptors (AChRs) and cholinesterase as a step toward determining agrin's mechanism of action. When agrin was added to the medium bathing chick myotubes small (less than 4 micron 2) aggregates of AChRs began to appear within 2 h and increased rapidly in number until 4 h. Over the next 12-20 h the number of aggregates per myotube decreased as the mean size of each aggregate increased to approximately 15 micron 2. The accumulation of AChRs into agrin-induced aggregates occurred primarily by lateral migration of AChRs already in the myotube plasma membrane at the time agrin was added to the cultures. Aggregates of AChRs and cholinesterase remained as long as agrin was present in the medium; if agrin was removed the number of aggregates declined slowly. The formation and maintenance of agrin-induced AChR aggregates required Ca++, Co++ and Mn++ inhibited agrin-induced AChR aggregation and increased the rate of aggregate dispersal. Mg++ and Sr++ could not substitute for Ca++. Agrin-induced receptor aggregation also was inhibited by phorbol 12-myristate 13-acetate, an activator of protein kinase C, and by inhibitors of energy metabolism. The similarities between agrin's effects on cultured myotubes and events that occur during formation of neuromuscular junctions support the hypothesis that axon terminals release molecules similar to agrin that induce the differentiation of the postsynaptic apparatus.

Agrin↗
PubMed1987

Identification of agrin, a synaptic organizing protein from Torpedo electric organ.

Extracts of the electric organ of Torpedo californica contain a proteinaceous factor that causes the formation of patches on cultured myotubes at which acetylcholine receptors (AChR), acetylcholinesterase (AChE), and butyrylcholinesterase (BuChE) are concentrated. Results of previous experiments indicate that this factor is similar to the molecules in the synaptic basal lamina that direct the aggregation of AChR and AChE at regenerating neuromuscular junctions in vivo. We have purified the active components in the extracts 9,000-fold. mAbs against four different epitopes on the AChR/AChE/BuChE-aggregating molecules each immunoprecipitated four polypeptides from electric organ extracts, with molecular masses of 150, 135, 95, and 70 kD. Gel filtration chromatography of electric organ extracts revealed two peaks of AChR/AChE/BuChE-aggregation activity; one comigrated with the 150-kD polypeptide, the other with the 95-kD polypeptide. The 135- and 70-kD polypeptides did not cause AChR/AChE/BuChE aggregation. Based on these molecular characteristics and on the pattern of staining seen in sections of muscle labeled with the mAbs, we conclude that the electric organ-aggregating factor is distinct from previously identified molecules, and we have named it "agrin."

Acetylcholinesterase↗
PubMed1987

Identification of agrin in electric organ extracts and localization of agrin-like molecules in muscle and central nervous system.

The portion of the muscle fibre's basal lamina that occupies the synaptic cleft at the neuromuscular junction contains molecules that cause the aggregation of acetylcholine receptors and acetylcholinesterase on regenerating muscle fibres. Agrin, which is extracted from basal lamina-containing fractions of the Torpedo electric organ and causes the formation of acetylcholine receptor and acetylcholinesterase aggregates on cultured myotubes, may be similar, if not identical, to the acetylcholine receptor- and acetylcholinesterase-aggregating molecules at the neuro-muscular junction. Here we summarize experiments which led to the identification of agrin and established that the basal lamina at the neuromuscular junction contains molecules antigenically similar to agrin. We also discuss results which raise the possibility that agrin-like molecules at the neuromuscular junction are produced by motor neurones.

Agrin↗
PubMed1987

Agrin.

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Agrin↗
PubMed1978

Modulation of transmission at an inhibitory synapse in the central nervous system of the leech.

The synaptic interactions among a group of cells in the leech C.N.S. that regulate the animal's heartbeat exhibit several remarkable features (Thompson & Stent, 1976 a, b, c). We have examined in detail the properties of the inhibitory synapse between two of these cells, the heart interneurone (HN cell) and the heart excitor motoneurone (HE cell). 1. Impulses in the presynaptic HN cell gave rise to monosynaptic i.p.s.p.s in the HE cell that were blocked by high concentrations of Mg and were reversed when the membrane potential of the post-synaptic motoneurone was hyperpolarized beyond--75 m V or when Cl was injected into the cell body. These i.p.s.p.s were chemically mediated, and involved an increase in chloride conductance. 2. In contrast to chemical synapses between sensory and motor cells in the leech C.N.S., little facilitation or depression of transmission occurred when the HN cell was stimulated at frequencies of 0.1--50 Hz. 3. Steady subthreshold depolarization of the presynaptic HN interneurone evoked a maintained hyperpolarization of the post-synaptic HE cell, indicating that currents injected into the HN cell body could spread to the terminals and cause continuous release of transmitter. 4. The size of the i.p.s.p. evoked in the HE motoneurone by an action potential in the HN interneurone varied with the resting membrane potential of the presynaptic cell. An impulse superimposed on a prolonged, subthreshold, depolarizing pulse produced a larger i.p.s.p.; conversely, prolonged hyperpolarization of the HN interneurone reduced the i.p.s.p. amplitude recorded in the HE cell. This effect was most obvious when the natural, rhythmical bursts of activity in the HN interneurone were interrupted by bathing the preparation in leech Ringer fluid containing elevated concentrations of Mg. Under these conditions a 10 mV depolarization of the HN cell increased the size of the i.p.s.p. in the HE cell approximately sixfold. Significant changes in i.p.s.p. amplitude occurred without any noticeable change in the amplitude and duration of the presynaptic action potential. With large presynaptic depolarizations, which produced the biggest i.p.s.p.s, there was some reduction in the amplitude and increase in the duration of the action potential. 5. Following a step depolarization of the presynaptic cell, the size of successive i.p.s.p.s increased with a time constant of about 1 sec. Upon repolarization the i.p.s.p.s decreased in amplitude to the original level. 6. stimulation of one HN cell also gives rise to an i.p.s.p. in its contralateral homologue (Thompson & Stent, 1976c). Trains of i.p.s.p.s produced in this way hyperpolarized at HN cell to such an extent that the size of the synaptic potential it evoked in an HE cell was reduced. 7. Thus, an HN interneurone inhibitis transmission between the contralateral HN and HE cells presynapitcally in addition to inhibiting directly the ipsilateral HE motoneurone.

Animals↗
PubMed1978

Quantal analysis of transmitter release at an inhibitory synapse in the central nervous system of the leech.

The quantal nature of transmitter release has been analysed at central inhibitory synapses in the leech nervous system between an interneurone (HN) and a motoneurone (HE) that regulate the heartbeat. 1. Ganglia were bathed in leech Ringer fluid containing 20 mM-Mg and 1.8 mM-Ca and the membrane of the presynaptic HN interneurone was hyperpolarized by current injection. Under these conditions successive inhibitory potentials in the HE motoneurone, evoked by impulses in the HN interneurone, showed striking fluctuations in amplitude. 2. Assuming a Poisson distribution of the i.p.s.p.s and estimating the number of failures from the amplitude histograms of the observed responses, the mean size of the quantal unit was estimated as 0.25 +/- 0.015 mV (S.E. of mean, n = 26). When m, the mean number of quanta released per trial, was varied by changing the membrane potential of the presynaptic HN cell (Nicholls & Wallace, 1978), the experimentally observed amplitude distributions could be predicted by the Poisson theory. 3. An independent estimate of the unit size was obtained by noise analysis. A long subthreshold depolarizing pulse applied to the presynaptic HN interneurone evoked a sustained hyperpolarization of the HE motoneurone, apparently caused by an increase in the rate of on-going release of quanta by the HN cell terminals. From the mean change in membrane potential and the increase in variance, the size of the unit was calculated as 0.21 +/- 0.039 mV (S.E. of mean, n = 11). For ten pairs of cells an estimate of unit amplitude was made both from the Poisson analysis and the analysis of variance, again with good agreement. For these cells the estimated unit sizes were 0.24 +/- 0.023 mV (S.E. of mean, n = 10) from the failures and 0.21 +/- 0.043 m V (S.E. of mean, n = 10) from the noise. 4. A similar analysis was made of the inhibitory synaptic potentials evoked in one HN interneurone by stimulation of its contralateral homologue. Transmission again appeared to be qualtal; the mean unit amplitude from Poisson analysis was 0.31 +/- 0.022 mV (S.E. of mean, n = 19) and from the noise 0.29 +/- 0.027 mV (S.E. of mean, n = 3). 5. We conclude that transmitter is released from the terminals of the HN interneurone in quantal units that evoke miniature i.p.s.p.s of about 0.25 mV in the post-synaptic cells. Furthermore, modulation of transmission proudced by variation in the presynaptic resting potential and during presynaptic inhibition results from changes in the mean number of quanta released by each impulse.

Animals↗
PubMed1976

Octopamine neurons in lobsters: location, morphology, release of octopamine and possible physiological role.

Octopamine cells are found along second thoracic roots, where they serve as neurosecretory neurons capable of releasing octopamine at two distinct points: one into the hemolymph immediately before it enters the gills; one into the hemolymph immediately after it leaves the gills. The octopamine cells receive a cholinergic synaptic input. We presume that this input is from processes of peripheral sensory cells bringing information to the CNS. Octopamine can increase the strength of contraction of exoskeletal muscles and, at higher concentrations, can induce contractures in these muscles. These effects can be interpreted as a resetting of the level of ionized calcium within muscle fibers (the contracture) to a higher value or a possible enhanced entry of calcium ions during nerve stimulation (increased strength of contraction). The observed effects are of a prolonged duration, outlasting the time of application of octopamine by some 20-40 minutes. We do not know if this effect on muscle tension production is the normal physiological role of octopamine. Other possible roles will be explored in the future. The pathway involving the octopamine neurons in lobsters may provide a model neurohumoral system that can be studied and understood in detail from the level of sensory input to the level of behavioral output.

Animals↗
PubMed1976

Modification and regeneration of synaptic connections in cultured leech ganglia.

Segmental ganglia of the central nervous system of the leech were maintained in culture medium outside the animal for several weeks in order to study the properties of synapses and regeneration by identified sensory and motor nerve cells. A variety of preparations were used, including single ganglia, chains of ganglia and ganglia connected to the areas of skin and muscle that they normally innervate in the animal. (1) For up to 10 weeks after removal from the animal, resting and action potentials recorded from sensory and motor neurons resembled those seen in normal ganglia. The same individual cell in a cultured ganglion could be recorded from with intracellular electrodes on a second occasion after an interval of a few days. (2) Sensory cells, identified as touch, pressure or nociceptive according to their morphology and electrical properties, continued to respond selectively to stimuli of the appropriate modality applied to their receptive fields in the skin; action potentials in motor cells caused contractions in the appropriate muscles. Culture of ganglia for more than 3 weeks caused the disappearance of synaptic potentials and a loss of transparency in ganglia. (3) Certain chemically mediated synaptic interactions between sensory and motor nerve cells became markedly changed in cultured ganglia. These changes appeared over the first 3 weeks and consisted of abnormally large excitatory and inhibitory synaptic potentials. The changes in synaptic transmission observed in culture were in many respects similar to those occurring in ganglia maintained within an animal after lesions have been made in the nervous system (Jansen et al. 1974). (4) The morphological appearances of sensory cells were compared in cultured and normal ganglia after injection of horseradish peroxidase. In cultured ganglia, the branching pattern appeared normal, but varicosities became more conspicuous. (5) When connectives linking cultured ganglia were crushed or cut, regeneration occurred. By 7 days, impulses propagated through the regenerated fibers and evoked synaptic potentials on cells within the next ganglion. The course taken by regenerating axons was observed in cells injected with horseradish peroxidase. The results again resembled those seen in animals with similar lesions. (6) The cultured ganglia provide preparations in which it is possible to analyze the mechanisms that underlie long-term changes similar to those seen in the leech central nervous system in situ.

Animals↗
PubMed1976

The association of octopamine with specific neurones along lobster nerve trunks.

Octapamine and its synthetic enzyme, tyramine beta-hydroxylase (TBH), are found in high concentrations at two points along second thoracic nerve roots in lobsters. The first is in the proximal section of the second root between the ventral nerve cord and the bifurcation of the root into medial (to flexor muscles) and lateral (to extensors) branches. The second region of high concentration is within a well known crustacean neurosecretory system, the pericardial organ, located close to the ends of the lateral branches of the roots. 2. With several different staining procedures, small clusters of nerve cell bodies are found within the connective tissue sheath in the proximal regions of the second roots. No cell bodies are seen in the pericardial organ regions. Cell bodies are variable in number and position between corresponding roots in the same animal and homologous roots among different animals. The average numbers of cell bodies, however, correlate well with TBH and octopamine content, and with the synthesis of octopamine in these same regions of roots. 3. Small clusters of root cell bodies dissected from preparations have greater than 500-fold higher activities of TBH than isolated efferent excitatory and inhibitory or afferent sensory axons. 4. Along with octopamine, the preferential synthesis of acetylcholine and serotonin is also seen in proximal segments of roots. Acetylcholine synthesis in these regions may represent transmitter synthesized in the nerve terminals innervating the root cells. The role of serotonin in these regions is not understood at this time but the amounts of endogenous serotonin found are only a tenth of the amounts of octopamine present. 5. Dopamine is not synthesized from tyrosine in second thoracic roots. However, if DOPA or dopamine are used as precursor compounds, then noradrenaline, which is usually not found in lobsters, can be accumulated in proximal segments of roots. 6. Phenolamines are converted to two further metabolites by lobster tissues. The compounds are unidentified and are named fast and slow product on the basis of their migration on electrophoresis at acid pH. Some partial characterization of slow product reveals that it is a mixture of compounds that can be converted on mild acid hydrolysis to fast product and the parent phenolamine. 7. The several lines of evidence presented suggest that nerve cells found in the proximal segments of the second thoracic roots contain and can synthesize octopamine. Since not all the cells in any single root have been analysed for octopamine or TBH, however, the possibility that one or more of the cells contain physiologically interesting substances other than octopamine is not eliminated.

Animals↗