The effect of postsynaptic block on development of the neuromuscular junction in postnatal rats

Summary The effects of reduced muscle activity on the ultra structural development of the rat neuromuscular junction (NMJ) have been studied. Soleus muscles of rats were treated with -bungarotoxin (BTx) in order to produce a postsynaptic block of activity between the ages of 10 and 12 days. Muscles of litter mates were treated with saline over the same period. The development of these control and BTx treated muscles was then compared to that of normal muscles from untreated litter mates. During the period between the tenth and twelfth days after birth, normal soleus NMJs show two major ultrastructural changes. 1. The average number of axon terminal profiles present at each endplate decreases. This is thought to reflect the withdrawal of superfluous axons from the endplates. 2. There is an increasing specialization of the postsynaptic structures of the junction: complexity of folding of the muscle junctional membrane increases, as does the accumulation of subjunctional sarcoplasm and muscle nuclei.In soleus muscles treated with BTx, the number of axon terminal profiles observed at the endplates does not decrease, suggesting that elimination of supernumerary axons does not occur. In addition, specialization of the postsynaptic structures of the NMJ is retarded during the period of ACh receptor block.     

Electron microscopic localization of [125I]alpha-bungarotoxin binding sites in the outer plexiform layer of the goldfish retina.

Summary Light and electron microscope autoradiography were performed on goldfish (Carassius auratus) retinas incubated in [¹²⁵I] labelled -bungarotoxin. The toxin was bound preferentially to membrane receptors in the inner and outer plexiform layers. Binding was suppressed by 10^–5 M nicotine or 10^–5 M native -bungarotoxin. Electron microscopic analysis of the outer plexiform layer (OPL) strongly suggested that -bungarotoxin binding sites were located on small bipolar cell dendritic processes that invaginated rod and cone synaptic terminals, and on large bipolar cell dendritic processes more proximally situated in the OPL. Large horizontal cell processes in the OPL and horizontal cell processes that invaginated rod and cone synaptic terminals did not appear to be labelled.     

The potentiating action of acetylcholine on the chemosensitivity of denervated skeletal muscle

The acetylcholine sensitivity of soleus and extensor digitorum longus muscles of young rats was studied in vitro by measuring the isometric tension and membrane depolarisation developed in response to bath application of acetylcholine. Contractures of denervated muscles to acetylcholine gradually increased when the same concentration of acetylcholine was applied at 10 min intervals up to 60 min incubation whilst contractures evoked by potassium sulphate did not change significantly. The twitch tension of the muscles elicited in response to direct electrical stimulation did not increase and direct electrical stimulation failed to increase the acetylcholine sensitivity. Increased responses in vitro to acetylcholine were observed only in muscles denervated between 2 and 10 days previously with an optimum increase in sensitivity at 4–6 days. The increases in sensitivity were due to an action of acetylcholine since muscles left untreated exhibited no increases. Responses to carbamylcholine chloride, which is not hydrolysed by acetylcholinesterase, also increased.Membrane depolarisations in response to acetylcholine increased in a similar manner to the contracture responses in both soleus and extensor digitorum longus muscles, whilst the resting membrane potentials did not change. Denervated muscles which had been treated with acetylcholine bound more¹²⁵I-labelled α-bungarotoxin than muscles which had not been treated with acetylcholine. Denervated soleus muscles which had been previously blocked with unlabelled α-bungarotoxin responded to applications of acetylcholine with a gradually increasing depolarisation response and subsequently bound more [¹²⁵I]α-bungarotoxin than muscles left without acetylcholine treatment.The possibility is discussed that acetylcholine acts upon the surface membranes of denervated muscles to increase the number of active acetylcholine receptors.     

Ion-induced release of LHRH,TRH and α-MSH from hypothalamic synaptosomes and its inhibition by vinblastine

Homogenates of rat hypothalamic tissue were fractionated by means of discontinuous sucrose density gradient centrifugation. Immunoreactive luteinizing hormone releasing hormone (LHRH), thyrotropin releasing hormone (TRH), and -melanocyte stimulating hormone (-MSH) were found to be concentrated in the synaptosome-enriched fraction. This fraction was suspended in 0.32 M sucrose and the release of the three peptides was investigated. After incubation, the synaptosomes were re-isolated by ultrafiltration, and the concentration of each peptide in the ultrafiltrate was determined by radioimmunoassay. When the synaptosomal fraction was incubated at 30° C in 0.32 M sucrose containing either 60 mM K⁺-2 mM Ca²⁺ or 140 mM Na⁺ alone a release of LHRH, TRH, and -MSH occurred. Of the total content 30–50% of LHRH but only about 10% of TRH and -MSH was releasable. When the synaptosome preparation was preincubated for 30 min at 30°C with 10^–4 M vinblastine. K⁺- as well as Na⁺-induced release of LHRH, THR, and -MSH was inhibited, and the stimulatory effect of each cation was almost totally blocked by preincubation with 5×10^–4 and 10^–3 M vinblastine. The inhibitory action of vinblastine (5×10^–4 M) did not affect the oxidation of glucose to CO₂ by the synaptosomes. The results of the present investigation demonstrate that synaptosome-enriched fractions of hypothalamic origin are more stable with respect to LHRH, TRH, and -MSH release during incubation in isotonic sucrose than they are in ionic solutions, and that the peptides are released by a vinblastine-sensitive mechanism.Supported by grants from the National Institute of Arthritis, Metabolism, and Digestive Diseases (AMO 1237), the National Institute of Child Health and Human Development (HDO8672), and the National Institutes of Health contract (5-P17-HL1487-06)Supported by Grant No. 512-6951 from the Danish Medical Research Council     

The effect of muscle extracts on the contracture response of skeletal muscle to acetylcholine

Summary Preincubation of normal rat soleus muscles in vitro with homogenates prepared from mixed leg muscles which had been denervated 4 days previously resulted in an increase in the contracture response to acetylcholine. After 30 min incubation a 1.5-fold increase was observed. Homogenates of normally innervated muscles did not increase the response. The active principles of the denervated muscles were found to reside in the cytosol fraction. An approximately 2-fold increase was observed upon incubation with the cytosol for 30 min; incubation for longer periods resulted in a subsequent decrease in the response. The effect of the denervated muscle cytosol was concentration-dependent and heat-labile. Normal muscle cytosol also increased the soleus muscle response to acetylcholine but this fraction was less effective than denervated muscle cytosol. The response of control muscles incubated in Krebs-Henseleit solution was found to decrease with time.Commercially obtained phospholipases C and D increased the response of normal soleus muscles approximatcly 2-fold. Phospholipase A, lipase, trypsin, collagenase and a bacterial protease had no effect, lysozyme produced a small but consistent increase in the response to acetylcholine.     

An α-mating-type-specific mutation causing specific defect in sexual agglutinability in the yeast Saccharomyces cerevisiae

We have identified a recessive -mating-type-specific gene agl causing agglutinability defect without significant effects on other sexual activities. a cells carrying agl showed sexual agglutination with cells but cells carrying agl showed sexual agglutination with neither cells nor a cells. cells carrying agl produced pheromone and responded to a pheromone just like wild cells. cells carrying agl showed a little decreased but significant mating ability when tested on solid media or membrane filter. The agl mutant is different from any -specific ste mutants found so far in many sexual activities. The agl gene is recessive, and unlinked to the mating type locus. Biological significance of the mating type agglutinability is discussed based on the results obtained with the mutant.     

Decrease in force potentiation and appearance of α-adrenergic mediated contracture in aging rat skeletal muscle

The effect of increasing age on contractile performance and catecholamine receptor activity was investigated in a distal, predominantly fast twitch oxidative glycolytic (FOG) muscle from the plantar surface of the rat hindfoot. The ability of the flexor digitorum brevis (FDB), isolated from anesthetized rats and maintained in vitro, to undergo post-tetanic potentiation and a staircase response declined with age. Potentiation following repetitive stimulation was reduced by 50% in 2 year old rats and eliminated in 3 year old animals. The rate of muscle fatigue during intermittent tetanic stimulation also increased in aging muscles. FDB, regardless of age, did not develop a positive inotropic response to 10^–6 M epinephrine applied in vitro, but 3 year old FDB generated a prolonged contracture. Contracture tension was approximately 25% of twitch tension and was maintained for 2–10 min in the continued presence of catecholamine. Contractures were eliminated by pretreatment with -adrenergic antagonists or by removing Ca²⁺ from the bathing medium. In addition to decreased contractile capacity, aging muscles acquire a population of -adrenergic receptors which may underlie some of the metabolic and structural changes associated with increasing age.     

Effect of cholinergic agonists on resting membrane potential of earthworm body wall muscle cells

Carbachol in a concentration of 5×10^-8 mol/liter does not hyperpolarize, and in a concentration of 5×10^-6 mol/liter depolarizes the membrane of somatic muscle cell in earthworm. d-Tubocurarine, -bungarotoxin, atropine, and hexamethonium added to the incubation medium did not abolish the carbachol-induced decrease in resting membrane potential. Each of these drugs alone had no effect on resting membrane potential in muscle cells. Presumably, the acetylcholine-sensitive receptor-channel membrane complex in earthworm muscle cell differs from acetylcholine receptor in skeletal muscle fibers and peripheral neurons of vertebrates.     

Spatial distribution and size of acetylcholine receptor clusters determined by motor nerves in developing chick muscles

Summary The size and distribution of acetylcholine receptor clusters (AChR-C) on normal and aneural developing muscle fibres of the chick wing were studied by labelling AChR with fluorescent conjugates of -bungarotoxin (-BGT). AChR-C of a size typical of initial synaptic contacts (5 m long) were present at 7 days incubation, shortly after the appearance of nerves, and were grouped in bands corresponding to muscle nerve branches. A regular distribution of large ( 5 m) AChR-C separated by 100–200 m had developed by 10–14 days in the slow-tonic anterior latissimus dorsi and ulnimetacarpalis dorsalis muscles. The role of motor innervation in the formation of AChR-C was assessed by removing the brachial neural tube at 2 days incubation in order to prevent nerves entering the wing. Neural-tube removal prevented the appearance of the large AChR-C normally associated with the early synaptic contacts. Small AChR-C (< 2 m long) appeared in aneural muscles, but these were not grouped into bands characteristic of the large AChR-C in normal muscles. The results suggest that the formation of junctional AChR-C is dependent on nerves.     

Structurally differentDrosophila striated muscles utilize distinct variants of Z-band-associated proteins

Summary Monoclonal antibodies raised against four proteins from insect asynchronous flight muscle have been used to characterize the cross-reacting proteins in synchronous muscle ofDrosophila melanogaster. Two proteins,-actinin and Z(400/600), are found at the Z-band of every muscle examined. A larger variant of-actinin is specific for the perforated Z-bands of supercontractile muscle. A third Z-band protein, Z(210), has a very limited distribution. It is found only in the asynchronous muscle and in the large cells of the jump muscle (tergal depressor of the trochanter). The absence of Z(210) from the anterior four small cells of the jump muscle demonstrates that cells within the same muscle do not have identical Z-band composition. The fourth protein, projectin, > 600 kDa polypeptide component of the connecting filaments in asynchronous muscle, is also detected in all synchronous muscles studied. Surprisingly, projectin is detected in the region of the thick filaments in synchronous muscles, rather than between the thick filaments and the Z-band, as in asynchronous muscles. Despite their different locations, the projectins of synchronous and asynchronous muscles are very similar, but not identical, as judged by SDS-PAGE and by peptide mapping. Projectin shows immunological cross-reactivity with twitchin, a nematode giant protein that is a component of the body wall A-band and shares similarities with vertebrate titin.     

Ablation of primary afferent terminals reduces nicotinic receptor expression and the nociceptive responses to nicotinic agonists in the spinal cord

A variety of studies indicate that spinal nicotinic acetylcholine receptors modulate the behavioral and autonomic responses elicited by afferent stimuli. To examine the location of and role played by particular subtypes of nicotinic receptors in mediating cardiovascular and nociceptive responses, we treated neonatal and adult rats with capsaicin to destroy C-fibers in primary afferent terminals. Reduction of C-fiber terminals was ascertained by the loss of isolectin B4, CGRP and vanilloid receptors as monitored by immunofluorescence. Receptor autoradiography shows a reduction in number of epibatidine binding sites following capsaicin treatment. The reduction is particularly marked in the dorsal horn and primarily affects the class of high affinity epibatidine binding sites thought to modulate nociceptive responses. Accompanying the loss of terminals and nicotinic binding sites were significant reductions in the expression of 3, 4, 5, 2 and 4 nicotinic receptor subunits in the superficial layers of the spinal cord as determined by antibody staining and confocal microscopy. The loss of nicotinic receptors that follows capsaicin treatment results in attenuation of the nociceptive responses to both spinal cytisine and epibatidine. Capsaicin treatment also diminishes the capacity of cytisine to desensitize nicotinic receptors mediating nociception, but it shows little effect on intrathecal nicotinic agonist elicited pressor and heart rate responses. Hence, our data suggest that 3, 4, 5, 2 and 4 subunits of nicotinic receptors are localized in the spinal cord on primary afferent terminals that mediate nociceptive input. A variety of convergent data based on functional studies and subunit expression suggest that 3 and 4, in combination with 2 and 5 subunits, form the majority of functional nicotinic receptors on C-fiber primary afferent terminals. Conversely, spinal nicotinic receptors not located on C-fibers play a primary role in the spinal pathways evoking spinally coordinated autonomic cardiovascular responses.     

Preexposure of mouse peritoneal macrophages to lipopolysaccharide and other stimuli enhances the nitric oxide response to secondary stimuli

The aim of this study was to compare the regulation of the production of tumor necrosis factor- (TNF-) and secondary nitric oxide (NO) in macrophages submitted to a sequence of two stimulations. Pre-exposure for 18h of mouse thioglycollate-elicited peritoneal macrophages to low doses (1–10 ng/ml) of lipopolysaccharide (LPS), in the presence or absence of serum, induces on one hand a desensitization (endotoxin tolerance) for secondary TNF- reponses to LPS and, on the other hand, a 4 fold increase (priming) of serondary NO responses. Preexposure to components from Gram-positive bacteria (lipoteichoic acid, peptidoglycan) and to a synthetic lipid structurally related to lipid A (compound M4), induced similar effects. In contrast to the desensitization for TNF- secretion, the priming for NO production was not mimicked by sodium nitroprusside, a generator of NO. The results suggest that concomitant but distinct activation pathways induced by LPS and other agents can be dissociated by serum-independent modulation processes elicited by preexposure of the cells to LPS itself, or to other stimuli.accepted by M. J. Parnham     

Endplates after esterase inactivationin vivo: correlation between esterase concentration, functional response and fine structure

Summary Mouse sternomastoid muscles were incubated with diisopropylfluorophosphate (DFP)in vivo, and the time course of recovery was studied using histochemistry, EM autoradiography and physiology. We found that: (1) the ability of the muscle to sustain tetanus in response to nerve stimulation is eliminated when the esterases at the neuromuscular junctions are saturated with DFP. This ability is regained partially when <10% of the DFP-binding sites have recovered. (2) There is a positive correlation between the frequency of stimulation at which the tetanic response can be maintained and the extent of acetylcholinesterase (AChE) recovery. (3) Tetanic responses at fusion frequency (about 100 Hz) appear indistinguishable from controls with only about 25% of normal AChE. (4) Butyrylcholinesterase (BuChE) possibly of Schwann cell origin recovers more rapidly than does AChE. (5) The muscle shows fine structural changes involving Z band dissolution and the breakdown of sarcoplasmic reticulum within hours after esterase inactivation. (6) This myopathy reaches a peak at three days after esterase inactivation and is almost fully recovered by two weeks. (7) It can be eliminated if, at the time of esterase inactivation, the nerve is cut or the acetylcholine receptors at the endplate are inactivated by -bungarotoxin.We suggest that the myopathy, seen after DFP, is mediated by Ca²⁺ fluxes due to prolonged action of acetylcholine (ACh) in the absence of esterases.     

Peroxisome proliferator-activated receptor-α and liver cancer: where do we stand?

The peroxisome proliferator-activated receptor- (PPAR), first identified in 1990 as a member of the nuclear receptor superfamily, has a central role in the regulation of numerous target genes encoding proteins that modulate fatty acid transport and catabolism. PPAR is the molecular target for the widely prescribed lipid-lowering fibrate drugs and the diverse class of chemicals collectively referred to as peroxisome proliferators. The lipid-lowering function of PPAR occurs across a number of mammalian species, thus demonstrating the essential role of this nuclear receptor in lipid homeostasis. In contrast, prolonged administration of PPAR agonists causes hepatocarcinogenesis, specifically in rats and mice, indicating that PPAR also mediates this effect. There is no strong evidence that the low-affinity fibrate ligands are associated with cancer in humans, but it still remains a possibility that chronic activation with high-affinity ligands could be carcinogenic in humans. It is now established that the species difference between rodents and humans in response to peroxisome proliferators is due in part to PPAR. The cascade of molecular events leading to liver cancer in rodents involves hepatocyte proliferation and oxidative stress, but the PPAR target genes that mediate this response are unknown. This review focuses on the current understanding of the role of PPAR in hepatocarcinogenesis and identifies future research directions that should be taken to delineate the mechanisms underlying PPAR agonist-induced hepatocarcinogenesis.     

The 3′-untranslated sequence of human skeletal muscle α-actin mRNA

Summary A human actin cDNA clone pGF3 isolated from a fetal skeletal muscle cDNA library is described. The insert cDNA is homologous to skeletal muscle -actin as judged by restriction mapping and nucleotide sequencing. The recombinant contains a substantial portion of the coding and the complete 3-untranslated region. Comparison of the 3 ends of human and rat skeletal muscle and human cardiac -actins reveals little homology between different types of actin genes in man but marked conservation of this region in the skeletal muscle actins of man and rat.     

Activation of the skeletal α-actin promoter during muscle regeneration

Little is known conerning promoter regulation of genes in regenerating skeletal muscles. In young rats, recovery of muscle mass and protein content is complete within 21 days. During the initial 5–10 days of regeneration, mRNA abundance for IGF-I, myogenin and MyoD have been shown to be dramatically increased. The skeletal -actin promoter contains E box and serum response element (SRE) regulatory regions which are directly or indirectly activated by myogenin (or MyoD) and IGF-I proteins, respectively. We hypothesized that the skeletal -actin promoter activity would increase during muscle regeneration, and that this induction would occur before muscle protein content returned to normal. Total protein content and the percentage content of skeletal -actin protein was diminished at 4 and 8 days and re-accumulation had largely occurred by 16 days post-bupivacaine injection. Skeletal - actin mRNA per whole muscle was decreased at day 8, and thereafter returned to control values. During regeneration at day 8, luciferase activity (a reporter of promoter activity) directed by –424 skeletal -actin and –99 skeletal -actin promoter constructs was increased by 700% and 250% respectively; however, at day 16, skeletal -actin promoter activities were similar to control values. Thus, initial activation of the skeletal - actin promoter is associated with regeneration of skeletal muscle, despite not being sustained during the later stages of regrowth. The proximal SRE of the skeletal -actin promoter was not sufficient to confer a regeneration-induced promoter activation, despite increased serum response factor protein binding to this regulatory element in electrophoretic mobility shift assays. Skeletal -actin promoter induction during regeneration is due to a combination of regulatory elements, at least including the SRE and E box. © Kluwer Academic Publishers.     

Identification of alpha-cardiac myosin heavy chain mRNA and protein in extraocular muscle of the adult rabbit

Summary Extraocular muscles contain both fast-twitch and multiply-innervated, tonic-contracting fibres. In rat, these fibres collectively express numerous myosin heavy chain isoforms including fast-type embryonic and neonatal, adult slow twitch type I and fast twitch type II, and a fast isoform unique to extraocular muscle. Immunocytochemical and Western blotting results are presented which suggest that, in rabbit, an additional species, the -cardiac myosin heavy chain, is present. The immunoreactive species is found in all rabbit extraocular muscles and in the rotatory extraocular muscles is expressed in almost all fibres which do not contain a fast myosin heavy chain. Positive identification of this isoform as the -cardiac myosin heavy chain was obtained by sequencing a cloned PCR product derived from extraocular muscle mRNA unique to the 3-end of rabbit -cardiac myosin heavy chain mRNA. This is the first unequivocal demonstration of -cardiac myosin heavy chain expression in extraocular muscle.     

Intrinsic membrane properties causing a bistable behaviour of α-motoneurones

Summary In decerebrate cats a train of impulses in Ia afferents may lead to a sustained increase in excitability of -motoneurones of homonymous and heteronymous muscles. It was previously suggested that this long-lasting excitability increase reflects a maintained synaptic input to the motoneurones from excitatory interneurones. With intracellular recording from motoneurones we here demonstrate that the sustained increase of -motoneurone activity is due to an all-or-none plateau depolarization. This plateau can be induced by a short train of excitatory synaptic potentials or a brief, intracellularly injected depolarizing current pulse and is terminated by a short train of inhibitory synaptic potentials or a hyperpolarizing current pulse. It is concluded that maintained motor unit firing triggered by a brief train of impulses in Ia afferent reflects an intrinsic bistable behaviour of -motoneurones.     

Neural and Mechanical Contributions to the Stretch Reflex: A Model Synthesis

A model for the soleus stretch reflex in the decerebrate cat was synthesized from models of the neural and muscular components, including the two proprioceptors (the muscle spindle and Golgi tendon organ) and their associated afferents (Ia, II, and Ib), the motoneuron pool with its reflex pathways, the branches of the motoneurons to the intrafusal muscles ( innervation), and the extrafusal muscle. Parameters for the muscle and receptor models were chosen independently to match their responses in isolation. Reflex gains and inputs were estimated to fit the response to stretch measured by Nichols and Houk. The chosen reflex gains and inputs are not unique; many different combinations reproduced the characteristic stretch response. With a single set of fixed parameters, the model predicted many mechanical properties of the stretch reflex, including linearization effects (when the stretch magnitude and direction are varied), as well as the dependence on operating force and initial muscle length. The model did not accurately predict the responses at higher stretch velocities, due to failure of the extrafusal muscle model. © 2002 Biomedical Engineering Society. PAC02: 8719Ff, 8719Rr, 8719La     

Macrophage inflammatory protein-1 alpha expression in non-neoplastic and neoplastic lung tissue

The chemokines are members of a bipartite superfamily of soluble proteins that have been implicated in a wide range of acute and chronic inflammatory processes, as well as other immunoregulatory functions. Macrophage inflammatory protein-1 alpha (MIP-1) belongs to the C-C subfamily of these chemokines and is primarily a potent chemoattractant and activator of monocytes. MIP-1 is also thought to play a role in host defence. We examined the expression of MIP-1 in normal lung, inflammatory lung tissue and lung cancer cells by the immunoperoxidase method using a MIP-1 monoclonal antibody. MIP-1 protein was found to be expressed not only by alveolar macrophages, but also by bronchial ciliated cells, hyperplastic alveolar type II cells and activated fibroblasts surrounding malignant tissue. Of 33 cases of lung cancer, 23 (70%) expressed MIP-1. These observations suggest that lung cancer cells, non-neoplastic alveolar type II cells and fibroblasts can participate in inflammatory cell recruitment via the production of MIP-1. Tumour derived MIP- may also affect the interaction between lung cancer and host inflammatory cells.