Cloning and characterization of fiber type-specific ryanodine receptor isoforms in skeletal muscles of fish

We have cloned agroup of cDNAs that encodes the skeletal ryanodine receptor isoform(RyR1) of fish from a blue marlin extraocular muscle library. The cDNAsencode a protein of 5,081 amino acids with a calculated molecular massof 576,302 Da. The deduced amino acid sequence shows strong sequenceidentity to previously characterized RyR1 isoforms. An RNA probederived from a clone of the full-length marlin RyR1 isoform hybridizesto RNA preparations from extraocular muscle and slow-twitch skeletalmuscle but not to RNA preparations from fast-twitch skeletal or cardiacmuscle. We have also isolated a partial RyR clone from marlin andtoadfish fast-twitch muscles that shares 80% sequence identity withthe corresponding region of the full-length RyR1 isoform, and a RNAprobe derived from this clone hybridizes to RNA preparations fromfast-twitch muscle but not to slow-twitch muscle preparations. Westernblot analysis of slow-twitch muscles in fish indicates the presence ofonly a single high-molecular-mass RyR proteincorresponding to RyR1. [³H]ryanodine bindingassays revealed the fish slow-twitch muscle RyR1 had a greatersensitivity for Ca²⁺ than thefast-twitch muscle RyR1. The results indicate that, in fish muscle,fiber type-specific RyR1 isoforms are expressed and the two proteinsare physiologically distinct.

     

Properties of synaptic inputs from myenteric neurons innervating submucosal S neurons in guinea pig ileum

This study examined synaptic inputs from myenteric neurons innervating submucosal neurons. Intracellular recordings were obtained from submucosal S neurons in guinea pig ileal preparations in vitro, and synaptic inputs were recorded in response to electrical stimulation of exposed myenteric plexus. Most S neurons received synaptic inputs [>80% fast (f) excitatory postsynaptic potentials (EPSP), >30% slow (s) EPSPs] from the myenteric plexus. Synaptic potentials were recorded significant distances aboral (fEPSPs, 25 mm; sEPSPs, 10 mm) but not oral to the stimulating site. When preparations were studied in a double-chamber bath that chemically isolated the stimulating "myenteric chamber" from the recording side "submucosal chamber," all fEPSPs were blocked by hexamethonium in the submucosal chamber, but not by a combination of nicotinic, purinergic, and 5-hydroxytryptamine-3 receptor antagonists in the myenteric chamber. In 15% of cells, a stimulus train elicited prolonged bursts of fEPSPs (>30 s duration) that were blocked by hexamethonium. These findings suggest that most submucosal S neurons receive synaptic inputs from predominantly anally projecting myenteric neurons. These inputs are poised to coordinate intestinal motility and secretion.     

Actions of acetylcholine on the salivary gland cells of the pond snail, Planorbis corneus

Intracellular recordings have been made from salivary gland cells of the pond snail Planorbis corneus. Gland cells produced a dose-dependent biphasic response to the bath application of acetylcholine (ACh), an initial depolarization being followed by a hyperpolarization. Nicotine and the nicotinic agonist tetramethylammonium had an excitatory action on the gland cells. The muscarinic agonists acetyl-beta-methyl choline and arecoline were also stimulants, but muscarine, bethanechol and pilocarpine produced no response from gland cells at 10(-3) M. A number of cholinergic antagonists, including atropine, hexamethonium and curare, effectively blocked the response to ACh. The depolarizing phase of the ACh response resulted from an increased membrane permeability to Na+ ions, though the participation of other ionic species cannot be ruled out. The hyperpolarizing phase of the ACh response was produced by the activity of an electrogenic Na+/K+ pump.     

Cholinergic interneurons in the feeding system of the pond snail Lymnaea stagnalis. II. N1 interneurons make cholinergic synapses with feeding motoneurons.

The N1 neurons are a population of interneurons active during the protraction phase of the feeding rhythm. All the N1 neurons are coupled by electrical synapses which persist in a high Mg/low Ca saline which blocks chemical synapses. Individual N1 spikes produce discrete electrotonic postsynaptic potentials (PSPS) in other N1 cells, but the coupling is not strong enough to ensure 1:1 firing. Bursts of N1 spikes generate compound PSPS in the feeding motoneurons. The sign (excitation or inhibition) of the N1 input corresponds with the synaptic barrage recorded during the protraction phase. Discrete PSPS are only resolved in a Hi-Di saline. Their variation in latency and number can be explained by variation in electrotonic propagation within the electrically coupled network of N1 cells. The excitatory postsynaptic potentials (ESPS) in the 1 cell are reduced by 0.5 mM antagonists hexamethonium (HMT), atropine (ATR), curare (d-TC) and by methylxylocholine (MeXCh), all of which block the excitatory cholinergic receptor (Elliott et al. (Phil. Trans. R. Soc. Lond. 336, 157-166 (Preceding paper.) (1992)). The 1 cell EPSPS were transiently blocked by phenyltrimethylammonium (PTMA), which is both an agonist and antagonist at the 1 cell excitatory acetylcholine (ACh) receptor (Elliott et al. 1992). The inhibitory postsynaptic potential (IPSP) in the 3 cell is blocked by bath applications of MeXCh and PTMA, which both abolish the response of the 3 cell to ACh (Elliott et. al. 1992). The effects of the cholinergic antagonists on the response of 4 cluster and 5 cells to N1 stimulation matches their response to ACh (Elliott et al. 1992). It is concluded that the population of N1 cells are multiaction, premotor cholinergic interneurons.     

Cholinergic interneurons in the feeding system of the pond snail Lymnaea stagnalis. I. Cholinergic receptors on feeding neurons.

All the identified feeding motoneurons of Lymnaea respond to bath or iontophoretically applied acetylcholine (ACh). Three kinds of receptors (one excitatory, one fast inhibitory and one slow inhibitory) were distinguished pharmacologically. The agonist TMA (tetramethylammonium) activates all three receptors, being weakest at the slow inhibitory receptor. PTMA (phenyltrimethylammonium) is less potent than TMA and is ineffective at the slow inhibitory receptor, which is the only receptor sensitive to arecoline. At 0.5 mM the antagonists HMT (hexamethonium) and ATR (atropine) selectively block the excitatory response, while PTMA reduces the response to ACh at all three receptors. d-TC (curare) antagonizes only the fast excitatory and the fast inhibitory responses, but MeXCh (methylxylocholine) blocks the fast excitatory and slow inhibitory responses solely. For each of the feeding motoneurons, the sign of the cholinergic response (excitation or inhibition) is the same as the synaptic input received in the N1 phase of the feeding rhythm.     

Smooth muscle expresses a cardiac/slow muscle isoform of the Ca2+-transport ATPase in its endoplasmic reticulum.

Smooth muscle expresses in its endoplasmic reticulum an isoform of the Ca2+-transport ATPase that is very similar to or identical with that of the cardiac-muscle/slow-twitch skeletal-muscle form. However, this enzyme differs from that found in fast-twitch skeletal muscle. This conclusion is based on two independent sets of observations, namely immunological observations and phosphorylation experiments. Immunoblot experiments show that two different antibody preparations against the Ca2+-transport ATPase of cardiac-muscle sarcoplasmic reticulum also recognize the endoplasmic-reticulum/sarcoplasmic-reticulum enzyme of the smooth muscle and the slow-twitch skeletal muscle whereas they bind very weakly or not at all to the sarcoplasmic-reticulum Ca2+-transport ATPase of the fast-twitch skeletal muscle. Conversely antibodies directed against the fast-twitch skeletal-muscle isoform of the sarcoplasmic-reticulum Ca2+-transport ATPase do not bind to the cardiac-muscle, smooth-muscle or slow-twitch skeletal-muscle enzymes. The phosphorylated tryptic fragments A and A1 of the sarcoplasmic-reticulum Ca2+-transport ATPases have the same apparent Mr values in cardiac muscle, slow-twitch skeletal muscle and smooth muscle, whereas the corresponding fragments in fast-twitch skeletal muscle have lower apparent Mr values. This analytical procedure is a new and easy technique for discrimination between the isoforms of endoplasmic-reticulum/sarcoplasmic-reticulum Ca2+-transport ATPases.     

Effects of acetylcholinesterase inhibitor paraoxon denote the possibility of non-quantal acetylcholine release in myocardium of different vertebrates

Effects of organophosphorous acetylcholinesterase inhibitor paraoxon were studied in the isolated atrial and ventricular myocardium preparations of a fish (cod), an amphibian (frog) and a mammal (rat) using the microelectrode technique. Incubation of isolated atrium with paraoxon (5 × 10⁻⁶–5 × 10⁻⁵ M) caused significant reduction of action potential duration and marked slowing of sinus rhythm. These effects were abolished by muscarinic blocker atropine and therefore are caused by acetylcholine, which accumulates in the myocardium due to acetylcholinesterase inhibition even in the absence of vagal input. Hemicholinium III is a blocker of high affinity choline-uptake transporters, which are believed to mediate non-quantal release of acetylcholine from cholinergic terminals in different tissues. In the atrial myocardium of all the three studied species, hemicholinium III (10⁻⁵ M) significantly suppressed all the effects of paraoxon. Blocker of parasympathetic ganglionic transmission hexamethonium bromide (10⁻⁴ M) and inhibitor of vesicular acetylcholine transporters vesamicol (10⁻⁵ M) failed to attenuate paraoxon effects. Among ventricular myocardium preparations of three species paraoxon provoked marked cholinergic effects only in frog, hemicholinium III abolished these effects effectively. We conclude that paraoxon stops degradation of acetylcholine in the myocardium and helps to reveal the effects of acetylcholine, which is continuously secreted from the cholinergic nerves in non-quantal manner. Thus, non-quantal release of acetylcholine in the heart is not specific only for mammals, but is also present in the hearts of different vertebrates.     

Action of morphine on guinea-pig myenteric plexus and mouse vas deferens studied by intracellular recording.

Morphine reduces the output of transmitter from the myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum and from the mouse vas deferens. Intracellular recordings were made from ganglion cells of the myenteric plexus and smooth muscle cells of the vas deferens. Synaptic transmission within the myenteric plexus was blocked by hexamethonium. Morphine did not change the properties of the ganglion cells, nor did it affect synaptic potentials. 5-Hydroxytryptamine inhibited acetylcholine release at intraganglionic synapses by an action which was unaffected by morphine. In the vas deferens, excitatory junction potentials were elicited by stimulation of postganglionic adrenergic nerve fibres. The junction potentials were depressed by morphine and levorphanol but not by dextrorphan. This depression was reversed by naloxone. The results indicate that morphine acts directly to reduce transmitter release at the neuro-effector junctions in the myenteric plexus-longitudinal muscle preparation and in the vas deferens in these species.     

Quantitative investigation of the neuromuscular junction of rat skeletal muscle fibres after double innervation

Summary In normal (untreated) rats the mean length ratio of postsynaptic to presynaptic membrane was 2.7±0.8 for neuromuscular junctions of slow-twitch soleus muscle fibres and 4.2±1.0 for neuromuscular junctions of fast-twitch extensor digitorum longus muscle fibres; this difference was significant (P<0.001). After experimental double innervation by fast and slow muscle nerves for four months, the ratio was (1) 2.9±0.8 for the original slow-twitch fibre end-plate and 2.8±0.8 for the newly established one, both not significantly different from that of the normal slow-twitch fibres; and (2) 2.2±0.5 for the original fast-twitch fibre end-plate and 2.2±0.7 for the newly established one, both significantly smaller than that of the normal fast-twitch fibres (P<0.001). This means that the double innervated slow-twitch muscle fibres retained their original neuromuscular junction type, whereas the doubly-innervated fast-twitch muscle fibres underwent a dramatic transformation of their neuromuscular junction from the fast-muscle to the slow-muscle type. In both doubly innervated fibres, the ultrastructural characteristics of neuromuscular junctions, whether altered or not, were identical at both end-plate regions.     

Chemosensitivity of crayfish slowly adapting stretch receptors to nicotine

It has recently been demonstrated that slowly adapting stretch receptors (SASRs) in the airways of the dog respond directly to nicotine (Federation Proc. 43: 318, 1984). The purpose of the present experiment was to investigate this chemical effect on an isolated stretch receptor. The crayfish muscle receptor organ was chosen, since crayfish muscle is reported to be insensitive to nicotine or acetylcholine and therefore permits the testing of any direct chemical effect of nicotine on the muscle stretch receptors. The tail was removed and pinned out in a tissue bath, and a stretch receptor organ was surgically isolated. Single-unit SASR extracellular nerve recordings were made while simultaneously measuring tension in the tail. Drugs were prepared in Van Harreveld's solution and administered into the bath kept at 18 degrees C. When resting muscle tension was essentially reduced to zero by cutting both ends of the receptor organ muscle, nicotine (0.07 microM) added to the bath increased receptor activity fourfold. This response was abolished by treatment with hexamethonium (690 microM). In a second group of animals in which the muscle was left intact, nicotine was shown to significantly increase receptor sensitivity to step changes in muscle tension. Once again hexamethonium blocked the response to nicotine. These results demonstrate that the sensitivity of mechanoreceptor can be altered by chemical interaction with nicotinic receptors, which dramatically alter sensory receptor activity.     

Neural mechanism generating firing patterns in jaw motoneurons during the food-induced response in Aplysia kurodai

1. In each right and left buccal ganglia of Aplysia kurodai, we identified 4 premotor neurons impinging on the ipsilateral jaw-closing and -opening motoneurons. Three of them (MA1 neurons) had features of multifunctional neurons. Current-induced spikes in the MA1 neurons produced excitatory junction potentials (EJPs) in the buccal muscle fibers. In addition, tactile stimulation of the buccal muscle surface produced a train of spikes in the MA1 neurons without synaptic input. The other neuron (MA2) had only a premotor function. 2. The MA1 and MA2 neurons had similar synaptic effects on the jaw-closing and -opening motoneurons. Current-induced spikes in the premotor neurons gave rise to monosynaptic inhibitory postsynaptic potentials (IPSPs) in the ipsilateral jaw-closing motoneurons. Simultaneously, spikes in one of the MA1 neurons and the MA2 also gave rise to monosynaptic excitatory postsynaptic potentials (EPSPs) in the ipsilateral jaw-opening motoneuron. 3. The IPSPs and the EPSPs induced by spikes in the premotor neurons were reversibly blocked by d-tubocurarine and hexamethonium, respectively, suggesting that the MA1 and MA2 neurons are cholinergic. 4. When depolarizing and hyperpolarizing current pulses were passed into one premotor neuron, attenuated but similar potential changes were produced in another randomly selected premotor neuron in the same ganglion, suggesting that they are electronically coupled.     

Identification of polypeptides associated with sarcolemmal vesicles enriched in orthogonal arrays

Electron microscopy of freeze-fracture replicas from the sarcolemmas of fast-twitch muscle fibers reveals orthogonal arrays of particles. The biochemical nature of macromolecules associated with the sarcolemmal orthogonal array was investigated using muscle fragments and isolated sarcolemmal vesicles. Muscle fragments incubated in vitro with the lectin concanavalin A exhibited a clustering of orthogonal arrays into local patches. Treatment with other lectins did not result in the clustering of arrays. Clustering was inhibited by the addition of alpha-methyl-D-mannoside, a ligand which also binds concanavalin A. These results suggest that the orthogonal arrays (or associated components) specifically bind concanavalin A. Sarcolemmal vesicles from rabbit sacrospinalis (SAC) and rat extensor digitorum longus (EDL) (both primarily fast-twitch) and rat soleus (SOL) (primarily slow-twitch) were obtained by a combination of low-salt fractionation and sucrose density gradient centrifugation. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins and glycoproteins solubilized from these vesicles revealed several bands. Four of these bands were present in gels from both the rabbit and rat fast-twitch muscle sarcolemmal preparations (that contained arrays), yet were absent in gels from rat slow-twitch muscle sarcolemmal preparations (not bearing arrays). An enrichment in vesicles containing arrays was achieved by binding SAC sarcolemmal vesicles to Con A-Sepharose 4B beads. SDS-PAGE analysis of array-enriched vesicles from the concanavalin A beads revealed enrichment of three major bands at Mr 93,000, 54,000 and 49,000. These enriched bands correlate with three of the four bands common to fast-twitch EDL and SAC, yet absent in slow-twitch SOL sarcolemmal preparations. We conclude that at least one macromolecular component associated with the sarcolemmal orthogonal array is a concanavalin A binding glycoprotein. We further conclude that three candidates for this component co-purify with the morphological array, and have approximate molecular weights of 93,000, 54,000 and 49,000.     

Responses of neuromuscular systems under gravity or microgravity environment.

Hindlimb suspension of rats induces induces fiber atrophy and type shift of muscle fibers. In contrast, there is no change in the cell size or oxidative enzyme activity of spinal motoneurons innervating muscle fibers. Growth-related increases in the cell size of muscle fibers and their spinal motoneurons are inhibited by hindlimb suspension. Exposure to microgravity induces atrophy of fibers (especially slow-twitch fibers) and shift of fibers from slow- to fast-twitch type in skeletal muscles (especially slow, anti-gravity muscles). In addition, a decrease in the oxidative enzyme activity of spinal motoneurons innervating slow-twitch fibers and of sensory neurons in the dorsal root ganglion is observed following exposure to microgravity. It is concluded that neuromuscular activities are important for maintaining metabolism and function of neuromuscular systems at an early postnatal development and that gravity effects both efferent and afferent neural pathways.     

Assessing the roles of glutamatergic and cholinergic synaptic drive in the control of fictive swimming frequency in young Xenopus tadpoles

This paper investigates the proposal that the frequency of the swimming central pattern generator in young Xenopus tadpoles is partly determined by the population of glutamatergic premotor interneurons active on each cycle. During fictive swimming spinal neurons also receive cholinergic and electrotonic excitation from motoneurons. As frequency changes during swimming we make two predictions: first, since most motoneurons fire very reliably at all frequencies, the electrotonic and nicotinic drive from motoneurons should remain constant, and second, when swimming frequency decreases, the glutamatergic drive should decrease as the number of active premotor excitatory interneurons decreases. We have tested these predictions by measuring the excitatory synaptic drive to motoneurons as frequency changes during fictive swimming. The components of synaptic drive were revealed by the local microperfusion of strychnine together with different excitatory antagonists. After blocking the nicotinic acetylcholine receptor, the mainly glutmatergic excitatory synaptic drive still changed with frequency. However, when glutamate receptors or all chemical transmission was blocked, excitation did not change with frequency. Our predictions are confirmed, suggesting that premotor excitatory interneurons are a major factor in frequency control in the tadpole central pattern generator and that motoneurons provide a stable background excitation. Accepted: 14 August 1998     

Electrophysiological properties of spinal motoneurons in the adult turtle

The purpose of this study was to develop a scheme for classifying turtle motoneurons, such that their properties could be compared to those of other vertebrate species, including, in particular, the cat. A 130-cell sample of turtle motoneurons was provisionally classified into four groups (1-4) on the basis of a cluster analysis of the cells' intracellularly recorded input resistance, rheobase, and slope of their stimulus current-spike frequency relation. These measurements, using sharp microelectrodes and an in vitro spinal cord slice preparation, were particularly robust. It is argued that the cat counterpart of our turtle type 1, 2, and 3 motoneurons innervate slow-twitch muscle fibers, fast-twitch-oxidative fibers, and fast-twitch-glycolytic fibers, respectively. Our turtle type 4 motoneuron is thought analogous to a particularly high-threshold cat and human cell that innervates highly fatigable fast-twitch muscle fibers in both species. Our turtle type 1 category may include cells that innervate non-twitch muscle fibers, which are found in other non-mammalian vertebrates. To advance comparative spinal cord neurobiology, the present results invite comparison to the motoneurons of other vertebrate species, which have yet to be subjected to similar or other classification procedures.     

Neuromuscular electrophysiology of the filarial helminth Dipetalonema viteae

1. A body wall preparation is described which permits intracellular recording from the somatic muscle cells of the small filarial nematode, Dipetalonema viteae. Using this preparation, resting membrane potentials were measured and spontaneous muscle depolarizations described. 2. Stimulatory effects noted upon the addition of acetylcholine, or the cholinergic agonists suggest the hypothesis that acetylcholine is the excitatory neurotransmitter. However, in contrast with vertebrate tissues, the cholinergic antagonists, d-tubocurarine, hexamethonium and pentolinium do not inhibit somatic muscle activity of the worm. 3. GABA inhibited somatic muscle depolarizations, suggesting the possibility that it may serve as an inhibitory neurotransmitter. 4. The anthelmintic drug, levamisole, produced a depolarizing block. Effects of other pharmacological agents are described, discussed and compared with effects on vertebrate muscles.     

Nicotinic action of anatoxin-a on guinea pig ileum antagonized by thymopentin

(+)-Anatoxin-a (ANTX) stimulated guinea pig ileum contraction with a potency similar to that of acetylcholine (ACh); the stimulation was blocked by tubocurarine, hexamethonium, or atropine. Although the contraction stimulated by ANTX was blocked by atropine, no specific inhibition of the binding of [3H]N-methylscopolamine to ileum membranes was observed in the presence of ANTX. Furthermore, ANTX failed to stimulate the secretion of alpha-amylase from pancreatic acinar cells, a process that is activated by cholinergic agonists at the muscarinic receptors. When the ileum itself was stimulated by ACh, the contraction was not blocked by either hexamethonium or tubocurarine. Preincubation of the ileum with hemicholinium caused a 50% reduction in the ability of ANTX to stimulate contraction. Based upon these data, it was inferred that ANTX binds to postganglionic synaptic nicotinic receptors in the ileum, thus releasing endogenous ACh, which in turn causes ileum contraction by interacting with the postsynaptic muscarinic receptors. It was also observed that thymopentin (TP-5), a pentapeptide corresponding to positions 32-36 of thymopoietin, blocked the stimulation of ileum contraction by ANTX.     

Cholinergic modulation of progesterone-induced maturation of Xenopus oocytes in vitro

Mixed and muscarinic cholinergic agonists (acetylcholine, carbamylcholine, methacholine, oxotremorine, and pilocarpine) accelerated in a dose-dependent manner the progesterone-induced maturation of Xenopus laevis oocytes. None of these agonists induced oocyte maturation in the absence of progesterone. The accelerating effect of cholinergic agonists was blocked in a dose-dependent manner by specific muscarinic antagonists (atropine and scopolamine) but not by specific nicotinic antagonists (d-tubocurarine and hexamethonium). The specific nicotinic agonist, dimethylphenylpiperazine, alone induced maturation in the absence of progesterone. The optimal promoting effect of acetylcholine was observed when oocytes were exposed to acetylcholine for 30 min, 5 min after the addition of progesterone, and was markedly better than when oocytes were exposed to acetylcholine throughout their incubation with progesterone. The effect of acetylcholine was observed in both follicle-enclosed and in defolliculated oocytes, indicating that follicular cells were not the target of the cholinergic drugs.