Presynaptic Purinergic Modulation of the Rat Neuro-Muscular Transmission.

ATP, being a well-known universal high-energy compound, plays an important role as a signaling molecule and together with its metabolite adenosine they both attenuate the release of acetylcholine in the neuro-muscular synapse acting through membrane P2 and P1 receptors, respectively. In this work, using a mechanomyographic method, we analyzed the presynaptic mechanisms by which ATP and adenosine can modulate the transduction in the rat m. soleus and m. extensor digitorum longus. N-ethylmaleimide, a G-protein antagonist, prevents the modulating effects of both ATP and adenosine. The action of ATP is abolished by chelerythrin, a specific phospholipase C inhibitor, while the inhibitory effect of adenosine is slightly increased by Rp-cAMPS, an inhibitor of protein kinase A, and by nitrendipine, a blocker of L-type Ca2+ channels. The addition of DPCPX, an A1 receptor antagonist, fully prevents the inhibitory action of adenosine in both muscles. Our data indicate that the inhibitory action of ATP involves metabotropic P2Y receptors and is mediated by phospholipase C dependent processes in rat motor neuron terminals. We suggest that the presynaptic effect of adenosine consists of negative and positive actions. The negative action occurs by stimulation of adenosine A1 receptors while the positive action is associated with the stimulation of adenosine A2A receptors, activation of protein kinase A and opening of L-type calcium channels. The combined mechanism of the modulating action of ATP and adenosine provides fine tuning of the synapse to fast changing conditions in the skeletal muscles.

P2 Receptor Signaling in Motor Units in Muscular Dystrophy.

The purine signaling system is represented by purine and pyrimidine nucleotides and nucleosides that exert their effects through the adenosine, P2X and P2Y receptor families. It is known that, under physiological conditions, P2 receptors play only a minor role in modulating the functions of cells and systems; however, their role significantly increases under some pathophysiological conditions, such as stress, ischemia or hypothermia, when they can play a dominant role as a signaling molecule. The diversity of P2 receptors and their wide distribution in the body make them very attractive as a target for the pharmacological action of drugs with a new mechanism of action. The review is devoted to the involvement of P2 signaling in the development of pathologies associated with a loss of muscle mass. The contribution of adenosine triphosphate (ATP) as a signal molecule in the pathogenesis of a number of muscular dystrophies (Duchenne, Becker and limb girdle muscular dystrophy 2B) is considered. To understand the processes involving the purinergic system, the role of the ATP and P2 receptors in several models associated with skeletal muscle degradation is also discussed.

Modulatory Roles of ATP and Adenosine in Cholinergic Neuromuscular Transmission.

A review of the data on the modulatory action of adenosine 5'-triphosphate (ATP), the main co-transmitter with acetylcholine, and adenosine, the final ATP metabolite in the synaptic cleft, on neuromuscular transmission is presented. The effects of these endogenous modulators on pre- and post-synaptic processes are discussed. The contribution of purines to the processes of quantal and non-quantal secretion of acetylcholine into the synaptic cleft, as well as the influence of the postsynaptic effects of ATP and adenosine on the functioning of cholinergic receptors, are evaluated. As usual, the P2-receptor-mediated influence is minimal under physiological conditions, but it becomes very important in some pathophysiological situations such as hypothermia, stress, or ischemia. There are some data demonstrating the same in neuromuscular transmission. It is suggested that the role of endogenous purines is primarily to provide a safety factor for the efficiency of cholinergic neuromuscular transmission.

The effects of ATP on the contractions of rat and mouse fast skeletal muscle.

INTRODUCTION: The aim of this study was to compare the effects of adenosine-5'-triphosphate (ATP) and adenosine on the contractility of rodent extensor digitorum longus (EDL) muscle at normal and low temperatures. METHODS: Contractions of rat and mouse isolated EDL were induced by either electrical stimulation (ES) or exogenous carbachol and recorded in the presence of ATP or adenosine (both at 100 µM). RESULTS: ATP at all temperatures caused a decrease of the contractions induced by carbachol in rat and mouse EDL and ES-induced contractions in rat EDL, while it potentiated the ES-induced contractions of mouse EDL. Adenosine reduced the contractility of rat and mouse EDL evoked by ES and did not affect the carbachol-induced contractions of rat and mouse EDL at any temperature. DISCUSSION: Under various temperature conditions, ATP inhibits pre- but potentiates postsynaptic processes in the mouse EDL; in the rat EDL ATP causes only inhibition of neuromuscular conduction. Muscle Nerve 59:509-516, 2019.

Effects of ATP and adenosine on contraction amplitude of rat soleus muscle at different temperatures.

INTRODUCTION: The aim of this study was to evaluate the effects of adenosine 5'-triphosphate (ATP) and adenosine on the contractility of mammalian skeletal muscle under hypothermic conditions. METHODS: Contractions of isolated rat soleus muscle were induced by either electrical stimulation (ES) or carbachol at physiological temperatures (37°C) and hypothermic conditions (30-14°C) and recorded in the presence of ATP, adenosine, suramin, and 8-(p-sulfophenyl)-theophylline (8-SPT). RESULTS: At 37°C, incubation of the muscles with ATP inhibited ES-induced contractions; the inhibitory effect of ATP disappeared at 14°C. Adenosine inhibited ES-induced contractions at all temperature levels; 8-SPT fully prevented the action of adenosine. ATP and adenosine did not significantly affect carbachol-induced contractions at 37°C, while at lower temperatures ATP potentiated them. Suramin fully prevented effects of ATP. CONCLUSIONS: ATP is involved in both pre- and postsynaptic regulation of rat soleus muscle contractility, and these processes are significantly more pronounced at low temperatures. Muscle Nerve 55: 417-423, 2017.

Extraneuronal toxicity of Alzheimer's ß-amyloid peptide: comparative study on vertebrate skeletal muscles.

INTRODUCTION: Alzheimer's ß-amyloid peptide (ßAP) is known to possess a wide range of toxic effects on neurons in vitro and in vivo; however, there is little information available regarding its impact on other excitable tissues such as skeletal muscles, which, apart from brain cells, are thought to also be targets of ßAP. METHODS: Utilizing the combination of electrophysiology and myography, we investigated whether ßAP also impairs the functioning of myocytes in frogs and mice. RESULTS: Although application of ßAP in the range of 10(-6) to 10(-8) M induced depolarization of muscle fibers in both species, it impaired contractility in frogs but not in mice, by reducing endplate potential amplitude and increasing the threshold potential. CONCLUSIONS: Unchanged contractility in the mouse in the presence of ßAP is due to a higher safety factor of neuromuscular transmission in mammals compared with amphibians. Possible clinical implications are discussed.

Alzheimer's beta-amyloid-induced depolarization of skeletal muscle fibers: implications for motor dysfunctions in dementia.

Numerous findings obtained over the last decades suggest that accumulation of beta-amyloid peptide (betaAP) plays the central role in the pathogenesis of Alzheimer's disease. It is well established that betaAP has wide range of toxic effects on neurons in vitro and in vivo, however the influence of betaAP in the periphery and on various other types of excitable tissues, eg. skeletal muscle cells, is almost unknown despite the many non-cognitive and other extra-neuronal symptoms associated with Alzheimer's dementia. Here we utilized conventional electrophysiological technique to investigate the effects and mechanisms of betaAP action on the resting membrane potential of frog skeletal muscle fibers. betaAP in the range of concentrations from 10(-6) to 10(-8)M produced slow, significant, reversible depolarization of muscle fiber membranes. The impact developed and was washed out faster at higher concentrations of betaAP (10(-6)-0(-7)M). The effect of betaAP was completely absent when applied in Na+-free Tris+ solutions. betaAP-mediated depolarization was also prevented by tetrodotoxin (10(-5)M) pre-treatment and rescued by tetrodotoxin after-treatment. These findings suggest that betaAP-induced depolarization of skeletal muscle plasma membranes can significantly disturb the functioning of skeletal muscles and therefore contribute to motor dysfunction observed in Alzheimer's disease and other disorders associated with betaAP accumulation.

Interaction of hydrocortisone with ATP and adenosine on nerve-mediated contractions of frog skeletal muscle.

The inhibitory effects of ATP and adenosine on the nerve-mediated contractile responses of isolated sartorius muscle of the frog, Rana ridibunda, evoked by electrical field stimulation (EFS) were studied using pharmacological organ-bath technique. The effects of hydrocortisone applied in vitro and in vivo on contractility of sartorius muscle were also examined. ATP (100 microM) significantly reduced the amplitude of contraction to EFS of sartorius muscle, while pyridoxalphosphate-6-azonphenyl-2',4'-disulfonic acid (PPADS; 10 microM), a P2 receptor antagonist, abolished inhibitory effect of ATP. A similar inhibitory effect of adenosine (100 microM) was fully antagonized by 8-(p-sulfophenyl)-theophylline (8-SPT, 100 microM), a P1 receptor antagonist. Incubation of the tissue with hydrocortisone (10 microM) caused a slight, but significant, decrease of muscle contractions. After incubation of muscle preparations with both hydrocortisone and ATP, no inhibition of muscle contractility was registered. A single injection of hydrocortisone (100 mg/kg) 12 h prior to experiments to frogs did not significantly change the nerve-mediated contractility of isolated sartorius muscle; however, it abolished the inhibitory action of ATP without changing inhibitory activity of adenosine. After treatment of frogs with hydrocortisone for 14 days (100 mg/kg/day), both ATP and adenosine retained their inhibitory action on EFS-induced contractions of the muscle, and their effects were antagonized by PPADS and 8-SPT, respectively. It is concluded that hydrocortisone has antagonistic actions against the inhibitory effects of ATP at the frog neuromuscular junction, although this effect is lost following long-term treatment with hydrocortisone.

The mechanisms of multi-component paired-pulse facilitation of neurotransmitter release at the frog neuromuscular junction.

We have studied the mechanisms of paired-pulse facilitation (PPF) of neurotransmitter release in isolated nerve-muscle preparations of the frog cutaneous pectoris muscle. In normal extracellular Ca(2+) concentration ([Ca(2+)](o), 1.8 mM), as the interpulse interval was increased from 5 to 500 ms, PPF decayed as a sum of two exponential components: a larger but shorter first component (F1) and a smaller but more prolonged second component (F2). In low [Ca(2+)](o) (0.5 mM), both F1 and F2 increased, and a third "early" component (Fe) appeared whose amplitude was larger and whose duration was shorter than F1 or F2. In the presence of the "fast" Ca(2+) buffer BAPTA-AM, Fe disappeared, whereas F1 and F2 decreased in amplitude and duration. In contrast, the "slow" Ca(2+) buffer EGTA-AM caused a decrease of Fe and reduction or complete blockade of F2, without any changes of F1. In solutions containing Sr(2+) (1 mM), the magnitude of Fe was decreased, F1 was significantly reduced and shortened, but F2 was unaffected. Application of the calmodulin inhibitor W-7 (10 microM) at normal [Ca(2+)](o) produced a marked decrease of F2, and at low [Ca(2+)](o), a complete blockade of Fe. These results suggest that PPF at frog motor nerve terminals is mediated by several specific for different PPF components intraterminal Ca(2+) binding sites, which trigger neurotransmitter release. These sites have a higher affinity for Ca(2+) ions and are located farther from the release-controlling Ca(2+) channels than the Ca (2+) sensor that mediates phasic release.

Ovalbumin-induced sensitization affects non-quantal acetylcholine release from motor nerve terminals and alters contractility of skeletal muscles in mice.

Skeletal muscles play key roles in the development of various pathologies, including bronchial asthma and several types of auto-immune disorders, e.g. polymyositis. Since most of these maladies have an immunological/allergic element, this paper is devoted to assessing the impact of immunobiological reorganization on the functional properties of isolated skeletal muscles in mice. A combination of two methods (myography and electrophysiology) was used to evaluate extensor digitorum longus (EDL) and diaphragmatic muscle (DM) in this regard. Conventional myographic technique showed that ovalbumin-induced sensitization (OS) produced different changes in the contractile properties of EDL and DM. The amplitudes of carbachol (CCh)-induced contractions increased in DM but decreased in EDL. Those changes were inversely related to OS-mediated changes of non-quantal acetylcholine (ACh) release intensity within the muscle endplate, as shown by the electrophysiologically measured H-effect. These results clearly show that OS-mediated changes of non-quantal ACh release alter the functional properties of postjunctional ACh receptors and therefore contribute to the disturbance of CCh-induced contractility of skeletal muscles. Other mechanisms of OS-mediated changes of skeletal muscle contractility are also proposed and discussed.

Different effects of ATP on the contractile activity of mice diaphragmatic and skeletal muscles.

Apart from acetyl-choline (Ach), adenosine-5'-trisphosphate (ATP) is thought to play a role in neuromuscular function, however little information is available on its cellular physiology. As such, effects of ATP and adenosine on contractility of mice diaphragmatic and skeletal muscles (m. extensor digitorum longa-MEDL) have been investigated in in vitro experiments. Application of carbacholine (CCh) in vitro in different concentrations led to pronounced muscle contractions, varying from 9.15+/-4.76 to 513.13+/-15.4 mg and from 44.65+/-5.01 to 101.46+/-9.11 mg for diaphragm and MEDL, respectively. Two hundred micromolars of CCh in both muscles caused the contraction with the 65% (diaphragm) to 75% (MEDL) of maximal contraction force-this concentration was thus used in further experiments. It was found that application of ATP (100 microM) increased the force of diaphragmatic contraction caused by CCh (200 microM) from 335.2+/-51.4 mg (n=21) in controls to 426.5+/-47.8 mg (n=10; P<0.05), but decreased the contractions of MEDL of CCh from 76.6+/-6.5mg (n=26) in control to 40.2+/-9.0mg (n=8; P<0.05). Application of adenosine (100 microM) had no effect on CCh-induced contractions of these muscles. Resting membrane potential (MP) measurements using sharp electrodes were done at 10, 20 and 30 min after the application of ATP and adenosine. Diaphragm showed depolarization from 75+/-0.6 down to 63.2+/-1.05, 57.2+/-0.96 and 53.6+/-1.1 mV after 10, 20 and 30 min of exposition, respectively (20 fibers from 4 muscles each, P<0.05 in all three cases). Adenosine showed no effect on diaphragmatic MP. Both agents were ineffective in case of MEDL. The effects of ATP in both tissues were abolished by suramin (100 microM), a P2-receptor antagonist, and chelerythrin (50 microM), a specific protein-kinase C (PKC) inhibitor, but were not affected by 1H-[1,2,4]-oxadiazolo-[4,3-alpha]-quinoxalin-1-one (ODQ, 1 microM), a guanylyl-cyclase inhibitor, or by adenosine-3,5-monophosphothioate (Rp-cAMP, 1 microM), a protein-kinase A (PKA) inhibitor. Besides the action on contractile activity, ATP (100 microM) led to a significant (P<0.001) depolarization of diaphragm muscle fibers from 74.5+/-2.3 down to 64+/-2.1, 58.2+/-2.2 and 54.3+/-2.4 mV after 10, 20 and 30 min of incubation, respectively. Incubation of MEDL with the same ATP concentration showed no significant change of MP. Denervation of the muscles for 28 days led to a decrease of CCh-induced contractions of diaphragm down to 171.1+/-34.5mg (n=11, P<0.05), but increased the contractile force of MEDL up to 723.9+/-82.3mg (n=9, P<0.01). Application of ATP elevated the contractility of denervated diaphragm caused by CCh up to normal values (311.1+/-79.7 mg, n=6, P>0.05 versus control), but did not significantly affect of contractility of MEDL, which became 848.1+/-62.7 mg (n=6). These results show that the effects of ATP on both diaphragmatic and skeletal muscles are mediated through P2Y receptors coupled to chelerytrin-sensitive protein-kinase C.

Evidences for calcium-dependent inactivation of calcium current at the frog motor nerve terminal.

Assessment of calcium-dependent inactivation of calcium current in nerve terminals is not feasible due to technical reasons. Perineural measurement of calcium-flow, however, might be utilized as indirect means to evaluate synaptic currents. Using perineural recording from frog neuromuscular junction, supra-threshold stimuli applied to motor nerve in paired-pulse manner with varying inter-pulse intervals (5-50 ms) are demonstrated in this study to cause paired-pulse depression (PPD) of Ca(2+)-current. PPD of Ca(2+)-flow was reduced at lower extracellular Ca(2+) concentrations, in BAPTA-AM and EGTA-AM treated preparations and after replacing extracellular Ca(2+) with Sr(2+). Using perineural measurement of calcium current as an indirect model to investigate synaptic ionic activity, our findings demonstrate that PPD may be attributed to calcium-dependent inactivation of Ca(2+)-current, which may serve as negative feedback in response to massive Ca(2+) entry to motor nerve terminals. A putative sensor of Ca(2+)-current is also proposed in this study.

The influence of hypothermia on P2 receptor-mediated responses of frog skeletal muscle.

The contractile responses of isolated Rana ridibunda frog sartorius muscle contractions evoked by electrical field stimulation (EFS) were studied at three temperature conditions of 17, 22 and 27 degrees C. Temperature-dependent increase of muscle contractility was found. ATP (10-100 microM) concentration dependently inhibited the electrical field stimulation-evoked contractions of sartorius muscle at all three temperatures; this effect was significantly more prominent at a temperature of 17 degrees C than at other two temperatures. Adenosine (100 microM) also caused inhibition of electrical field stimulation-evoked contractions which was statistically identical at all three temperature conditions tested. A P2 receptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 10 microM) reduced the inhibitory effect of ATP at all three temperatures but did not affect inhibitory action of adenosine. In contrast, 8-(p-sulfophenyl)theophylline (8-SPT, 100 microM), a nonselective P1 receptor antagonist, abolished inhibitory effects of adenosine at all three temperature conditions but did not antagonize inhibition caused by ATP. In electrophysiological experiments, ATP (100 microM) and adenosine (100 microM) temperature dependently reduced end-plate currents recorded in sartorius neuromuscular junction by voltage-clamp technique. The inhibitory effects of both agonists were enhanced with the decrease of temperature. 8-SPT (100 microM) abolished the inhibitory effect of adenosine but not ATP on end-plate currents. Suramin (100 microM), a nonselective P2 receptor antagonist, inhibited the action of ATP but not adenosine, while PPADS (10 microM) had no influence on the effects of either ATP or adenosine. It is concluded from this study that the effectiveness of P2 receptor-mediated inhibition of frog skeletal muscle contraction in contrast to that of adenosine is dependent on the temperature conditions.