Opposite effect of ATP on contraction force of tonic and phasic skeletal muscles in frogs.

Experiments in vitro showed that ATP and adenosine equally suppressed contractions of frog m. sartorius, which belongs to the phasic type muscles. Adenosine receptors antagonist 8-SPT abolished the effect of adenosine, but did not change the effect of ATP. This fact proves the independence of signaling pathways of these purines. ATP produced an opposite effect on the tonic muscle m. cruralis and increased the force of its contraction. Adenosine produced an inhibitory effect on the force of m. cruralis contration. In this case, 8-SPT also eliminated the effect of adenosine, but did not change the effect of ATP. The potentiating effect of ATP was blocked by suramin, a nonselective antagonist of P2 receptors, which attests to their involvement into the effects of this purine. The opposite effects of purinergic regulation reflect fundamental differences in functional organization of phasic and tonic muscular systems. It was hypothesized that the increase in contraction force under the effect of ATP is a mechanism providing maitenance of the contracted state of tonic muscle without appreciable metabolic costs.

Possible mechanisms for the effect of protein sensitization on functional properties of the isolated m. soleus and m. EDL from mice.

We studied possible involvement of ATP in the influence of protein sensitization on contractile function and non-quantum secretion in the end-plate of isolated skeletal muscles from mouse leg. The dynamic vector of muscle contraction force was shown to correlate with changes in non-quantum secretion of acetylcholine under various conditions of experimental pathology. However, the degree of these changes was lower in sensitized animals. It can be hypothesized that the ATP-induced variability in functional properties of slow muscles during protein sensitization reflects the development of resistance to external loads. The adaptive changes in fast muscles during protein sensitization are not associated with the ATP-mediated mechanisms of excitation.

Possible mechanisms for the effect of protein sensitization on contractile function of fast and slow muscles in mice.

We studied the mechanisms underlying the effect of immunobiological reorganization of the organism on contractile function of isolated skeletal muscles from mouse leg (fast muscle, m. extensor digitorum longus; and slow muscle, m. soleus). Protein sensitization was accompanied by changes in contractile properties of fast and slow skeletal muscles. These changes were differently directed in muscles with various phenotypes. The force of carbachol-induced contraction (cholinergic agonist) increased in the slow muscle, but decreased in the fast muscle. The direction of changes in the force of carbachol-induced contractions under conditions of protein sensitization in skeletal muscles correlates with changes in non-quantal secretion of acetylcholine in the endplate (H-effect). Opposite changes in functional properties of fast and slow muscles from mouse leg during protein sensitization are related to choline-mediated excitation of the muscle fiber membrane. Our results suggest that changes in contractile function of skeletal muscles during protein sensitization are associated with variations in choline-mediated excitation of the muscle fiber membrane and modification of electromechanical coupling.

The presynaptic effects of arachidonic acid and prostaglandin E2 at the frog neuromuscular junction.

Arachidonic acid and prostaglandin E2 decreased the frequency of miniature endplate potentials with producing any changes in the their amplitude-time parameters. Arachidonic acid and prostaglandin E2 decreased the quantum composition of endplate currents and the amplitude of the third phase of the nerve ending response, which reflects currents though potential-dependent K+ channels. A perineural method was used to demonstrate that arachidonic acid and prostaglandin E2 suppressed the nerve ending Ca2+ current. The cyclooxygenase blocker indomethacin increased neurotransmitter secretion and decreased the third phase of the nerve ending response. The effects of arachidonic acid and prostaglandin E2 on evoked neurotransmitter release were not seen in the presence of indomethacin, while the third phase of the response continued to show a reduction. It is suggested that prostaglandin E2 mediates the effects of arachidonic acid on spontaneous and evoked neurotransmitter secretion, Ca2+ currents, and Ca2+ -dependent K+ currents. In addition, arachidonic acid and prostaglandin E2 had their own effects on potential-dependent K+ currents in nerve endings.

The effects of carbachol on the proximal and distal parts of frog motor nerve endings.

Cholinomimetics not only activate postsynaptic cholinoreceptors in neuromuscular synapses, but also alter the process of acetylcholine secretion from nerve endings. However, the mechanism of action of cholinomimetics on the secretory process remains unidentified. We approached the question of the mechanism of the presynaptic action of cholinomimetics in the present study by investigating the effects of the n,m-cholinomimetic carbachol on nerve ending currents and postsynaptic membrane currents. Carbachol induced decreases in the postsynaptic response, without affecting the duration and amplitude of the nerve ending current in both the central and distal part of the nerve ending. However, carbachol increased the time between the arrival of the presynaptic action potential and the start of transmitter secretion. This effect on synaptic delay was more marked in the distal parts of the ending. The action of another potential modulator, extracellular potassium, was accompanied by decreases in presynaptic currents and also by increases in synaptic delay. These data provide evidence for the suppressive effect of carbachol on acetylcholine secretion acting via presynaptic metabotropic cholinoreceptors which control the level and time course of secretion of neurotransmitter quanta.

Role of protein kinase C in the effect of ATP on contractile function of the isolated strip from mouse diaphragm.

We studied the effects of adenosine and ATP on contractile function of the isolated strip from mouse diaphragm. ATP significantly increased the strength of muscle contraction induced by carbachol. Adenosine had no effect on carbachol-induced muscle contraction. P2 receptor antagonist suramin abolished the effect of ATP. The positive chronotropic effect of ATP was not observed after treatment with specific protein kinase C inhibitor chelerythrine. Our results indicate that the effect of ATP on contractile function of mouse diaphragm is realized via protein kinase C.

Effect of GABAergic and adrenergic agents on activity of Na+/K+ pump and Cl(-)-cotransport in somatic muscle cells of earthworm Lumbricus Terrestris.

GABA, baclofen, epinephrine, and norepinephrine hyperpolarized the membrane of earthworm somatic cells. This effect was prevented by furosemide, removal of Cl- from the medium, or activation of Na+/K+ pump by 3-fold increase external potassium concentration. It was hypothesized that GABA, baclofen, epinephrine, and norepinephrine stimulate Na+/K+ transport via specific receptor inputs, but their effect on resting potential can be realized only under conditions of working Cl- symport.

Mechanism of N-ethylmaleimide-induced contraction of the frog sartorius muscle.

We studied parameters of the frog sartorius muscle contraction initiated by ryanodine receptor agonists in the presence of ROS donors. We hypothesized that sodium nitroprusside and hydrogen peroxide inhibit initiation of contractions by N-ethylmaleimide and that this effect of ROS donors on parameters of N-ethylmaleimide-induced contractions is due to a direct effects of sodium nitroprusside and hydrogen peroxide on N-ethylmaleimide, but not to inactivation of ryanodine receptors in the sarcoplasmatic reticulum of frog skeletal muscle.

Mechanisms of hyperpolarizing effect of GABA on resting potential of the Lumbricus terrestris muscular wall somatic cells.

GABA, baclofen, isoguvacine increase, and cis-4-aminocrotonic acid does not modify resting membrane potential of muscle cells. Bicuculline, phaclofen, N-ethylmaleimide, chlorpromazine, verapamil, and removal of Ca2+ from bathing solution abolished the effect of baclofen, while U73122 and D609 were ineffective in this respect. The authors conclude that the Lumbricus terrestris muscle cells contain GABAergic structures similar to a- and b-receptors. Activation of GABA receptors induced Cl- inward current and Ca2+ entry with subsequent activation of calmodulin-like proteins, which causes membrane hyperpolarization by increasing the effect of "pumping potential" on resting membrane potential.

[Presynaptic effects of arachidonic acid and prostaglandin E2 in the frog neuromuscular synapse].

Arachidonic acid and prostaglandin E2 decreased the frequency of miniature endplate currents without changing their amplitude-temporary parameters. They also reduced the evoked transmitter release and the amplitude of the 3rd phase of nerve ending response corresponding to the voltage-dependent K(+)-current. Using perineural recording, It was shown that arachidonic acid and prostaglandin E2 decreased the Ca2+ currents of nerve endings. Indometacin: inhibitor of cyclooxygenase, enhanced the evoked transmitter release and decreased the 3rd phase of nerve ending response. Indometacin prevented the effects of arachidonic acid on evoked transmitter release, whereas the effects of arachidonic acid on the 3rd phase was preserved. Prostaglandin E2 seems to mediate the effects of arachidonic acid on spontaneous and evoked transmitter release, Ca(2+)- and Ca(2+)-activated K(+)-currents. Moreover, the arachidonic acid and prostaglandin E2 exerted their own effects upon voltage-dependent potassium current of motor nerve ending.

Reactive oxygen species contribute to the presynaptic action of extracellular ATP at the frog neuromuscular junction.

During normal cell metabolism the production of intracellular ATP is associated with the generation of reactive oxygen species (ROS), which appear to be important signalling molecules. Both ATP and ROS can be released extracellularly by skeletal muscle during intense activity. Using voltage clamp recording combined with imaging and biochemical assay of ROS, we tested the hypothesis that at the neuromuscular junction extracellular ATP generates ROS to inhibit transmitter release from motor nerve endings. We found that ATP produced the presynaptic inhibitory action on multiquantal end-plate currents. The inhibitory action of ATP (but not that of adenosine) was significantly reduced by several antioxidants or extracellular catalase, which breaks down H2O2. Consistent with these data, the depressant effect of ATP was dramatically potentiated by the pro-oxidant Fe2+. Exogenous H2O2 reproduced the depressant effects of ATP and showed similar sensitivity to anti- and pro-oxidants. While NO also inhibited synaptic transmission, inhibitors of the NO-producing cascade did not prevent the depressant action of ATP. The ferrous oxidation in xylenol orange assay showed the increase of ROS production by ATP and 2-MeSADP but not by adenosine. Suramin, a non-selective antagonist of P2 receptors, and pertussis toxin prevented the action of ATP on ROS production. Likewise, imaging with the ROS-sensitive dye carboxy-2',7'-dichlorodihydrofluorescein revealed increased production of ROS in the muscle treated with ATP or ADP while UTP or adenosine had no effect. Thus, generation of ROS contributed to the ATP-mediated negative feedback mechanism controlling quantal secretion of ACh from the motor nerve endings.

[Effect of hydrocortisone on purine actions in the nerve-muscle preparation]. / Vliianie gidrokortizona na moduliruiushchie éffekty purinov v nervno-myshechnom soedinenii.

Daily administration of hydrocortisone first increased and then decreased amplitude of multiquantal end-plate currents induced by motor nerve stimulation. The initial facilitating phase of the hormone action was accompanied by elimination of the ATP pre-synaptic effect. Later the inhibitory effect of the ATP becomes restored. The counteraction of the ATP effect was reproduced in the isolated muscle bathed in the saline with hydrocortisone which suggests a non-genomic action of the hormone on pre-synaptic P2 receptor. The data obtained suggest that prevention of the ATP inhibitory action might be a component of a facilitating acute stress reaction.

[Effect of carbacholine on proximal and distal regions of motor nerve terminals in the frog]. / Vliianie karbakholina na proksimal'nye i distal'nye uchastki dvigatel'nykh nervnykh okonchanii liagushki.

Carbacholine depressed postsynaptic currents in the frog m. sartorius leaving intact presynaptic currents in proximal and distal portions of the motor nerve ending. The carbacholine depressing action was followed by an increase in the time gap between the beginning of presynaptic depolarisation and subsequent quantal release. This effect was considerably more obvious in the distal portions of the nerve endings. Effect of extracellular potassium was evident in a diminishing of presynaptic currents due to membrane depolarisation. The data obtained suggest that carbacholine presynaptically depresses synaptic transmission via metabotropic cholinergic receptors controlling the time course of the transmitter release.

Effects of some transmitters on resting membrane potential of somatic cell in Lumbricus terrestris muscle wall.

Serotonin, glutamate, glycine, ATP, and muscarine had no effect on resting membrane potential of muscle cell in earthworm Lumbricus terrestris. Nicotine depolarizes and GABA hyperpolarizes the muscle membrane. Removal of K(+), Cl(-) and addition of ouabaine and strychnine to the solution abolished the effect of GABA. The authors conclude that the Lumbricus terrestris myocyte membrane contains nicotine receptors and GABAergic receptors sensitive to strychnine. Stimulation of these receptors activates sarcolemmal ionic pumps and causes membrane hyperpolarization.

ATP but not adenosine inhibits nonquantal acetylcholine release at the mouse neuromuscular junction.

The postsynaptic membrane of the neuromuscular synapse treated with antiacetylcholinesterase is depolarized due to nonquantal release of acetylcholine (ACh) from the motor nerve ending. This can be demonstrated by the hyperpolarization produced by the application of curare (H-effect). ATP (1 x 10-5 M) decreased the magnitude of the H-effect from 5 to 1.5 mV. The membrane input resistance and the ACh sensitivity were unchanged, and so changes in these cannot explain the ATP effect. Adenosine alone was without effect on the nonquantal release. On the other hand, both ATP and adenosine depressed the frequency of spontaneous miniature endplate potentials, to 56% and 43% respectively. The protein kinase A inhibitor Rp-cAMP or the guanylyl cyclase inhibitor 1H-[1,2,4]oxidiazolo[4,3-a]quinoxalin-1-one did not affect the inhibitory influence of ATP on the H-effect, whereas staurosporine, an inhibitor of protein kinase C, completely abolished the action of ATP. Suramin, an ATP antagonist, enhanced the H-effect to 8.6 mV and, like staurosporine, prevented the inhibitory effect of ATP. ATP thus suppresses the nonquantal release via a direct action on presynaptic metabotropic P2 receptors coupled to protein kinase C, whilst adenosine exerts its action mainly by affecting the mechanisms underlying quantal release. These data, together with earlier evidence, show that nonquantal release of ACh can be modulated by several distinct regulatory pathways, in particular by endogenous substances which may or may not be present in the synaptic cleft at rest or during activity.