Interaction of glutamate- and adenosine-induced decrease of acetylcholine quantal release at frog neuromuscular junction.

In a frog neuromuscular preparation of m. sartorius, glutamate had a reversible dose-dependent inhibitory effect on both spontaneous miniature endplate potentials (MEPP) and nerve stimulation-evoked endplate potentials (EPP). The effect of glutamate on MEPP and EPP is caused by the activation of metabotropic glutamate receptors, as it was eliminated by MCPG, an inhibitor of group I metabotropic glutamate receptors. The depression of evoked EPP, but not MEPP frequency was removed by inhibiting the NO production in the muscle by L-NAME and by ODQ that inhibits the soluble NO-sensitive guanylyl cyclase. The glutamate-induced depression of the frequency of spontaneous MEPP is apparently not caused by the stimulation of the NO cascade. The particular glutamate-stimulated NO cascade affecting the evoked EPP can be down-regulated also by adenosine receptors, as the glutamate and adenosine actions are not additive and application of adenosine partially prevents the further decrease of quantal content by glutamate. On the other hand, there is no obvious interaction between the glutamate-mediated inhibition of EPP and inhibitory pathways triggered by carbacholine and ATP. The effect of glutamate on the evoked EPP release might be due to NO-mediated modulation (phosphorylation) of the voltage-dependent Ca2+ channels at the presynaptic release zone that are necessary for evoked quantal release and open during EPP production.

Adenosine triphosphoric acid as a factor of nervous regulation of Na+/K+/2Cl- cotransport in rat skeletal muscle fibers.

Exogenous adenosine triphosphoric acid produces a biphasic effect on the resting membrane potential of muscle fibers in rat diaphragm. Depolarization of the sarcolemma observed 10 min after application of adenosine triphosphoric acid results from activation of Na(+)/K(+)/2Cl(-) cotransport. The increase in chloride cotransport is related to activation of postsynaptic P2Y receptors and protein kinase C. Repolarization of the membrane develops 40 min after treatment with adenosine triphosphoric acid and after 50 min the resting membrane potential almost returns the control level. This increase in the resting membrane potential of the sarcolemma is probably associated with activation of the Na(+)/K(+) pump and increase in membrane permeability for chlorine ions in response to long-term activity of Cl(-) cotransport. Thus, adenosine triphosphoric acid co-secreted with acetylcholine in the neuromuscular synapse probably plays a role in the regulation resting membrane potential and cell volume of muscle fibers.

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.

SNAP25 is a pre-synaptic target for the depressant action of reactive oxygen species on transmitter release.

Reactive oxygen species (ROS) participate in various physiological and pathological processes in the nervous system, but the specific pathways that mediate ROS signalling remain largely unknown. Using electrophysiological techniques and biochemical evaluation of isolated fusion proteins, we explored the sensitivity to standard oxidative stress of the entire synapse, the pre-synaptic machinery and essential fusion proteins underlying transmitter exocytosis. Oxidative stress induced by H(2)O(2) plus Fe(2+) inhibited both evoked and spontaneous quantal release from frog or mouse motor nerve endings, while it left post-synaptic sensitivity unchanged. The depressant effect of H(2)O(2) on acetylcholine release was pertussis toxin-insensitive, ruling out G-protein cascades. Experiments with ionomycin, a Ca(2+) ionophore, revealed that ROS directly impaired the function of releasing machinery. In line with this, SNAP25, one of three essential fusion proteins, showed a selectively high sensitivity to the oxidative signals. Several ROS scavengers enhanced evoked synaptic transmission, consistent with tonic inhibition by endogenous ROS. Our data suggest that ROS-induced impairment of releasing machinery is mediated by SNAP25, which appears to be a pre-synaptic ROS sensor. This mechanism of ROS signalling could have widespread implications in the nervous system and might contribute to the pathogenesis of neurodegenerative diseases.

Negative cross-talk between presynaptic adenosine and acetylcholine receptors.

Functional interactions between presynaptic adenosine and acetylcholine (ACh) autoreceptors were studied at the frog neuromuscular junction by recording miniature end-plate potentials (MEPPs) during bath or local application of agonists. The frequency of MEPPs was reduced by adenosine acting on presynaptic adenosine A1 receptors (EC(50) = 1.1 microm) or by carbachol acting on muscarinic M2 receptors (EC(50) = 1.8 microm). However, carbachol did not produce the depressant effect when it was applied after the action of adenosine had reached its maximum. This phenomenon implied that the negative cross-talk (occlusion) had occurred between A1 and M2 receptors. Moreover, the occlusion was receptor-specific as ATP applied in the presence of adenosine continued to depress MEPP frequency. Muscarinic antagonists [atropine or 1-[[2-[(diethylamino)methyl)-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido [2,3-b][1,4]benzodiazepine-6-one) (AFDX-116)] had no effect on the inhibitory action of adenosine and adenosine antagonists [8-(p-sulfophenyl)theophylline (8-SPT) or 1,3-dipropyl-8-cyclopentylxanthine (DPCPX)] had no effect on the action of carbachol. These data suggested that membrane-delimited interactions did not occur between A1 and M2 receptors. Both carbachol and adenosine similarly inhibited quantal release triggered by high potassium, ionomycin or sucrose. These results indicated a convergence of intracellular pathways activated by M2 and A1 receptors to a common presynaptic effector located downstream of Ca(2+) influx. We propose that the negative cross-talk between two major autoreceptors could take place during intense synaptic activity and thereby attenuate the presynaptic inhibitory effects of ACh and adenosine.

Effects of carbachol and adenosine on neurotransmitter secretion induced by potassium chloride, ionomycin, and sucrose.

We compared the effects of adenosine and cholinergic agonist carbachol on spontaneous secretion during local application of K+, ionomycin, and sucrose increasing Ca2+ concentration in the nerve terminal. Adenosine and carbachol had no effect on Ca2+ entry, but modulated later stages of exocytosis.

Mechanisms of ATP action on motor nerve terminals at the frog neuromuscular junction.

We have shown previously that ATP inhibits transmitter release at the neuromuscular junction through the action on metabotropic P2Y receptors coupled to specific second messenger cascades. In the present study we recorded K(+) or Ca(2+) currents in motor nerve endings or blocked K(+) or Ca(2+) channels in order to explore the nature of downstream presynaptic effectors. Endplate currents were presynaptically depressed by ATP. Blockers of Ca(2+)-activated K(+)-channels, such as iberiotoxin, apamin or tetraethylammonium, did not change the depressant action of ATP. By contrast, K(+) channel blocker 4-aminopyridine (4-AP) and raised extracellular Ca(2+) attenuated the effect of ATP. However, these effects of 4-AP and high Ca(2+) were reversed by Mg(2+), suggesting Ca(2+)-dependence of the ATP action. Ba(2+) promoted the depressant action of ATP as did glibenclamide, a blocker of ATP-sensitive K(+) channels, or mild depolarization produced by 7.5 mm K(+). None of the K(+) channel blockers affected the depressant action of adenosine. Focal recording revealed that neither ATP nor adenosine affected the fast K(+) currents of the motor nerve endings. However, unlike adenosine, ATP or UTP, an agonist of P2Y receptors, reversibly reduced the presynaptic Ca(2+)-current. This effect was abolished by suramin, an antagonist of P2 receptors. Depressant effect of ATP on the endplate and Ca(2+)-currents was mimicked by arachidonate, which precluded the action of ATP. ATP reduced acetylcholine release triggered by ionomycin or sucrose, suggesting inhibition of release machinery. Thus, the presynaptic depressant action of ATP is mediated by inhibition of Ca(2+) channels and by mechanism acting downstream of Ca(2+) entry.

Distinct receptors and different transduction mechanisms for ATP and adenosine at the frog motor nerve endings.

Corelease of ATP with ACh from motor endings suggests a physiological role for ATP in synaptic transmission. We previously showed that, on skeletal muscle, ATP directly inhibited ACh release via presynaptic P2 receptors. The receptor identification (P2X or P2Y) and its transduction mechanism remained, however, unknown. In the present study using the voltage-clamp technique we analyzed the properties of presynaptic ATP receptors and subsequent effector mechanisms. ATP or adenosine presynaptically depressed multiquantal end-plate currents, with longer latency for ATP action. ATPgammaS, agonist at P2X receptors, or Bz-ATP, agonist at P2X7 receptors, were ineffective. The action of ATP was prevented by suramin and unchanged by PPADS or TNP-ATP, antagonists of P2X receptors, or RB-2, a blocker of certain P2Y receptors. The depressant action of ATP was reproduced by UTP, metabotropic P2Y receptor agonist. Pertussis toxin (PTX), antagonist of Gi/o-proteins, and inhibitors of phosphatidylcholine specific PLC (D609) and PKC (staurosporine or chelerythrine) prevented the effect of ATP while blockers of PLA2 (OBAA) and COX (aspirin or indomethacin) attenuated it. Inhibitors of phosphatidylinositide-specific PLC (U73122), guanylylcyclase (ODQ), PKA (Rp-cAMPS) or PLD (1-butanol) did not affect the action of ATP. No inhibitor of second messengers (except PTX) changed the action of adenosine. Our data indicate, for motor nerve endings, the existence of inhibitory P2Y receptors coupled to multiple intracellular cascades including phosphatidylinositide-specific PLC/PKC/PLA2/COX. This divergent presynaptic P2 signalling (unlike the single effector mechanism for P1 receptors) could provide feedback inhibition of transmitter release and perhaps be involved in presynaptic plasticity.

Serotonin modulates neuromuscular transmission in frogs.

The effect of 5-hydroxytryptamine (serotonin) on neuromuscular transmission in frog skeletal muscle was studied using voltage clamp technique. Serotonin produced no effect on end-plate currents during low frequency electrical stimulation of the motor nerve, but increased the amplitude depression of multiquantal currents during high-frequency stimulation similar to motor commands fired by motoneurons. It was shown that the inhibitory effect of serotonin on neuromuscular transmission is determined by slow potential-dependent block of open ionic channels in the postsynaptic membrane accumulating during rhythmic activation of the synapse.