Endogenous purines modulate K+ -evoked ACh secretion at the mouse neuromuscular junction.

At the mouse neuromuscular junction, adenosine triphosphate (ATP) is co-released with the neurotransmitter acetylcholine (ACh), and once in the synaptic cleft, it is hydrolyzed to adenosine. Both ATP/adenosine diphosphate (ADP) and adenosine modulate ACh secretion by activating presynaptic P2Y13 and A1 , A2A , and A3 receptors, respectively. To elucidate the action of endogenous purines on K+ -dependent ACh release, we studied the effect of purinergic receptor antagonists on miniature end-plate potential (MEPP) frequency in phrenic diaphragm preparations. At 10 mM K+ , the P2Y13 antagonist N-[2-(methylthio)ethyl]-2-[3,3,3-trifluoropropyl]thio-5'-adenylic acid, monoanhydride with (dichloromethylene)bis[phosphonic acid], tetrasodium salt (AR-C69931MX) increased asynchronous ACh secretion while the A1 , A3 , and A2A antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), (3-Ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1, 4-(±)-dihydropyridine-3,5-, dicarboxylate (MRS-1191), and 2-(2-Furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (SCH-58261) did not modify neurosecretion. The inhibition of equilibrative adenosine transporters by S-(p-nitrobenzyl)-6-thioinosine provoked a reduction of 10 mM K+ -evoked ACh release, suggesting that the adenosine generated from ATP is being removed from the synaptic space by the transporters. At 15 and 20 mM K+ , endogenous ATP/ADP and adenosine bind to inhibitory P2Y13 and A1 and A3 receptors since AR-C69931MX, DPCPX, and MRS-1191 increased MEPP frequency. Similar results were obtained when the generation of adenosine was prevented by using the ecto-5'-nucleotidase inhibitor α,ß-methyleneadenosine 5'-diphosphate sodium salt. SCH-58261 only reduced neurosecretion at 20 mM K+ , suggesting that more adenosine is needed to activate excitatory A2A receptors. At high K+ concentration, the equilibrative transporters appear to be saturated allowing the accumulation of adenosine in the synaptic cleft. In conclusion, when motor nerve terminals are depolarized by increasing K+ concentrations, the ATP/ADP and adenosine endogenously generated are able to modulate ACh secretion by sequential activation of different purinergic receptors.

P2Y13 receptors mediate presynaptic inhibition of acetylcholine release induced by adenine nucleotides at the mouse neuromuscular junction.

It is known that adenosine 5'-triphosphate (ATP) is released along with the neurotransmitter acetylcholine (ACh) from motor nerve terminals. At mammalian neuromuscular junctions (NMJs), we have previously demonstrated that ATP is able to decrease ACh secretion by activation of P2Y receptors coupled to pertussis toxin-sensitive Gi/o protein. In this group, the receptor subtypes activated by adenine nucleotides are P2Y12 and P2Y13. Here, we investigated, by means of pharmacological and immunohistochemical assays, the P2Y receptor subtype that mediates the modulation of spontaneous and evoked ACh release in mouse phrenic nerve-diaphragm preparations. First, we confirmed that the preferential agonist for P2Y12-13 receptors, 2-methylthioadenosine 5'-diphosphate trisodium salt hydrate (2-MeSADP), reduced MEPP frequency without affecting MEPP amplitude as well as the amplitude and quantal content of end-plate potentials (EPPs). The effect on spontaneous secretion disappeared after the application of the selective P2Y12-13 antagonists AR-C69931MX or 2-methylthioadenosine 5'-monophosphate triethylammonium salt hydrate (2-MeSAMP). 2-MeSADP was more potent than ADP and ATP in reducing MEPP frequency. Then we demonstrated that the selective P2Y13 antagonist MRS-2211 completely prevented the inhibitory effect of 2-MeSADP on MEPP frequency and EPP amplitude, whereas the P2Y12 antagonist MRS-2395 failed to do this. The preferential agonist for P2Y13 receptors inosine 5'-diphosphate sodium salt (IDP) reduced spontaneous and evoked ACh secretion and MRS-2211 abolished IDP-mediated modulation. Immunohistochemical studies confirmed the presence of P2Y13 but not P2Y12 receptors at the end-plate region. Disappearance of P2Y13 receptors after denervation suggests the presynaptic localization of the receptors. We conclude that, at motor nerve terminals, the Gi/o protein-coupled P2Y receptors implicated in presynaptic inhibition of spontaneous and evoked ACh release are of the subtype P2Y13. This study provides new insights into the types of purinergic receptors that contribute to the fine-tuning of cholinergic transmission at mammalian neuromuscular junction.

Differential effect of serum from bipolar versus schizophrenic patients on spontaneous acetylcholine release at mammalian neuromuscular junction.

OBJECTIVE: The diagnosis of bipolar disease frequently requires a long time since the age of onset, especially because the disease is misdiagnosed with schizophrenia. The aim of the present work was to investigate whether sera from bipolar patients have an active substance that allows making a fast identification of the disease. METHODS: Sera from healthy volunteers, euthymic and non-stabilized bipolar patients, and schizophrenic patients were passively transferred into CF1 mice and after 2 day injections, MEPP frequency from diaphragm muscles was recorded. The same procedure was performed with sera fraction of high and low MW (cut-off 3000). RESULTS: Sera from non-stabilized bipolar patients induced a decreased MEPP frequency and occluded the presynaptic inhibitory effect of the specific adenosine A(1) receptor agonist 2-chloro-N(6)-cyclopentyl-adenosine (CCPA) in the recipient mice, while in the euthymic bipolar group spontaneous secretion reached control values although the action of CCPA was still prevented. Similar results were obtained with low MW sera fraction from euthymic and non-stabilized bipolar patients. The addition of adenosine deaminase to the sera fraction prevented the modification of spontaneous ACh release. In mice injected with sera from schizophrenic patients, MEPP frequency was within control values and CCPA induced its typical inhibitory action. CONCLUSIONS: These results indicate that bipolar patients contain in their blood an active substance compatible with adenosine, which was able to modify spontaneous ACh release in the recipient mice. This effect was not observed with sera from healthy volunteers and schizophrenic patients. The increase of adenosine concentration may result from synaptic hyperactivity that presumably plays a role in the symptoms of bipolar disorder and/or may derive from peripheral cells through a more general mechanism. SIGNIFICANCE: The different results obtained with bipolar and schizophrenic sera raise the possibility that the passive transfer model could be used as a diagnostic test in the future.