Adriana Losavio - Academia.edu (original) (raw)
Papers by Adriana Losavio
Neurología Argentina, 2017
Journal of Neuroscience Research
At the mouse neuromuscular junction, adenosine triphosphate (ATP), which is co‐released with the ... more At the mouse neuromuscular junction, adenosine triphosphate (ATP), which is co‐released with the neurotransmitter acetylcholine (ACh), and its metabolite adenosine, modulate neurotransmitter release by activating presynaptic inhibitory P2Y13 receptors (a subtype of ATP/adenosine diphosphate [ADP] receptor), inhibitory A1 and A3 adenosine receptors, and excitatory A2A adenosine receptors. To study the effect of endogenous purines, when phrenic‐diaphragm preparations are depolarized by different nerve stimulation patterns, we analyzed the effect of the antagonists for P2Y13, A1, A3, and A2A receptors (AR‐C69931MX, 8‐cyclopentyl‐1,3‐dipropylxanthine, MRS‐1191, and SCH‐58261, respectively) on the amplitude of the end‐plate potentials of the trains, and contrasted these results with those obtained with the selective agonists of these receptors (2‐methylthioadenosine 5′‐diphosphate trisodium salt hydrate, 2‐chloro‐N6‐cyclopentyl‐adenosine, inosine, and PSB‐0777, respectively). During cont...
The American Journal of Tropical Medicine and Hygiene, 1989
To search for the sequential compromise of the spinal cord, nerves, and skeletal muscle in mice c... more To search for the sequential compromise of the spinal cord, nerves, and skeletal muscle in mice chronically infected with Trypanosoma cruzi, animals were subjected to electromyographic investigation, end-plate recordings, and histological studies at 7, 15, 37, 60, 90, 120, 180, 270, and 360 days postinfection. Electromyographic studies showed signs of motor unit remodeling as early as 15 days postinfection, when diminished duration and amplitude of motor unit potentials pointing to a primary muscle involvement were found. Thereafter, certain features of denervation, reinnervation, and primary muscle involvement were often found to coexist. Low miniature end-plate potentials with normal frequency and acetylcholine quantum content were found in end-plate recordings made at the phrenic-diaphragm in vitro. Double end-plate potentials were observed in most of the tested muscle fibers from day 90 postinfection. All these features suggest post-synaptic damage of the end-plate and the presence of reinnervation after day 90 postinfection. Histological studies disclosed inflammatory infiltrates consisting of lymphocytes and macrophages, with vasculitis as the main lesion in the hamstring muscles; intracellular parasites were seen in 25% of the cases. Neuropathic features, as expressed by type fiber grouping and grouped muscle fiber atrophy, were found. On nerve examination epineural, perineural, and endoneural vasculitis were seen. Digestion chambers and myelin ovoids (axonal degeneration) were observed. In teased fiber preparations, segmental internodal and paranodal demyelination and remyelination were found. The lumbar inflammatory spinal cord failed to show grey or white matter infiltrates. However, spinal roots and dorsal root ganglia were densely affected by inflammatory cells.
Journal of Neuroscience Research
At the mouse neuromuscular junction, adenosine triphosphate (ATP), which is co‐released with the ... more At the mouse neuromuscular junction, adenosine triphosphate (ATP), which is co‐released with the neurotransmitter acetylcholine (ACh), and its metabolite adenosine, modulate neurotransmitter release by activating presynaptic inhibitory P2Y13 receptors (a subtype of ATP/adenosine diphosphate [ADP] receptor), inhibitory A1 and A3 adenosine receptors, and excitatory A2A adenosine receptors. To study the effect of endogenous purines, when phrenic‐diaphragm preparations are depolarized by different nerve stimulation patterns, we analyzed the effect of the antagonists for P2Y13, A1, A3, and A2A receptors (AR‐C69931MX, 8‐cyclopentyl‐1,3‐dipropylxanthine, MRS‐1191, and SCH‐58261, respectively) on the amplitude of the end‐plate potentials of the trains, and contrasted these results with those obtained with the selective agonists of these receptors (2‐methylthioadenosine 5′‐diphosphate trisodium salt hydrate, 2‐chloro‐N6‐cyclopentyl‐adenosine, inosine, and PSB‐0777, respectively). During cont...
Life Sciences, May 1, 2000
Regulation of neurotransmitter release is thought to involve modulation of the release probabilit... more Regulation of neurotransmitter release is thought to involve modulation of the release probability by protein phosphorylation. Activation of the CAMP-protein kinase A (PKA) pathway has been shown to facilitate synaptic transmission in mammalian neuromuscular synapses, although the relevant phosphorylation targets are mostly unknown. We found that the inhibitor of the phosphodiesterase aminophylline (1 m.M AMIN), the membrane-permeable analog of CAMP, 8-Br-CAMP (5 mM) and, the diect adenylate cyclase activator, forskolin (20 PM), induced an increase of miniature end-plate potentials (MEPPs) frequency in rat neuromuscular junctions. We investigated the possible involvement of the voltagedependent calcium channels (VDCC), since these proteins are known to be phosphorylated by PKA. But this possibility was ruled out, since the increase in MEPPs frequency was not attenuated by the VDCC blocker Cd" (100 pM) and it was observed when AMIN was studied on hyperosmotic response, which is independent of [Ca2'10 and of Ca2' infhrx through the VDCC. The lack of action of AMIN on MEPPs frequency when [Ca'+]i was diminished by exposing the preparations to zero Ca2'-EGTA solution (isotonic condition) or when nerve terminals were loaded with a permeant Ca2' chelator (BAPTA-AM) (hypertonic condition), indicate that cAMPmediated presynaptic facilitation is a function of nerve terminal Ca2" concentration We also found that AMIN exerted a comparable increase in MEPPs f%equency in control and high K" (10 and 15 mM), suggesting a single mechanism of action for spontaneous and K+-induced secretion.
American Journal of Physiology-cell Physiology, Dec 1, 1997
Spontaneous secretion of the neurotransmitter acetylcholine in mammalian neuromuscular synapsis d... more Spontaneous secretion of the neurotransmitter acetylcholine in mammalian neuromuscular synapsis depends on the Ca 2ϩ content of nerve terminals. The Ca 2ϩ electrochemical gradient favors the entry of this cation. We investigated the possible involvement of three voltage-dependent Ca 2ϩ channels (VDCC) (L-, N-, and P/Q-types) on spontaneous transmitter release at the rat neuromuscular junction. Miniature end-plate potential (MEPP) frequency was clearly reduced by 5 µM nifedipine, a blocker of the L-type VDCC, and to a lesser extent by the N-type VDCC blocker,-conotoxin GVIA (-CgTx, 5 µM). On the other hand, nifedipine and-CgTx had no effect on K ϩ-induced transmitter secretion.-Agatoxin IVA (100 nM), a P/Q-type VDCC blocker, prevents acetylcholine release induced by K ϩ depolarization but failed to affect MEPP frequency in basal conditions. These results suggest that in the mammalian neuromuscular junction Ca 2ϩ enters nerve terminals through at least three different channels, two of them (L-and N-types) mainly related to spontaneous acetylcholine release and the other (P/Q-type) mostly involved in depolarization-induced neurotransmitter release. Ca 2ϩ-binding molecule-related spontaneous release apparently binds Ca 2ϩ very rapidly and would probably be located very close to Ca 2ϩ channels, since the fast Ca 2ϩ chelator (BAPTA-AM) significantly reduced MEPP frequency, whereas EGTA-AM, exhibiting slower kinetics, had a lower effect. The increase in MEPP frequency induced by exposing the preparation to hypertonic solutions was affected by neither external Ca 2ϩ concentration nor L-, N-, and P/Q-type VDCC blockers, indicating that extracellular Ca 2ϩ is not necessary to produce hyperosmotic neurosecretion. On the other hand, MEPP frequency was diminished by BAPTA-AM and EGTA-AM to the same extent, supporting the view that hypertonic response is promoted by ''bulk'' intracellular Ca 2ϩ concentration increases.
Experimental Neurology, Apr 1, 1985
We studied the ionic and water content and the resting membrane potential of rat extensor digitor... more We studied the ionic and water content and the resting membrane potential of rat extensor digitorum longus EDL muscles at different times after unilateral nerve crush. Intracellular potassium concentration decreased progressively during the 1st week after nerve crush whereas intracellular sodium concentration increased significantly. At about day 10, when functional reinnervation (presence of end-plate potentials and miniature end-plate potentials) was detected, the above changes tended to return to control values. In addition, there was a significant difference between muscles with long and short nerve stumps. These results suggest a neurogenic dependency of muscle hydroelectrolytic composition. The decrease in resting membrane potential was greatest after 6.5 days of denervation when changes in the internal ionic concentration were maximum; however, these ionic changes contributed little to the decrease. The recovery of the resting membrane potential commenced at least 48 h before the first signs of functional reinnervation (10th day). This finding suggested an important contribution of some neurotrophic material in early stages of the reinnervation when nerve-muscle contacts were already established. Later, the contribution of mechanical activity to the restoration of the RMP became apparent (20th day); fibrillation potentials had disappeared by that time.
Neuroscience, 2011
The mechanism of action of the A2A adenosine receptor agonist 2-p-(2-carboxyethyl) phenethylamino... more The mechanism of action of the A2A adenosine receptor agonist 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS-21680) in the facilitation of spontaneous (isotonic and hypertonic condition) and K+-evoked acetylcholine (ACh) release was investigated in the mouse diaphragm muscles. At isotonic condition, the CGS-21680-induced excitatory effect on miniature end-plate potential (MEPP) frequency was not modified in the presence of CdCl2 and in a medium free of Ca2+ (0Ca2+-EGTA), but it was abolished after buffering the rise of intracellular Ca2+ with 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra(acetoxy-methyl) (BAPTA-AM) and when the Ca2+-ATPase inhibitor thapsigargin was used to deplete intracellular Ca2+ stores. CGS-21680 did not have a direct effect on the Ca2+-independent neurotransmitter-releasing machinery, since the modulatory effect on the hypertonic response was also occluded by BAPTA-AM and thapsigargin. CGS-21680 facilitation on K+-evoked ACh release was not altered by the P/Q-type voltage-dependent calcium channel (VDCC) blocker ω-Agatoxin IVA, but it was completely prevented by both, the L-type VDCC blocker nitrendipine (which is known to immobilize their gating charges), or thapsigargin, suggesting that the effects of CGS-21680 on L-type VDCC and thapsigargin-sensitive internal stores are associated. We found that the VDCC pore blocker Cd2+ (2 mM Ca2+ or 0Ca2+-EGTA) failed to affect the CGS-21680 effect in high K+ whereas nitrendipine in 0Ca2+-EGTA+Cd2+ occluded its action. The blockade of Ca2+ release from endoplasmic reticulum with ryanodine antagonized the facilitating effect of CGS-21680 in control and high K+ concentration. It is concluded that, at the mouse neuromuscular junction, activation of A2A receptors facilitates spontaneous and K+-evoked ACh release by an external Ca2+-independent mechanism but that involves mobilization of Ca2+ from internal stores: during spontaneous ACh release stimulating directly the ryanodine-sensitive stores and, at high K+, probably modulating the L-type VDCCs which may cause the opening of the ryanodine receptors that would be directly coupled to the channels. In both cases, Ca2+ released from the endoplasmic reticulum would be capable of activating the exocytotic machinery, thus producing facilitation of ACh release.
Annals of the New York Academy of Sciences, May 1, 1998
Clinical Neurophysiology, Dec 1, 2006
Objective: The diagnosis of bipolar disease frequently requires a long time since the age of onse... more 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.
Journal of Neuroscience Research, Feb 13, 2018
At the mouse neuromuscular junction, adenosine triphosphate (ATP) is co-released with the neurotr... more 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 P2Y and A , A , and A 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 P2Y 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 A , A , and A 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 P2Y and A and A 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,β-methyleneadenosine 5'-diphosphate sodium salt. SCH-58261 only reduced neurosecretion at 20 mM K , suggesting that more adenosine is needed to activate excitatory A 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.
Neuroscience, Jul 1, 2008
At the mouse neuromuscular junction, activation of adenosine A 1 and P2Y receptors inhibits acety... more At the mouse neuromuscular junction, activation of adenosine A 1 and P2Y receptors inhibits acetylcholine release by an effect on voltage dependent calcium channels related to spontaneous and evoked secretion. However, an effect of purines upon the neurotransmitter-releasing machinery downstream of Ca 2؉ influx cannot be ruled out. An excellent tool to study neurotransmitter exocytosis in a Ca 2؉-independent step is the hypertonic response. Intracellular recordings were performed on diaphragm fibers of CF1 mice to determine the action of the specific adenosine A 1 receptor agonist 2-chloro-N 6-cyclopentyl-adenosine (CCPA) and the P2Y 12-13 agonist 2-methylthio-adenosine 5=-diphosphate (2-MeSADP) on the hypertonic response. Both purines significantly decreased such response (peak and area under the curve), and their effect was prevented by specific antagonists of A 1 and P2Y 12-13 receptors, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and N-[2-(methylthioethyl)]-2-[3,3,3-trifluoropropyl]thio-5=-adenylic acid, monoanhydride with dichloromethylenebiphosphonic acid, tetrasodium salt (AR-C69931MX), respectively. Moreover, incubation of preparations only with the antagonists induced a higher response compared with controls, suggesting that endogenous ATP/ADP and adenosine are able to modulate the hypertonic response by activating their specific receptors. To search for the intracellular pathways involved in this effect, we studied the action of CCPA and 2-MeSADP in hypertonicity in the presence of inhibitors of several pathways. We found that the effect of CPPA was prevented by the calmodulin antagonist N-(6aminohexil)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) while that of 2-MeSADP was occluded by the protein kinase C antagonist chelerythrine and W-7. On the other hand, the inhibitors of protein kinase A (N-(2[pbromocinnamylamino]ethyl)-5-isoquinolinesulfonamide, H-89) and phosphoinositide-3 kinase (PI3K) (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride, LY-294002) did not modify the modulatory action in hypertonicity of both purines. Our results provide evidence that activation of A 1 and P2Y 12-13 receptors by CCPA and 2-MeSADP inhibits ACh release from mammalian motor nerve terminals through an effect on a Ca 2؉-independent step in the cascade of the exocytotic process. Since presynaptic calcium channels are intimately associated with components of the synaptic vesicle docking and fusion processes, further experiments could clarify if the actions of purines on calcium channels and on secretory machinery are related.
British Journal of Pharmacology, May 1, 2004
At the mouse neuromuscular junction, adenosine (AD) and the A 1 agonist 2-chloro-N 6cyclopentyl-a... more At the mouse neuromuscular junction, adenosine (AD) and the A 1 agonist 2-chloro-N 6cyclopentyl-adenosine (CCPA) induce presynaptic inhibition of spontaneous acetylcholine (ACh) release by activation of A 1 AD receptors through a mechanism that is still unknown. To evaluate whether the inhibition is mediated by modulation of the voltage-dependent calcium channels (VDCCs) associated with tonic secretion (L-and N-type VDCCs), we measured the miniature endplate potential (mepp) frequency in mouse diaphragm muscles. 2 Blockade of VDCCs by Cd 2 þ prevented the effect of the CCPA. Nitrendipine (an L-type VDCC antagonist) but not o-conotoxin GVIA (an N-type VDCC antagonist) blocked the action of CCPA, suggesting that the decrease in spontaneous mepp frequency by CCPA is associated with an action on L-type VDCCs only. 3 As A 1 receptors are coupled to a G i/o protein, we investigated whether the inhibition of PKA or the activation of PKC is involved in the presynaptic inhibition mechanism. Neither N-(2[p-bromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide (H-89, a PKA inhibitor), nor 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine (H-7, a PKC antagonist), nor phorbol 12-myristate 13-acetate (PHA, a PKC activator) modified CCPA-induced presynaptic inhibition, suggesting that these second messenger pathways are not involved. 4 The effect of CCPA was eliminated by the calmodulin antagonist N-(6-aminohexil)-5-chloro-1naphthalenesulfonamide hydrochloride (W-7) and by ethylene glycol-bis(b-aminoethyl ether)-N,N,N 0 ,N 0-tetraacetic acid-acetoxymethyl ester e6TD-BM, which suggests that the action of CCPA to modulate L-type VDCCs may involve Ca 2 þ-calmodulin. 5 To investigate the action of CCPA on diverse degrees of nerve terminal depolarization, we studied its effect at different external K þ concentrations. The effect of CCPA on ACh secretion evoked by 10 mM K þ was prevented by the P/Q-type VDCC antagonist o-agatoxin IVA. 6 CCPA failed to inhibit the increases in mepp frequency evoked by 15 and 20 mM K þ. We demonstrated that, at high K þ concentrations, endogenous AD occupies A1 receptors, impairing the action of CCPA, since incubation with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, an A 1 receptor antagonist) and adenosine deaminase (ADA), which degrades AD into the inactive metabolite inosine, increased mepp frequency compared with that obtained in 15 and 20 mM K þ in the absence of the drugs. Moreover, CCPA was able to induce presynaptic inhibition in the presence of ADA. It is concluded that, at high K þ concentrations, the activation of A 1 receptors by endogenous AD prevents excessive neurotransmitter release.
Neuroscience, Sep 1, 2006
At the neuromuscular junction, ATP is co-released with the neurotransmitter acetylcholine (ACh) a... more At the neuromuscular junction, ATP is co-released with the neurotransmitter acetylcholine (ACh) and once in the synaptic space, it is degraded to the presynaptically active metabolite adenosine. Intracellular recordings were performed on diaphragm fibers of CF1 mice to determine the action of extracellular ATP (100 M) and the slowly hydrolysable ATP analog 5=-adenylylimidodiphosphate lithium (␥imido ATP) (30 M) on miniature end-plate potential (MEPP) frequency. We found that application of ATP and ␥-imido ATP decreased spontaneous secretion by 45.3% and 55.9% respectively. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective A 1 adenosine receptor antagonist and ␣,-methylene ADP sodium salt (␣-MeADP), which is an inhibitor of ecto-5=-nucleotidase, did not prevent the inhibitory effect of ATP, demonstrating that the nucleotide is able to modulate spontaneous ACh release through a mechanism independent of the action of adenosine. Blockade of Ca 2؉ channels by both, Cd 2؉ or the combined application of nitrendipine and-conotoxin GVIA (-CgTx) (L-type and N-type Ca 2؉ channel antagonists, respectively) prevented the effect of ␥-imido ATP, indicating that the nucleotide modulates Ca 2؉ influx through the voltage-dependent Ca 2؉ channels related to spontaneous secretion. ␥-Imido ATP-induced modulation was antagonized by the non-specific P2 receptor antagonist suramin and the P2Y receptor antagonist 1-amino-4-[[4-[[4chloro-6-[[3(or4)-sulfophenyl] amino]-1,3,5-triazin-2-yl]amino]-3-sulfophenyl] amino]-9,10-dihydro-9,10-dioxo-2-anthracenesulfonic acid (reactive blue-2), but not by pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt (PPADS), which has a preferential antagonist effect on P2X receptors. Pertussis toxin and N-ethylmaleimide (NEM), which are blockers of G i/o proteins, prevented the action of the nucleotide, suggesting that the effect is mediated by P2Y receptors coupled to G i/o proteins. The protein kinase C (PKC) antagonist chelerythrine and the calmodulin antagonist N-(6-aminohexil)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) occluded the effect of ␥-imido ATP, while the protein kinase A (PKA) antagonist KT-5720 and the inhibitor of the calcium/ calmodulin-dependent protein kinase II (CAMKII) KN-62 failed to do so. ␥-Imido ATP did not affect 10, 15 and 20 mM K ؉-evoked release and application of reactive blue-2 before incubation in high K ؉ induced a higher asynchronous secretion. Thus, our results show that at mammalian neuromuscular junctions, ATP induces presynaptic inhibition of spontaneous ACh release due to the modulation of Ca 2؉ channels related to tonic secretion through the activation of P2Y receptors coupled to G i/o proteins. We also demonstrated that at increasing degrees of membrane depolarization evoked by K ؉ , endogenously released ATP induces presynaptic inhibition as a means of preventing excessive neurotransmitter secretion.
Life Sciences, Feb 1, 1996
We studied the effect of aminophylline (1mM) and 9-aminoacridine (100 microM) on the contraction ... more We studied the effect of aminophylline (1mM) and 9-aminoacridine (100 microM) on the contraction threshold (CT) of rat diaphragm fibers (25 degrees C). The CT was measured by direct visualization (200 X) of the fiber under current-clamp conditions. The main findings are the following: 1) Aminophylline lowers the CT toward more negative values of the resting membrane potential (Vm). 2) 9-aminoacridine, a drug that diminishes Ca2+ release from the sarcoplasmic reticulum (SR), shifts the CT toward more positive values: 3) this effect is overcome by aminophylline. We suggest that the displacement in the CT to more negative Vm plays an important role in the potentiating effect of aminophylline. This could be the result of an enhancement of Ca2+ release from the SR.
Parasite Immunology, Oct 1, 2001
Pathology of chronic Chagas' disease involves peripheral nervous system (PNS) compromise.... more Pathology of chronic Chagas' disease involves peripheral nervous system (PNS) compromise. A high prevalence of antibodies reacting with nervous system antigens has been found in the sera of patients and infected animals, although their physiological role in mediating PNS tissue damage is unknown. Here, we demonstrate that epineural injection of sera from Trypanosoma cruzi infected mice affects the characteristics of the sciatic nerve action potential (SNAP) depending on the parasite strain. Sera from mice infected with the reticulotropic/neurotropic RA strain with reactivity against sciatic nerve (RA/Ne+ sera) induced delays on latency and diminished amplitudes 4 days after injection. Sera from mice infected with the myotropic CA-I strain failed to affect SNAP. Purified immunoglobulin (Ig)G from RA/Ne+ also diminished the amplitude of SNAP. Deposits of IgG labelling axonal fibres and/or myelin sheaths were detected in nerves injected with RA/Ne+ sera. No major histological damage or parasite DNA was found in those nerves. The SNAP changes after sera injection were similar to those observed in mice injected with trypomastigotes in the epineurum 17 days before and in chronically infected animals. This investigation suggests that autoantibodies triggered as a consequence of T. cruzi infection are able to mediate, at least in part, the electrophysiological abnormalities observed in PNS during the course of Chagas' disease.
British Journal of Pharmacology, Jul 26, 2013
BACKGROUND AND PURPOSE The role of inosine at the mammalian neuromuscular junction (NMJ) has not ... more BACKGROUND AND PURPOSE The role of inosine at the mammalian neuromuscular junction (NMJ) has not been clearly defined. Moreover, inosine was classically considered to be the inactive metabolite of adenosine. Hence, we investigated the effect of inosine on spontaneous and evoked ACh release, the mechanism underlying its modulatory action and the receptor type and signal transduction pathway involved. EXPERIMENTAL APPROACH End-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) were recorded from the mouse phrenic-nerve diaphragm preparations using conventional intracellular electrophysiological techniques. KEY RESULTS Inosine (100 μM) reduced MEPP frequency and the amplitude and quantal content of EPPs; effects inhibited by the selective A3 receptor antagonist MRS-1191. Immunohistochemical assays confirmed the presence of A3 receptors at mammalian NMJ. The voltage-gated calcium channel (VGCC) blocker Cd 2+ , the removal of extracellular Ca 2+ and the L-type and P/Q-type VGCC antagonists, nitrendipine and ω-agatoxin IVA, respectively, all prevented inosine-induced inhibition. In the absence of endogenous adenosine, inosine decreased the hypertonic response. The effects of inosine on ACh release were prevented by the Gi/o protein inhibitor N-ethylmaleimide, PKC antagonist chelerytrine and calmodulin antagonist W-7, but not by PKA antagonists, H-89 and KT-5720, or the inhibitor of CaMKII KN-62. CONCLUSION AND IMPLICATIONS Our results suggest that, at motor nerve terminals, inosine induces presynaptic inhibition of spontaneous and evoked ACh release by activating A3 receptors through a mechanism that involves L-type and P/Q-type VGCCs and the secretory machinery downstream of calcium influx. A3 receptors appear to be coupled to Gi/o protein. PKC and calmodulin may be involved in these effects of inosine.
Neuroscience, Jun 1, 2016
It is known that adenosine 5 0-triphosphate (ATP) is released along with the neurotransmitter ace... more It is known that adenosine 5 0-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 G i/o protein. In this group, the receptor subtypes activated by adenine nucleotides are P2Y 12 and P2Y 13. 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 P2Y 12-13 receptors, 2-methylthioadenosine 5 0-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 P2Y 12-13 antagonists AR-C69931MX or 2-methylthioadenosine 5 0-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 P2Y 13 antagonist MRS-2211 completely prevented the inhibitory effect of 2-MeSADP on MEPP frequency and EPP amplitude, whereas the P2Y 12 antagonist MRS-2395 failed to do this. The preferential agonist for P2Y 13 receptors inosine 5 0-diphosphate sodium salt (IDP) reduced spontaneous and evoked ACh secretion and MRS-2211 abolished IDP-mediated modulation. Immunohistochemical studies confirmed the presence of P2Y 13 but not P2Y 12 receptors at the end-plate region. Disappearance of P2Y 13 receptors after denervation suggests the presynaptic localization of the receptors. We conclude that, at motor nerve terminals, the G i/o proteincoupled P2Y receptors implicated in presynaptic inhibition of spontaneous and evoked ACh release are of the subtype P2Y 13. This study provides new insights into the types of purinergic receptors that contribute to the fine-tuning of cholinergic transmission at mammalian neuromuscular junction.
Neurología Argentina, 2017
Journal of Neuroscience Research
At the mouse neuromuscular junction, adenosine triphosphate (ATP), which is co‐released with the ... more At the mouse neuromuscular junction, adenosine triphosphate (ATP), which is co‐released with the neurotransmitter acetylcholine (ACh), and its metabolite adenosine, modulate neurotransmitter release by activating presynaptic inhibitory P2Y13 receptors (a subtype of ATP/adenosine diphosphate [ADP] receptor), inhibitory A1 and A3 adenosine receptors, and excitatory A2A adenosine receptors. To study the effect of endogenous purines, when phrenic‐diaphragm preparations are depolarized by different nerve stimulation patterns, we analyzed the effect of the antagonists for P2Y13, A1, A3, and A2A receptors (AR‐C69931MX, 8‐cyclopentyl‐1,3‐dipropylxanthine, MRS‐1191, and SCH‐58261, respectively) on the amplitude of the end‐plate potentials of the trains, and contrasted these results with those obtained with the selective agonists of these receptors (2‐methylthioadenosine 5′‐diphosphate trisodium salt hydrate, 2‐chloro‐N6‐cyclopentyl‐adenosine, inosine, and PSB‐0777, respectively). During cont...
The American Journal of Tropical Medicine and Hygiene, 1989
To search for the sequential compromise of the spinal cord, nerves, and skeletal muscle in mice c... more To search for the sequential compromise of the spinal cord, nerves, and skeletal muscle in mice chronically infected with Trypanosoma cruzi, animals were subjected to electromyographic investigation, end-plate recordings, and histological studies at 7, 15, 37, 60, 90, 120, 180, 270, and 360 days postinfection. Electromyographic studies showed signs of motor unit remodeling as early as 15 days postinfection, when diminished duration and amplitude of motor unit potentials pointing to a primary muscle involvement were found. Thereafter, certain features of denervation, reinnervation, and primary muscle involvement were often found to coexist. Low miniature end-plate potentials with normal frequency and acetylcholine quantum content were found in end-plate recordings made at the phrenic-diaphragm in vitro. Double end-plate potentials were observed in most of the tested muscle fibers from day 90 postinfection. All these features suggest post-synaptic damage of the end-plate and the presence of reinnervation after day 90 postinfection. Histological studies disclosed inflammatory infiltrates consisting of lymphocytes and macrophages, with vasculitis as the main lesion in the hamstring muscles; intracellular parasites were seen in 25% of the cases. Neuropathic features, as expressed by type fiber grouping and grouped muscle fiber atrophy, were found. On nerve examination epineural, perineural, and endoneural vasculitis were seen. Digestion chambers and myelin ovoids (axonal degeneration) were observed. In teased fiber preparations, segmental internodal and paranodal demyelination and remyelination were found. The lumbar inflammatory spinal cord failed to show grey or white matter infiltrates. However, spinal roots and dorsal root ganglia were densely affected by inflammatory cells.
Journal of Neuroscience Research
At the mouse neuromuscular junction, adenosine triphosphate (ATP), which is co‐released with the ... more At the mouse neuromuscular junction, adenosine triphosphate (ATP), which is co‐released with the neurotransmitter acetylcholine (ACh), and its metabolite adenosine, modulate neurotransmitter release by activating presynaptic inhibitory P2Y13 receptors (a subtype of ATP/adenosine diphosphate [ADP] receptor), inhibitory A1 and A3 adenosine receptors, and excitatory A2A adenosine receptors. To study the effect of endogenous purines, when phrenic‐diaphragm preparations are depolarized by different nerve stimulation patterns, we analyzed the effect of the antagonists for P2Y13, A1, A3, and A2A receptors (AR‐C69931MX, 8‐cyclopentyl‐1,3‐dipropylxanthine, MRS‐1191, and SCH‐58261, respectively) on the amplitude of the end‐plate potentials of the trains, and contrasted these results with those obtained with the selective agonists of these receptors (2‐methylthioadenosine 5′‐diphosphate trisodium salt hydrate, 2‐chloro‐N6‐cyclopentyl‐adenosine, inosine, and PSB‐0777, respectively). During cont...
Life Sciences, May 1, 2000
Regulation of neurotransmitter release is thought to involve modulation of the release probabilit... more Regulation of neurotransmitter release is thought to involve modulation of the release probability by protein phosphorylation. Activation of the CAMP-protein kinase A (PKA) pathway has been shown to facilitate synaptic transmission in mammalian neuromuscular synapses, although the relevant phosphorylation targets are mostly unknown. We found that the inhibitor of the phosphodiesterase aminophylline (1 m.M AMIN), the membrane-permeable analog of CAMP, 8-Br-CAMP (5 mM) and, the diect adenylate cyclase activator, forskolin (20 PM), induced an increase of miniature end-plate potentials (MEPPs) frequency in rat neuromuscular junctions. We investigated the possible involvement of the voltagedependent calcium channels (VDCC), since these proteins are known to be phosphorylated by PKA. But this possibility was ruled out, since the increase in MEPPs frequency was not attenuated by the VDCC blocker Cd" (100 pM) and it was observed when AMIN was studied on hyperosmotic response, which is independent of [Ca2'10 and of Ca2' infhrx through the VDCC. The lack of action of AMIN on MEPPs frequency when [Ca'+]i was diminished by exposing the preparations to zero Ca2'-EGTA solution (isotonic condition) or when nerve terminals were loaded with a permeant Ca2' chelator (BAPTA-AM) (hypertonic condition), indicate that cAMPmediated presynaptic facilitation is a function of nerve terminal Ca2" concentration We also found that AMIN exerted a comparable increase in MEPPs f%equency in control and high K" (10 and 15 mM), suggesting a single mechanism of action for spontaneous and K+-induced secretion.
American Journal of Physiology-cell Physiology, Dec 1, 1997
Spontaneous secretion of the neurotransmitter acetylcholine in mammalian neuromuscular synapsis d... more Spontaneous secretion of the neurotransmitter acetylcholine in mammalian neuromuscular synapsis depends on the Ca 2ϩ content of nerve terminals. The Ca 2ϩ electrochemical gradient favors the entry of this cation. We investigated the possible involvement of three voltage-dependent Ca 2ϩ channels (VDCC) (L-, N-, and P/Q-types) on spontaneous transmitter release at the rat neuromuscular junction. Miniature end-plate potential (MEPP) frequency was clearly reduced by 5 µM nifedipine, a blocker of the L-type VDCC, and to a lesser extent by the N-type VDCC blocker,-conotoxin GVIA (-CgTx, 5 µM). On the other hand, nifedipine and-CgTx had no effect on K ϩ-induced transmitter secretion.-Agatoxin IVA (100 nM), a P/Q-type VDCC blocker, prevents acetylcholine release induced by K ϩ depolarization but failed to affect MEPP frequency in basal conditions. These results suggest that in the mammalian neuromuscular junction Ca 2ϩ enters nerve terminals through at least three different channels, two of them (L-and N-types) mainly related to spontaneous acetylcholine release and the other (P/Q-type) mostly involved in depolarization-induced neurotransmitter release. Ca 2ϩ-binding molecule-related spontaneous release apparently binds Ca 2ϩ very rapidly and would probably be located very close to Ca 2ϩ channels, since the fast Ca 2ϩ chelator (BAPTA-AM) significantly reduced MEPP frequency, whereas EGTA-AM, exhibiting slower kinetics, had a lower effect. The increase in MEPP frequency induced by exposing the preparation to hypertonic solutions was affected by neither external Ca 2ϩ concentration nor L-, N-, and P/Q-type VDCC blockers, indicating that extracellular Ca 2ϩ is not necessary to produce hyperosmotic neurosecretion. On the other hand, MEPP frequency was diminished by BAPTA-AM and EGTA-AM to the same extent, supporting the view that hypertonic response is promoted by ''bulk'' intracellular Ca 2ϩ concentration increases.
Experimental Neurology, Apr 1, 1985
We studied the ionic and water content and the resting membrane potential of rat extensor digitor... more We studied the ionic and water content and the resting membrane potential of rat extensor digitorum longus EDL muscles at different times after unilateral nerve crush. Intracellular potassium concentration decreased progressively during the 1st week after nerve crush whereas intracellular sodium concentration increased significantly. At about day 10, when functional reinnervation (presence of end-plate potentials and miniature end-plate potentials) was detected, the above changes tended to return to control values. In addition, there was a significant difference between muscles with long and short nerve stumps. These results suggest a neurogenic dependency of muscle hydroelectrolytic composition. The decrease in resting membrane potential was greatest after 6.5 days of denervation when changes in the internal ionic concentration were maximum; however, these ionic changes contributed little to the decrease. The recovery of the resting membrane potential commenced at least 48 h before the first signs of functional reinnervation (10th day). This finding suggested an important contribution of some neurotrophic material in early stages of the reinnervation when nerve-muscle contacts were already established. Later, the contribution of mechanical activity to the restoration of the RMP became apparent (20th day); fibrillation potentials had disappeared by that time.
Neuroscience, 2011
The mechanism of action of the A2A adenosine receptor agonist 2-p-(2-carboxyethyl) phenethylamino... more The mechanism of action of the A2A adenosine receptor agonist 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS-21680) in the facilitation of spontaneous (isotonic and hypertonic condition) and K+-evoked acetylcholine (ACh) release was investigated in the mouse diaphragm muscles. At isotonic condition, the CGS-21680-induced excitatory effect on miniature end-plate potential (MEPP) frequency was not modified in the presence of CdCl2 and in a medium free of Ca2+ (0Ca2+-EGTA), but it was abolished after buffering the rise of intracellular Ca2+ with 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra(acetoxy-methyl) (BAPTA-AM) and when the Ca2+-ATPase inhibitor thapsigargin was used to deplete intracellular Ca2+ stores. CGS-21680 did not have a direct effect on the Ca2+-independent neurotransmitter-releasing machinery, since the modulatory effect on the hypertonic response was also occluded by BAPTA-AM and thapsigargin. CGS-21680 facilitation on K+-evoked ACh release was not altered by the P/Q-type voltage-dependent calcium channel (VDCC) blocker ω-Agatoxin IVA, but it was completely prevented by both, the L-type VDCC blocker nitrendipine (which is known to immobilize their gating charges), or thapsigargin, suggesting that the effects of CGS-21680 on L-type VDCC and thapsigargin-sensitive internal stores are associated. We found that the VDCC pore blocker Cd2+ (2 mM Ca2+ or 0Ca2+-EGTA) failed to affect the CGS-21680 effect in high K+ whereas nitrendipine in 0Ca2+-EGTA+Cd2+ occluded its action. The blockade of Ca2+ release from endoplasmic reticulum with ryanodine antagonized the facilitating effect of CGS-21680 in control and high K+ concentration. It is concluded that, at the mouse neuromuscular junction, activation of A2A receptors facilitates spontaneous and K+-evoked ACh release by an external Ca2+-independent mechanism but that involves mobilization of Ca2+ from internal stores: during spontaneous ACh release stimulating directly the ryanodine-sensitive stores and, at high K+, probably modulating the L-type VDCCs which may cause the opening of the ryanodine receptors that would be directly coupled to the channels. In both cases, Ca2+ released from the endoplasmic reticulum would be capable of activating the exocytotic machinery, thus producing facilitation of ACh release.
Annals of the New York Academy of Sciences, May 1, 1998
Clinical Neurophysiology, Dec 1, 2006
Objective: The diagnosis of bipolar disease frequently requires a long time since the age of onse... more 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.
Journal of Neuroscience Research, Feb 13, 2018
At the mouse neuromuscular junction, adenosine triphosphate (ATP) is co-released with the neurotr... more 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 P2Y and A , A , and A 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 P2Y 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 A , A , and A 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 P2Y and A and A 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,β-methyleneadenosine 5'-diphosphate sodium salt. SCH-58261 only reduced neurosecretion at 20 mM K , suggesting that more adenosine is needed to activate excitatory A 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.
Neuroscience, Jul 1, 2008
At the mouse neuromuscular junction, activation of adenosine A 1 and P2Y receptors inhibits acety... more At the mouse neuromuscular junction, activation of adenosine A 1 and P2Y receptors inhibits acetylcholine release by an effect on voltage dependent calcium channels related to spontaneous and evoked secretion. However, an effect of purines upon the neurotransmitter-releasing machinery downstream of Ca 2؉ influx cannot be ruled out. An excellent tool to study neurotransmitter exocytosis in a Ca 2؉-independent step is the hypertonic response. Intracellular recordings were performed on diaphragm fibers of CF1 mice to determine the action of the specific adenosine A 1 receptor agonist 2-chloro-N 6-cyclopentyl-adenosine (CCPA) and the P2Y 12-13 agonist 2-methylthio-adenosine 5=-diphosphate (2-MeSADP) on the hypertonic response. Both purines significantly decreased such response (peak and area under the curve), and their effect was prevented by specific antagonists of A 1 and P2Y 12-13 receptors, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and N-[2-(methylthioethyl)]-2-[3,3,3-trifluoropropyl]thio-5=-adenylic acid, monoanhydride with dichloromethylenebiphosphonic acid, tetrasodium salt (AR-C69931MX), respectively. Moreover, incubation of preparations only with the antagonists induced a higher response compared with controls, suggesting that endogenous ATP/ADP and adenosine are able to modulate the hypertonic response by activating their specific receptors. To search for the intracellular pathways involved in this effect, we studied the action of CCPA and 2-MeSADP in hypertonicity in the presence of inhibitors of several pathways. We found that the effect of CPPA was prevented by the calmodulin antagonist N-(6aminohexil)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) while that of 2-MeSADP was occluded by the protein kinase C antagonist chelerythrine and W-7. On the other hand, the inhibitors of protein kinase A (N-(2[pbromocinnamylamino]ethyl)-5-isoquinolinesulfonamide, H-89) and phosphoinositide-3 kinase (PI3K) (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride, LY-294002) did not modify the modulatory action in hypertonicity of both purines. Our results provide evidence that activation of A 1 and P2Y 12-13 receptors by CCPA and 2-MeSADP inhibits ACh release from mammalian motor nerve terminals through an effect on a Ca 2؉-independent step in the cascade of the exocytotic process. Since presynaptic calcium channels are intimately associated with components of the synaptic vesicle docking and fusion processes, further experiments could clarify if the actions of purines on calcium channels and on secretory machinery are related.
British Journal of Pharmacology, May 1, 2004
At the mouse neuromuscular junction, adenosine (AD) and the A 1 agonist 2-chloro-N 6cyclopentyl-a... more At the mouse neuromuscular junction, adenosine (AD) and the A 1 agonist 2-chloro-N 6cyclopentyl-adenosine (CCPA) induce presynaptic inhibition of spontaneous acetylcholine (ACh) release by activation of A 1 AD receptors through a mechanism that is still unknown. To evaluate whether the inhibition is mediated by modulation of the voltage-dependent calcium channels (VDCCs) associated with tonic secretion (L-and N-type VDCCs), we measured the miniature endplate potential (mepp) frequency in mouse diaphragm muscles. 2 Blockade of VDCCs by Cd 2 þ prevented the effect of the CCPA. Nitrendipine (an L-type VDCC antagonist) but not o-conotoxin GVIA (an N-type VDCC antagonist) blocked the action of CCPA, suggesting that the decrease in spontaneous mepp frequency by CCPA is associated with an action on L-type VDCCs only. 3 As A 1 receptors are coupled to a G i/o protein, we investigated whether the inhibition of PKA or the activation of PKC is involved in the presynaptic inhibition mechanism. Neither N-(2[p-bromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide (H-89, a PKA inhibitor), nor 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine (H-7, a PKC antagonist), nor phorbol 12-myristate 13-acetate (PHA, a PKC activator) modified CCPA-induced presynaptic inhibition, suggesting that these second messenger pathways are not involved. 4 The effect of CCPA was eliminated by the calmodulin antagonist N-(6-aminohexil)-5-chloro-1naphthalenesulfonamide hydrochloride (W-7) and by ethylene glycol-bis(b-aminoethyl ether)-N,N,N 0 ,N 0-tetraacetic acid-acetoxymethyl ester e6TD-BM, which suggests that the action of CCPA to modulate L-type VDCCs may involve Ca 2 þ-calmodulin. 5 To investigate the action of CCPA on diverse degrees of nerve terminal depolarization, we studied its effect at different external K þ concentrations. The effect of CCPA on ACh secretion evoked by 10 mM K þ was prevented by the P/Q-type VDCC antagonist o-agatoxin IVA. 6 CCPA failed to inhibit the increases in mepp frequency evoked by 15 and 20 mM K þ. We demonstrated that, at high K þ concentrations, endogenous AD occupies A1 receptors, impairing the action of CCPA, since incubation with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, an A 1 receptor antagonist) and adenosine deaminase (ADA), which degrades AD into the inactive metabolite inosine, increased mepp frequency compared with that obtained in 15 and 20 mM K þ in the absence of the drugs. Moreover, CCPA was able to induce presynaptic inhibition in the presence of ADA. It is concluded that, at high K þ concentrations, the activation of A 1 receptors by endogenous AD prevents excessive neurotransmitter release.
Neuroscience, Sep 1, 2006
At the neuromuscular junction, ATP is co-released with the neurotransmitter acetylcholine (ACh) a... more At the neuromuscular junction, ATP is co-released with the neurotransmitter acetylcholine (ACh) and once in the synaptic space, it is degraded to the presynaptically active metabolite adenosine. Intracellular recordings were performed on diaphragm fibers of CF1 mice to determine the action of extracellular ATP (100 M) and the slowly hydrolysable ATP analog 5=-adenylylimidodiphosphate lithium (␥imido ATP) (30 M) on miniature end-plate potential (MEPP) frequency. We found that application of ATP and ␥-imido ATP decreased spontaneous secretion by 45.3% and 55.9% respectively. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective A 1 adenosine receptor antagonist and ␣,-methylene ADP sodium salt (␣-MeADP), which is an inhibitor of ecto-5=-nucleotidase, did not prevent the inhibitory effect of ATP, demonstrating that the nucleotide is able to modulate spontaneous ACh release through a mechanism independent of the action of adenosine. Blockade of Ca 2؉ channels by both, Cd 2؉ or the combined application of nitrendipine and-conotoxin GVIA (-CgTx) (L-type and N-type Ca 2؉ channel antagonists, respectively) prevented the effect of ␥-imido ATP, indicating that the nucleotide modulates Ca 2؉ influx through the voltage-dependent Ca 2؉ channels related to spontaneous secretion. ␥-Imido ATP-induced modulation was antagonized by the non-specific P2 receptor antagonist suramin and the P2Y receptor antagonist 1-amino-4-[[4-[[4chloro-6-[[3(or4)-sulfophenyl] amino]-1,3,5-triazin-2-yl]amino]-3-sulfophenyl] amino]-9,10-dihydro-9,10-dioxo-2-anthracenesulfonic acid (reactive blue-2), but not by pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt (PPADS), which has a preferential antagonist effect on P2X receptors. Pertussis toxin and N-ethylmaleimide (NEM), which are blockers of G i/o proteins, prevented the action of the nucleotide, suggesting that the effect is mediated by P2Y receptors coupled to G i/o proteins. The protein kinase C (PKC) antagonist chelerythrine and the calmodulin antagonist N-(6-aminohexil)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) occluded the effect of ␥-imido ATP, while the protein kinase A (PKA) antagonist KT-5720 and the inhibitor of the calcium/ calmodulin-dependent protein kinase II (CAMKII) KN-62 failed to do so. ␥-Imido ATP did not affect 10, 15 and 20 mM K ؉-evoked release and application of reactive blue-2 before incubation in high K ؉ induced a higher asynchronous secretion. Thus, our results show that at mammalian neuromuscular junctions, ATP induces presynaptic inhibition of spontaneous ACh release due to the modulation of Ca 2؉ channels related to tonic secretion through the activation of P2Y receptors coupled to G i/o proteins. We also demonstrated that at increasing degrees of membrane depolarization evoked by K ؉ , endogenously released ATP induces presynaptic inhibition as a means of preventing excessive neurotransmitter secretion.
Life Sciences, Feb 1, 1996
We studied the effect of aminophylline (1mM) and 9-aminoacridine (100 microM) on the contraction ... more We studied the effect of aminophylline (1mM) and 9-aminoacridine (100 microM) on the contraction threshold (CT) of rat diaphragm fibers (25 degrees C). The CT was measured by direct visualization (200 X) of the fiber under current-clamp conditions. The main findings are the following: 1) Aminophylline lowers the CT toward more negative values of the resting membrane potential (Vm). 2) 9-aminoacridine, a drug that diminishes Ca2+ release from the sarcoplasmic reticulum (SR), shifts the CT toward more positive values: 3) this effect is overcome by aminophylline. We suggest that the displacement in the CT to more negative Vm plays an important role in the potentiating effect of aminophylline. This could be the result of an enhancement of Ca2+ release from the SR.
Parasite Immunology, Oct 1, 2001
Pathology of chronic Chagas' disease involves peripheral nervous system (PNS) compromise.... more Pathology of chronic Chagas' disease involves peripheral nervous system (PNS) compromise. A high prevalence of antibodies reacting with nervous system antigens has been found in the sera of patients and infected animals, although their physiological role in mediating PNS tissue damage is unknown. Here, we demonstrate that epineural injection of sera from Trypanosoma cruzi infected mice affects the characteristics of the sciatic nerve action potential (SNAP) depending on the parasite strain. Sera from mice infected with the reticulotropic/neurotropic RA strain with reactivity against sciatic nerve (RA/Ne+ sera) induced delays on latency and diminished amplitudes 4 days after injection. Sera from mice infected with the myotropic CA-I strain failed to affect SNAP. Purified immunoglobulin (Ig)G from RA/Ne+ also diminished the amplitude of SNAP. Deposits of IgG labelling axonal fibres and/or myelin sheaths were detected in nerves injected with RA/Ne+ sera. No major histological damage or parasite DNA was found in those nerves. The SNAP changes after sera injection were similar to those observed in mice injected with trypomastigotes in the epineurum 17 days before and in chronically infected animals. This investigation suggests that autoantibodies triggered as a consequence of T. cruzi infection are able to mediate, at least in part, the electrophysiological abnormalities observed in PNS during the course of Chagas' disease.
British Journal of Pharmacology, Jul 26, 2013
BACKGROUND AND PURPOSE The role of inosine at the mammalian neuromuscular junction (NMJ) has not ... more BACKGROUND AND PURPOSE The role of inosine at the mammalian neuromuscular junction (NMJ) has not been clearly defined. Moreover, inosine was classically considered to be the inactive metabolite of adenosine. Hence, we investigated the effect of inosine on spontaneous and evoked ACh release, the mechanism underlying its modulatory action and the receptor type and signal transduction pathway involved. EXPERIMENTAL APPROACH End-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) were recorded from the mouse phrenic-nerve diaphragm preparations using conventional intracellular electrophysiological techniques. KEY RESULTS Inosine (100 μM) reduced MEPP frequency and the amplitude and quantal content of EPPs; effects inhibited by the selective A3 receptor antagonist MRS-1191. Immunohistochemical assays confirmed the presence of A3 receptors at mammalian NMJ. The voltage-gated calcium channel (VGCC) blocker Cd 2+ , the removal of extracellular Ca 2+ and the L-type and P/Q-type VGCC antagonists, nitrendipine and ω-agatoxin IVA, respectively, all prevented inosine-induced inhibition. In the absence of endogenous adenosine, inosine decreased the hypertonic response. The effects of inosine on ACh release were prevented by the Gi/o protein inhibitor N-ethylmaleimide, PKC antagonist chelerytrine and calmodulin antagonist W-7, but not by PKA antagonists, H-89 and KT-5720, or the inhibitor of CaMKII KN-62. CONCLUSION AND IMPLICATIONS Our results suggest that, at motor nerve terminals, inosine induces presynaptic inhibition of spontaneous and evoked ACh release by activating A3 receptors through a mechanism that involves L-type and P/Q-type VGCCs and the secretory machinery downstream of calcium influx. A3 receptors appear to be coupled to Gi/o protein. PKC and calmodulin may be involved in these effects of inosine.
Neuroscience, Jun 1, 2016
It is known that adenosine 5 0-triphosphate (ATP) is released along with the neurotransmitter ace... more It is known that adenosine 5 0-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 G i/o protein. In this group, the receptor subtypes activated by adenine nucleotides are P2Y 12 and P2Y 13. 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 P2Y 12-13 receptors, 2-methylthioadenosine 5 0-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 P2Y 12-13 antagonists AR-C69931MX or 2-methylthioadenosine 5 0-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 P2Y 13 antagonist MRS-2211 completely prevented the inhibitory effect of 2-MeSADP on MEPP frequency and EPP amplitude, whereas the P2Y 12 antagonist MRS-2395 failed to do this. The preferential agonist for P2Y 13 receptors inosine 5 0-diphosphate sodium salt (IDP) reduced spontaneous and evoked ACh secretion and MRS-2211 abolished IDP-mediated modulation. Immunohistochemical studies confirmed the presence of P2Y 13 but not P2Y 12 receptors at the end-plate region. Disappearance of P2Y 13 receptors after denervation suggests the presynaptic localization of the receptors. We conclude that, at motor nerve terminals, the G i/o proteincoupled P2Y receptors implicated in presynaptic inhibition of spontaneous and evoked ACh release are of the subtype P2Y 13. This study provides new insights into the types of purinergic receptors that contribute to the fine-tuning of cholinergic transmission at mammalian neuromuscular junction.