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1.
Br J Pharmacol ; 169(8): 1810-23, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23731236

RESUMO

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.


Assuntos
Acetilcolina/metabolismo , Inosina/farmacologia , Junção Neuromuscular/efeitos dos fármacos , Receptor A3 de Adenosina/efeitos dos fármacos , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Carbazóis/farmacologia , Diafragma/inervação , Di-Hidropiridinas/farmacologia , Etilmaleimida/farmacologia , Feminino , Masculino , Camundongos , Junção Neuromuscular/metabolismo , Nervo Frênico , Pirróis/farmacologia , Receptor A3 de Adenosina/metabolismo , Receptores Purinérgicos P1
2.
Neuroscience ; 172: 164-76, 2011 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-20971164

RESUMO

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.


Assuntos
Acetilcolina/metabolismo , Agonistas do Receptor A2 de Adenosina/farmacologia , Adenosina/análogos & derivados , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/metabolismo , Fenetilaminas/farmacologia , Potássio/fisiologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Adenosina/farmacologia , Animais , Feminino , Masculino , Camundongos , Técnicas de Cultura de Órgãos , Receptor A2A de Adenosina/efeitos dos fármacos , Receptor A2A de Adenosina/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia
3.
Neuroscience ; 154(4): 1324-36, 2008 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-18534762

RESUMO

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-(6-aminohexil)-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.


Assuntos
Acetilcolina/metabolismo , Agonistas do Receptor A1 de Adenosina , Junção Neuromuscular/metabolismo , Agonistas do Receptor Purinérgico P2 , Purinas/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Eletrofisiologia , Exocitose/fisiologia , Camundongos , Transmissão Sináptica/fisiologia , Tionucleotídeos/metabolismo
4.
Neuroscience ; 142(1): 71-85, 2006 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-16843602

RESUMO

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 muM) and the slowly hydrolysable ATP analog 5'-adenylylimidodiphosphate lithium (betagamma-imido ATP) (30 muM) on miniature end-plate potential (MEPP) frequency. We found that application of ATP and betagamma-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 alpha,beta-methylene ADP sodium salt (alphabeta-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 omega-conotoxin GVIA (omega-CgTx) (L-type and N-type Ca(2+) channel antagonists, respectively) prevented the effect of betagamma-imido ATP, indicating that the nucleotide modulates Ca(2+) influx through the voltage-dependent Ca(2+) channels related to spontaneous secretion. betagamma-Imido ATP-induced modulation was antagonized by the non-specific P2 receptor antagonist suramin and the P2Y receptor antagonist 1-amino-4-[[4-[[4-chloro-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 betagamma-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. betagamma-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.


Assuntos
Acetilcolina/metabolismo , Inibição Neural/fisiologia , Junção Neuromuscular/fisiologia , Terminações Pré-Sinápticas/fisiologia , Receptores Purinérgicos P2/fisiologia , Trifosfato de Adenosina/farmacologia , Adenilil Imidodifosfato/farmacologia , Análise de Variância , Animais , Relação Dose-Resposta a Droga , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Inibição Neural/efeitos dos fármacos , Junção Neuromuscular/efeitos dos fármacos , Potássio/farmacologia , Antagonistas do Receptor Purinérgico P2 , Suramina/farmacologia , Xantinas/farmacologia
5.
Parasite Immunol ; 23(10): 533-9, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11696164

RESUMO

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.


Assuntos
Doença de Chagas/imunologia , Nervo Isquiático/fisiologia , Potenciais de Ação , Animais , Antígenos de Protozoários , Axônios/imunologia , Doença de Chagas/complicações , Doença de Chagas/parasitologia , DNA de Protozoário/análise , Modelos Animais de Doenças , Humanos , Soros Imunes , Imunoglobulina G/análise , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C3H , Bainha de Mielina/imunologia , Doenças do Sistema Nervoso Periférico/etiologia , Nervo Isquiático/parasitologia , Trypanosoma cruzi/genética
6.
Life Sci ; 66(26): 2543-56, 2000 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-10883732

RESUMO

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 mM AMIN), the membrane-permeable analog of cAMP, 8-Br-cAMP (5 mM) and, the direct adenylate cyclase activator, forskolin (20 microM), induced an increase of miniature end-plate potentials (MEPPs) frequency in rat neuromuscular junctions. We investigated the possible involvement of the voltage-dependent 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 Cd2+ (100 microM) and it was observed when AMIN was studied on hyperosmotic response, which is independent of [Ca2+]o and of Ca2+ influx through the VDCC. The lack of action of AMIN on MEPPs frequency when [Ca2+]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 cAMP-mediated presynaptic facilitation is a function of nerve terminal Ca2+ concentration. We also found that AMIN exerted a comparable increase in MEPPs frequency in control and high K+ (10 and 15 mM), suggesting a single mechanism of action for spontaneous and K+-induced secretion.


Assuntos
8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Acetilcolina/metabolismo , Junção Neuromuscular/efeitos dos fármacos , Animais , Canais de Cálcio/metabolismo , Diafragma/efeitos dos fármacos , Diafragma/metabolismo , Técnicas In Vitro , Ativação do Canal Iônico , Junção Neuromuscular/metabolismo , Concentração Osmolar , Potássio/farmacologia , Ratos , Ratos Wistar
8.
Muscle Nerve ; 20(6): 674-8, 1997 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9149073

RESUMO

Recent reports have shown that patients with Lambert-Eaton myasthenic syndrome (LEMS) improve transiently after high-dose intravenous immunoglobulin (IVIG) administration. Information about the usefulness of IVIG for long-term treatment is rather scanty. Our findings demonstrate the efficacy of monthly IVIG courses at a dose of 0.4 g/kg/day for 5 days, in a 41-year-old patient with LEMS without detectable malignancy. Improvement in limb strength, peak expiratory flow rate, and electrophysiological parameters, as well as clinical signs following IVIG, was evident as early as 7 days after the first course and is still maintained at 24-months follow-up.


Assuntos
Imunoglobulinas Intravenosas/uso terapêutico , Síndrome Miastênica de Lambert-Eaton/terapia , Adulto , Feminino , Seguimentos , Humanos , Fatores de Tempo
9.
Am J Physiol ; 273(6): C1835-41, 1997 12.
Artigo em Inglês | MEDLINE | ID: mdl-9435487

RESUMO

Spontaneous secretion of the neurotransmitter acetylcholine in mammalian neuromuscular synapsis depends on the Ca2+ content of nerve terminals. The Ca2+ electrochemical gradient favors the entry of this cation. We investigated the possible involvement of three voltage-dependent Ca2+ 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 microM nifedipine, a blocker of the L-type VDCC, and to a lesser extent by the N-type VDCC blocker, omega-conotoxin GVIA (omega-CgTx, 5 microM). On the other hand, nifedipine and omega-CgTx had no effect on K(+)-induced transmitter secretion. omega-Agatoxin IVA (100 nM), a P/Q-type VDCC blocker, prevents acetylcholine release induced by K+ depolarization but failed to affect MEPP frequency in basal conditions. These results suggest that in the mammalian neuromuscular junction Ca2+ 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(2+)-binding molecule-related spontaneous release apparently binds Ca2+ very rapidly and would probably be located very close to Ca2+ channels, since the fast Ca2+ 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 Ca2+ concentration nor L-, N-, and P/Q-type VDCC blockers, indicating that extracellular Ca2+ 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 Ca2+ concentration increases.


Assuntos
Acetilcolina/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo N , Canais de Cálcio/fisiologia , Junção Neuromuscular/fisiologia , Animais , Cálcio/fisiologia , Diafragma , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Técnicas In Vitro , Cinética , Nifedipino/farmacologia , Peptídeos/farmacologia , Ratos , Ratos Wistar , Venenos de Aranha/farmacologia , ômega-Agatoxina IVA , ômega-Conotoxina GVIA
10.
Life Sci ; 58(13): 1031-7, 1996.
Artigo em Inglês | MEDLINE | ID: mdl-8622555

RESUMO

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.


Assuntos
Aminacrina/farmacologia , Aminofilina/farmacologia , Contração Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/fisiologia , Animais , Cálcio/metabolismo , Diafragma , Relação Dose-Resposta a Droga , Estimulação Elétrica , Técnicas In Vitro , Cinética , Fibras Musculares Esqueléticas/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Ratos , Ratos Wistar , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Tetrodotoxina/farmacologia , Fatores de Tempo
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