Inosine induces presynaptic inhibition of acetylcholine release by activation of A3 adenosine receptors at the mouse neuromuscular junction.

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.

Excitatory effect of the A2A adenosine receptor agonist CGS-21680 on spontaneous and K+-evoked acetylcholine release at the mouse neuromuscular junction.

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.

Long-term follow-up of Lambert-Eaton syndrome treated with intravenous immunoglobulin.

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.

The effect of aminophylline on the contraction threshold of rat diaphragm fibers and its modification by 9-aminoacridine.

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.

Barium resistant potassium current in mammalian skeletal muscle following denervation.

Inward rectifier potassium channels are thought to be related to resting membrane potential and in innervated skeletal muscle they are specially sensitive to the blocking action of Ba2+ ions. After denervation other channels are known to become resistant to their blockers. We study the effect of Ba2+ upon the inward rectifier potassium channels after denervation. Rat extensor digitorum longus fibers were equilibrated for 150 minutes in 150 mM KCl; when they were returned to 5 mM KCl the resting potential went back to its original level with a half time of 35 minutes. This repolarization was blocked by 5 mM BaCl2 in innervated muscles and in muscles denervated for 7 days, but failed to do so after 14 days of denervation. Voltage-clamp experiments performed in lumbricalis denervated muscle showed a lack of effect of Ba2+ upon potassium current after 18 days of denervation. This results suggest that the inward rectifier potassium channels become resistant to Ba2+ ions after denervation, indicating a neural influence.

Functional changes of the sciatic nerve in mice chronically infected with Trypanosoma cruzi

Estudios histológicos previos, realizados en el nervio ciático de ratone chagásicos crónicos, demostraron degeneración axonal y desmielinización. Con el fin de investigar los cambios funcionales en el nervio ciático y en músculos inervados por él, se estudiarón 14 ratones infectados, 12 meses antes de los experimentos, con tripomastigotes (clon K-98 de CA-I). Se exploró la actividad electromiográfica en los músculos isquiotibiales y la latencia y amplitud del potencial de nervio "in vivo". Como grupo control se emplearon 13 ratos de igual edad y peso. El electromiograma mostró que una parte de las unidades motoras funcionantes habían aumentado su amplitud, duración y número de fases de sus potenciales, señalando aumento del tamaño de sus territorios, hecho que sugiere reinervación muscular a partir del envío de colaterales axónicas hacia fibras musculares previamente denervadas. El potencial de acción del nervio ciático evidenció, en los animales infectados, disminución de su amplitud y prolongación de su latencia. Esta observación señala reducción del número de axones funcionantes en el nervio y desmielinización de las fibras remantes. Los hallazgos electrofisiológicos hechos en el nervio coinciden con las descripciones histológicas anteriores y proveen evidencia adicional del estado funcional de las fibras que lo integran en el modelo experimental de esta parasitosis (AU)

Functional changes of the sciatic nerve in mice chronically infected with Trypanosoma cruzi

Estudios histológicos previos, realizados en el nervio ciático de ratone chagásicos crónicos, demostraron degeneración axonal y desmielinización. Con el fin de investigar los cambios funcionales en el nervio ciático y en músculos inervados por él, se estudiarón 14 ratones infectados, 12 meses antes de los experimentos, con tripomastigotes (clon K-98 de CA-I). Se exploró la actividad electromiográfica en los músculos isquiotibiales y la latencia y amplitud del potencial de nervio "in vivo". Como grupo control se emplearon 13 ratos de igual edad y peso. El electromiograma mostró que una parte de las unidades motoras funcionantes habían aumentado su amplitud, duración y número de fases de sus potenciales, señalando aumento del tamaño de sus territorios, hecho que sugiere reinervación muscular a partir del envío de colaterales axónicas hacia fibras musculares previamente denervadas. El potencial de acción del nervio ciático evidenció, en los animales infectados, disminución de su amplitud y prolongación de su latencia. Esta observación señala reducción del número de axones funcionantes en el nervio y desmielinización de las fibras remantes. Los hallazgos electrofisiológicos hechos en el nervio coinciden con las descripciones histológicas anteriores y proveen evidencia adicional del estado funcional de las fibras que lo integran en el modelo experimental de esta parasitosis

Functional changes of the sciatic nerve in mice chronically infected with Trypanosoma cruzi.

Early histological studies carried out in the sciatic nerve of mice chronically infected with Trypanosoma cruzi showed demyelination and scanty axonal degeneration. The experiments reported in this paper were designed to assess the functional state of the sciatic nerve and of some of the muscles it supplies. For these purposes 14 mice were infected with trypomastigotes (clon K-98, CA-I strain) 12 months before the investigation. Results were compared with 13 normal mice matched by age and weight. Hamstring muscles were studied electromyographically by means of a fine coaxial needle electrode and the sciatic nerve action potential characteristics were recorded with surface electrodes. All the experiments were carried out in vivo. In the infected mice the electromyogram showed that some motor unit potentials has enlarged amplitude and duration and increased number of phases, suggesting that the size of their territories had been enlarged, probably through axonal collateral sproutings and reinnervation of muscle fibers previously relinquished by their original innervation. The sciatic nerve action potential of the infected animals showed diminished amplitude and prolonged latency. These features signal reduced number of functional axons within the nerve and demyelination of the remaining conducting fibers. These findings are in line with the histological evidences of the involvement of the peripheral nervous system in Chagas disease and give additional information about the functional state of the peripheral nerves in the experimental model.

Functional changes of the sciatic nerve in mice chronically infected with Trypanosoma cruzi.

Early histological studies carried out in the sciatic nerve of mice chronically infected with Trypanosoma cruzi showed demyelination and scanty axonal degeneration. The experiments reported in this paper were designed to assess the functional state of the sciatic nerve and of some of the muscles it supplies. For these purposes 14 mice were infected with trypomastigotes (clon K-98, CA-I strain) 12 months before the investigation. Results were compared with 13 normal mice matched by age and weight. Hamstring muscles were studied electromyographically by means of a fine coaxial needle electrode and the sciatic nerve action potential characteristics were recorded with surface electrodes. All the experiments were carried out in vivo. In the infected mice the electromyogram showed that some motor unit potentials has enlarged amplitude and duration and increased number of phases, suggesting that the size of their territories had been enlarged, probably through axonal collateral sproutings and reinnervation of muscle fibers previously relinquished by their original innervation. The sciatic nerve action potential of the infected animals showed diminished amplitude and prolonged latency. These features signal reduced number of functional axons within the nerve and demyelination of the remaining conducting fibers. These findings are in line with the histological evidences of the involvement of the peripheral nervous system in Chagas disease and give additional information about the functional state of the peripheral nerves in the experimental model.

Blockers of potassium current and resting membrane potential in rat muscle fibers.

Rat diaphragm fibers were equilibrated for several hours in 150 mM KCl; when they were returned to 5 mM KCl the resting potential went back to its original level with a half time of 17 min. This repolarization was blocked by 5 mM BaCl2, a blocker of the inward rectifier K channel. On the other hand, 0.1 mM apamin and 0.02 mM glibenclamide which block the Ca-dependent and ATP sensitive K channels, respectively, and 0.1 mM 9-AC a blocker of the Cl- channel did not affect the repolarization. 5 mM barium decreased the K conductance measured under current-clamp conditions in diaphragm muscle fibers. The possible role of the inward rectifier system in the repolarization following return to normal [K]o is discussed.

Effect of aminophylline on the contraction threshold of rat diaphragm fibers.

We studied the effect of aminophylline (0.1-1 mM) on the contraction threshold (CT) of rat diaphragm fibers (25 degrees C). The CT was measured by direct visualization (x200) of the fiber under current-clamp conditions. The main findings are the following: 1) Aminophylline lowers the CT, in a dose-dependent manner, toward more negative values of the resting membrane potential (Vm). 2) Dibutyryl adenosine 3',5'-cyclic monophosphate (2 mM) shifts the CT, although this change is smaller than in the presence of xanthine. 3) Tetracaine (1 mM), a drug that diminishes Ca release from the sarcoplasmic reticulum, reduces the shift induced by 1 mM aminophylline; this is partially overcome by increasing aminophylline concentration to 5 mM. 4) Hyperpolarization of the fibers shifts the CT to more negative Vm. 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 Ca release from the sarcoplasmic reticulum.

Tools to differentiate immunologic and non-immunologic myasthenia gravis in infancy.

Twelve patients between 15 months and 13 years of age with clinical and pharmacological features of myasthenia gravis were studied. Repetitive nerve stimulation did not offer valuable information; the patients demonstrated either inspecific muscular decremental response or had normal behavior. Two clear groups of patients were identified after measurements of acetylcholine receptor (AChR) antibodies and MEPP amplitude recorded in the diaphragm of mice injected with sera from those patients. The first group included patients with positive AChR antibodies titers and decreased MEPP amplitude. The second one had negative AChR antibodies titers and MEPPs with normal amplitude. These data strongly suggest immunologic and non-immunologic mechanisms for the former and later respectively.

Influence of trophic substances in the regulation of resting membrane potential and ionic concentration in skeletal muscle.

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.