Amplification of neuromuscular transmission by methylprednisolone involves activation of presynaptic facilitatory adenosine A2A receptors and redistribution of synaptic vesicles.

The mechanisms underlying improvement of neuromuscular transmission deficits by glucocorticoids are still a matter of debate despite these compounds have been used for decades in the treatment of autoimmune myasthenic syndromes. Besides their immunosuppressive action, corticosteroids may directly facilitate transmitter release during high-frequency motor nerve activity. This effect coincides with the predominant adenosine A2A receptor tonus, which coordinates the interplay with other receptors (e.g. muscarinic) on motor nerve endings to sustain acetylcholine (ACh) release that is required to overcome tetanic neuromuscular depression in myasthenics. Using myographic recordings, measurements of evoked [(3)H]ACh release and real-time video microscopy with the FM4-64 fluorescent dye, results show that tonic activation of facilitatory A2A receptors by endogenous adenosine accumulated during 50 Hz bursts delivered to the rat phrenic nerve is essential for methylprednisolone (0.3 mM)-induced transmitter release facilitation, because its effect was prevented by the A2A receptor antagonist, ZM 241385 (10 nM). Concurrent activation of the positive feedback loop operated by pirenzepine-sensitive muscarinic M1 autoreceptors may also play a role, whereas the corticosteroid action is restrained by the activation of co-expressed inhibitory M2 and A1 receptors blocked by methoctramine (0.1 µM) and DPCPX (2.5 nM), respectively. Inhibition of FM4-64 loading (endocytosis) by methylprednisolone following a brief tetanic stimulus (50 Hz for 5 s) suggests that it may negatively modulate synaptic vesicle turnover, thus increasing the release probability of newly recycled vesicles. Interestingly, bulk endocytosis was rehabilitated when methylprednisolone was co-applied with ZM241385. Data suggest that amplification of neuromuscular transmission by methylprednisolone may involve activation of presynaptic facilitatory adenosine A2A receptors by endogenous adenosine leading to synaptic vesicle redistribution.

Antagonism by hemoglobin of effects induced by L-arginine in neuromuscular preparations from rats.

Nitric oxide (NO)-synthase is present in diaphragm, phrenic nerve and vascular smooth muscle. It has been shown that the NO precursor L-arginine (L-Arg) at the presynaptic level increases the amplitude of muscular contraction (AMC) and induces tetanic fade when the muscle is indirectly stimulated at low and high frequencies, respectively. However, the precursor in muscle reduces AMC and maximal tetanic fade when the preparations are stimulated directly. In the present study the importance of NO synthesized in different tissues for the L-Arg-induced neuromuscular effects was investigated. Hemoglobin (50 nM) did not produce any neuromuscular effect, but antagonized the increase in AMC and tetanic fade induced by L-Arg (9.4 mM) in rat phrenic nerve-diaphragm preparations. D-Arg (9.4 mM) did not produce any effect when preparations were stimulated indirectly at low or high frequency. Hemoglobin did not inhibit the decrease of AMC or the reduction in maximal tetanic tension induced by L-Arg in preparations previously paralyzed with d-tubocurarine and directly stimulated. Since only the presynaptic effects induced by L-Arg were antagonized by hemoglobin, the present results suggest that NO synthesized in muscle acts on nerve and skeletal muscle. Nevertheless, NO produced in nerve and vascular smooth muscle does not seem to act on skeletal muscle.

Antagonism by hemoglobin of effects induced by L-arginine in neuromuscular preparations from rats

Nitric oxide (NO)-synthase is present in diaphragm, phrenic nerve and vascular smooth muscle. It has been shown that the NO precursor L-arginine (L-Arg) at the presynaptic level increases the amplitude of muscular contraction (AMC) and induces tetanic fade when the muscle is indirectly stimulated at low and high frequencies, respectively. However, the precursor in muscle reduces AMC and maximal tetanic fade when the preparations are stimulated directly. In the present study the importance of NO synthesized in different tissues for the L-Arg-induced neuromuscular effects was investigated. Hemoglobin (50 nM) did not produce any neuromuscular effect, but antagonized the increase in AMC and tetanic fade induced by L-Arg (9.4 mM) in rat phrenic nerve-diaphragm preparations. D-Arg (9.4 mM) did not produce any effect when preparations were stimulated indirectly at low or high frequency. Hemoglobin did not inhibit the decrease of AMC or the reduction in maximal tetanic tension induced by L-Arg in preparations previously paralyzed with d-tubocurarine and directly stimulated. Since only the presynaptic effects induced by L-Arg were antagonized by hemoglobin, the present results suggest that NO synthesized in muscle acts on nerve and skeletal muscle. Nevertheless, NO produced in nerve and vascular smooth muscle does not seem to act on skeletal muscle

The neuromuscular transmission fade (Wedensky inhibition) induced by L-arginine in neuromuscular preparations from rats.

L-Arginine (4.7-18.8 mM) and 3-(4-morpholinyl)-sydonone imine hydrochloride (SIN-1; 1.15 mM) induced an increase in tetanic fade caused by indirect stimulation (180-200 Hz) of muscle. However, Wedensky inhibition, different from control, was not observed when the preparations treated with d-tubocurarine were directly stimulated by the same frequency. D-Arginine (9.4 mM) was ineffective in changing R values caused by indirect stimulation (180-200 Hz) of muscle. N(omega)-Nitro-L-arginine (73 mM) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM) did not produce any effect on Wedensky inhibition but did antagonize the tetanic fade induced by L-arginine (9.4 mM). The SIN-1 effect was antagonized by previous administration of ODQ (108 microM), which alone did not produce any effects on R values. These results indicate that NO acting at the presynaptic level increases the Wedensky inhibition induced by high frequency of stimulation applied on motor nerves, and its effect may be produced through the cGMP-GC pathway.

Reversal by methylene blue of tetanic fade induced in cats by nitric oxide.

Previous data from our laboratory have indicated that nitric oxide (NO) acting at the presynaptic level increases the amplitude of muscular contraction (AMC) of the phrenic-diaphragm preparations isolated from indirectly stimulated rats, but, by acting at the postsynaptic level, it reduces the AMC when the preparations are directly stimulated. In the present study we investigated the effects induced by NO when tetanic frequencies of stimulation were applied to in vivo preparations (sciatic nerve-anterior tibial muscle of the cat). Intra-arterial injection of NO (0.75-1.5 mg/kg) induced a dose-dependent increase in the Wedensky inhibition produced by high frequencies of stimulation applied to the motor nerve. Intra-arterial administration of 7.2 micrograms/kg methylene blue did not produce any change in AMC at low frequencies of nerve stimulation (0.2 Hz), but antagonized the NO-induced Wedensky inhibition. The experimental data suggest that NO-induced Wedensky inhibition may be mediated by the guanylate cyclase-cGMP pathway.

Neuromuscular facilitation and blockade induced by L-arginine and nitric oxide in the rat isolated diaphragm.

1. L-Arginine (4.7-9.5 mM) induced an increase in the amplitude of muscular contraction (AMC) evoked by nerve stimulation of rat diaphragm preparations, but produced a reduction of the AMC evoked by direct stimulation of muscles previously treated with d-tubocurarine. The facilitatory dose of L-arginine was ineffective in changing the twitch tension evoked by retrograde injection of acetylcholine. 2. N omega-nitro-L-arginine (18 mM) antagonized the increase in AMC induced by L-arginine in preparations indirectly stimulated, and a similar effect was obtained against the depression induced by L-arginine in directly stimulated muscle preparations. D-Arginine (4.5-9.5 mM) was ineffective in changing the AMC evoked by direct or indirect stimulation of the diaphragm. 3. NO (8.6 mM) induced an increase of the AMC evoked by indirect stimulation of the muscle and was ineffective in changing the twitch tension evoked by retrograde injection of acetylcholine. NO (8.6 mM) produced an increase followed by a reduction of the AMC evoked by direct stimulation of muscles, but the muscular facilitatory effect induced by NO was smaller than the neuromuscular facilitatory effect. 4. These results indicate that NO increases the AMC when it interacts at the presynaptic level and decreases the AMC when it interacts at the postsynaptic level.