Celia Regina Ambiel

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.

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.

Paradoxical neostigmine-induced TOFfade: on the role of presynaptic cholinergic and adenosine receptors.

Neuromuscular transmission is clinically monitored using the train-of-four ratio (TOFratio), which is the quotient between twitch tension produced by the fourth (T4) and by the first (T1) stimulus within a train-of-four stimulation at 2Hz. Neostigmine causes a paradoxical depression of the TOFratio (TOFfade) that is amplified by intra-arterial atropine in cats. This led us to question the usefulness of the TOFratio as a sole testing element to control neostigmine-induced reversal of neuromuscular transmission block caused by non-depolarizing agents. We hypothesized that the inhibition of cholinesterase activity might increase acetylcholine bioavailability and consequently cholinoceptor activation, leading to concomitant adenosine release from nerve endings and skeletal muscle fibers. The involvement of presynaptic muscarinic and adenosine receptors in neostigmine-induced TOFfade in the rat phrenic nerve diaphragm was investigated. Blockade of adenosine A2A receptors with ZM241385 and of muscarinic M2 receptors with methoctramine or atropine amplified neostigmine-induced TOFfade. Notwithstanding TOFfade amplification, the blockade of M2 or A2A receptors increased both T1 and T4 twitch tensions above control during the first 3min of neostigmine application. Beyond that period, the T1 twitch tension returned to baseline, whereas T4 decreased further until the control value with neostigmine alone. Blockade of M1 receptors by pirenzepine did not change neostigmine-induced TOFfade, unless A2A receptors were concurrently blocked with ZM241385. Data indicate that the paradoxical neostigmine-induced fade involves presynaptic mechanisms that regulate transmitter release and synaptic adenosine accumulation, including the activation of adenosine A2A and muscarinic M2 receptors.

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.

Apamin reduces neuromuscular transmission by activating inhibitory muscarinic M2 receptors on motor nerve terminals

This study was undertaken to investigate the mechanism by which the toxin from the bee venom, apamin, might exert beneficial effects in patients suffering from myotonic dystrophy. The effects of apamin were compared with those produced by another potassium channel blocker, 4-aminopyridine, on rat hemidiaphragm preparations stimulated at a 100 Hz frequency via the phrenic nerve. Apamin and 4-aminopyridine increased nerve-evoked tetanic fade without changing the maximal tetanic tension. The inhibitory effect of apamin was mimicked by acetylcholine. In contrast with apamin, 4-aminopyridine increased the amplitude of muscle contractions induced by nerve stimulation at 0.2 Hz frequency. All these compounds were devoid of effect when diaphragm muscle fibres were stimulated directly in the presence of the neuromuscular blocker, D-tubocurarine. The muscarinic M(2) receptor antagonist, methoctramine, prevented the inhibitory effects of both apamin and acetylcholine. Blockade of presynaptic facilitatory muscarinic M(1) and nicotinic receptors respectively with pirenzepine and hexamethonium increased apamin-induced tetanic fade. Data suggest that apamin inhibits neuromuscular transmission by a mechanism independent of the blockade of Ca(2+)-activated K(+) channels, which might involve the activation of inhibitory muscarinic M(2) receptors on motor nerve terminals. Such a mechanism may be the origin of the beneficial effect of apamin controlling muscle excitability in patients suffering from myotonic diseases.