Wilson Alves-Do-Prado

Gastric antiulcerogenic effects of Stryphnodendron adstringens in rats

The antiulcer activity of the total extract and the fractions of Stryphnodendron adstringens was studied in rats and compared with that of cimetidine. Ulcers were induced in rats by means of three experimental models: acute stress, acidified‐ethanol and indomethacin. The total extract and the fractions were found to have significant antiulcer activity in the case of the acute stress and acidified‐ethanol models. These findings support the use of S. adstringens extracts in the treatment of gastric lesions. Copyright

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.

Reversal by atropine of tetanic fade induced in cats by antinicotinic and anticholinesterase agents

The tetanic fade induced by intraarterial administration of neostigmine or by subparalyzing doses of d-tubocurarine or hexamethonium was studied in cat anterior tibial muscle preparations. The prior administration of atropine significantly reduced the time for recovery from the tetanic fade induced by the above mentioned drugs. A similar response was seen when atropine was administered prior to neostigmine administration for reversal of paralyzing doses of d-tubocurarine. We interpret these results to show that presynaptic muscarinic receptors participate in a negative feedback affecting acetylcholine release at the neuromuscular junction.

Presynaptic M1, M2, and A1 receptors play roles in tetanic fade induced by pancuronium or cisatracurium

PurposeWe investigated whether presynaptic facilitatory M1 and/or inhibitory M2 muscarinic receptors contributed to pancuronium- and cisatracurium-induced tetanic fade.MethodsPhrenic nerve-diaphragm muscle preparations of rats were indirectly stimulated with tetanic frequency (75 ± 3.3 Hz; mean ± SD). Doses of pancuronium, cisatracurium, hexamethonium, and d-tubocurarine for producing approximately 25% fade were determined. The effects of pirenzepine and methoctramine, blockers of presynaptic M1 and M2 receptors, respectively, on the tetanic fade were investigated.ResultsThe concentrations required for approximately 25% fade were 413 µM for hexamethonium (26.8 ± 2.4% 4% fade), 55 nM for d-tubocurarine (28.7 ± 2.55% fade), 0.32 µM for pancuronium (25.4 ± 2.2% fade), and 0.32 µM for cisatracurium (24.7 ± 0.8% fade). Pirenzepine or methoctramine alone did not produce the fade. Methoctramine, 1 µM, attenuated the fade induced by hexamethonium (to 16.0 ± 2.5% fade), d-tubocurarine (to 6.0 ± 1.6 fade), pancuronium (to 8.0 ± 4.0% fade), and cisatracurium (to 11.0 ± 3.3% fade). 10 nM pirenzepine attenuated only the fades produced by pancuronium (to 5.0 ± 0.11% fade) and cisatracurium (to 13.3 ± 5.3% fade). Cisatracurium (0.32 µM) showed antiacetylcholinesterase activity (in plasma, 14.2 ± 1.6%; 6%; in erythrocyt 17.2 ± 2.66%) similar to that of pancuronium (0.32 µM). The selective A1 receptor blocker, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 2.5 nM), also attenuated the fades induced by pancuronium and cisatracurium.ConclusionThe tetanic fades produced by pancuronium and cisatracurium depend on the activation of presynaptic inhibitory M2 receptors; these agents also have anticholinesterase activities. The fades induced by these agents also depend on the activation of presynaptic inhibitory A1 receptors through the activation of stimulatory M1 receptors by acetylcholine.

L‐Citrulline inhibits [3H]acetylcholine release from rat motor nerve terminals by increasing adenosine outflow and activation of A1 receptors

Nitric oxide (NO) production and depression of neuromuscular transmission are closely related, but little is known about the role of L‐citrulline, a co‐product of NO biosynthesis, on neurotransmitter release.

Tuning adenosine A1 and A2A receptors activation mediates l-citrulline-induced inhibition of [3H]-acetylcholine release depending on nerve stimulation pattern

The influence of nerve stimulation pattern on transmitter release inhibition by L-citrulline, the co-product of NO biosynthesis by nitric oxide synthase (NOS), was studied in the rat phrenic nerve-hemidiaphragm. We also investigated the putative interactions between NOS pathway and the adenosine system. L-citrulline (10-470 microM), the NOS substrate L-arginine (10-470 microM) and the NO donor 3-morpholinylsydnoneimine (SIN-1, 1-10 microM), concentration-dependently inhibited [(3)H]-acetylcholine ([(3)H]-ACh) release from rat motor nerve endings. Increasing stimulus frequency from 5 Hz-trains to 50 Hz-bursts enhanced [(3)H]-ACh release inhibition by l-arginine (47 microM) and L-citrulline (470 microM), whereas the effect of SIN-1 (10 microM) remained unchanged. NOS inhibition with N(omega)-nitro-L-arginine (100 microM) prevented the effect of L-arginine, but not that of L-citrulline. Adenosine deaminase (2.5 U/ml) and the adenosine transport inhibitor, S-(p-nitrobenzyl)-6-thioinosine (10 microM), attenuated release inhibition by L-arginine and L-citrulline. With 5 Hz-trains, blockade of A(1) receptors with 1,3-dipropyl-8-cyclopentyl xanthine (2.5 nM), but not of A(2A) receptors with ZM241385 (10nM), reduced the inhibitory action of l-arginine and L-citrulline; the opposite was verified with 50 Hz-bursts. Blockade of muscarinic M(2) autoreceptors with AF-DX116 (10 nM) also attenuated the effects of L-arginine and L-citrulline with 50 Hz-bursts. L-citrulline (470 microM) increased basal adenosine outflow via the equilibrative nucleoside transport system sensitive to NBTI (10 microM), without significantly (P>0.05) changing the nucleoside release subsequent to nerve stimulation. Data indicate that NOS-derived L-citrulline negatively modulates [(3)H]-ACh release by increasing adenosine outflow channelling to A(1) and A(2A) receptors activation depending on the stimulus paradigm. While adenosine acts predominantly at inhibitory A(1) receptors during 5 Hz-trains, inhibition of ACh release by L-citrulline at 50 Hz-bursts depends on the interplay between adenosine A(2A) and muscarinic M(2) receptors.

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.

Presynaptic facilitatory adenosine A2A receptors mediate fade induced by neuromuscular relaxants that exhibit anticholinesterase activity

1. Pancuronium, cisatracurium and vecuronium are antinicotinic agents that, in contrast with d‐tubocurarine and hexamethonium, exhibit anticholinesterase activity. Pancuronium‐, cisatracurium‐ and vecuronium‐induced fade results from blockade of facilitatory nicotinic receptors on motor nerves, but fade produced by such agents also depends on the presynaptic activation of inhibitory muscarinic M2 receptors by acetylcholine released from motor nerve terminals and activation of inhibitory adenosine A1 receptors by adenosine released from motor nerves and muscles. The participation of presynaptic facilitatory A2A receptors in fade caused by pancuronium, cisatracurium and vecuronium has not yet been investigated. In the present study, we determined the effects of ZM 241385, an antagonist of presynaptic facilitatory A2A receptors, on fade produced by these neuromuscular relaxants in the rat phrenic nerve–diaphragm (PND) preparation.

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.

Presynaptic muscarinic and adenosine receptors are involved in 2 Hz-induced train-of-four fade caused by antinicotinic neuromuscular relaxants in the rat.

1. Train‐of‐four fade (TOFfade) is a clinically useful parameter to monitor the degree of block of neuromuscular transmission in curarized patients. Experimentally, TOFfade has been attributed to the blockade of facilitatory nicotinic receptors on motor nerve terminals. There is less information regarding the involvement of coexistent presynaptic receptors (e.g. muscarinic M1 and M2, adenosine A1 and A2A) in the TOFfade produced by antinicotinic agents.