Maria A. Timóteo

Relative contribution of ecto‐ATPase and ecto‐ATPDase pathways to the biphasic effect of ATP on acetylcholine release from myenteric motoneurons

Background and purpose:  The relative contribution of distinct ecto‐nucleotidases to the modulation of purinergic signalling may depend on differential tissue distribution and substrate preference.

A2A adenosine receptor facilitation of neuromuscular transmission : Influence of stimulus paradigm on calcium mobilization

Abstract: The influence of stimulus pulse duration on calcium mobilization triggering facilitation of evoked [3H]acetylcholine ([3H]ACh) release by the A2A adenosine receptor agonist CGS 21680C was studied in the rat phrenic nerve‐hemidiaphragm. The P‐type calcium channel blocker ω‐agatoxin IVA (100 nM) decreased [3H]ACh release evoked with pulses of 0.04‐ms duration, whereas nifedipine (1 μM) inhibited transmitter release with pulses of 1‐ms duration. Depletion of intracellular calcium stores by thapsigargin (2 μM) decreased [3H]ACh release evoked by pulses of 1 ms, an effect observed even in the absence of extracellular calcium. With short (0.04‐ms) stimulation pulses, when P‐type calcium influx triggered transmitter release, facilitation of [3H]ACh release by CGS 21680C (3 nM) was attenuated by both thapsigargin (2 μM) and nifedipine (1 μM). With longer stimuli (1 ms), a situation in which both thapsigargin‐sensitive internal stores and L‐type channels are involved in ACh release, pretreatment with either ω‐agatoxin IVA (100 nM) or nifedipine (1 μM) reduced the facilitatory effect of CGS 21680C (3 nM). The results suggest that A2A receptor activation facilitates ACh release from motor nerve endings through alternatively mobilizing the available calcium pools (thapsigargin‐sensitive internal stores and/or P‐ or L‐type channels) that are not committed to the release process in each stimulation condition.

Tetanic failure due to decreased endogenous adenosine A2A tonus operating neuronal Cav1 (L-type) influx in Myasthenia gravis

J. Neurochem. (2011) 117, 797–811.

Negative crosstalk between M1 and M2 muscarinic autoreceptors involves endogenous adenosine activating A1 receptors at the rat motor endplate

At the rat motor nerve terminals, activation of muscarinic M(1) receptors negatively modulates the activity of inhibitory muscarinic M(2) receptors. The present work was designed to investigate if the negative crosstalk between muscarinic M(1) and M(2) autoreceptors involved endogenous adenosine tonically activating A(1) receptors on phrenic motor nerve terminals. The experiments were performed on rat phrenic nerve-hemidiaphragm preparations loaded with [(3)H]-choline (2.5 microCi/ml). Selective activation of muscarinic M(1) and adenosine A(1) receptors with 4-(N-[3-clorophenyl]-carbamoyloxy)-2-butyryltrimethylammonium (McN-A-343, 3 microM) and R-N(6)-phenylisopropyladenosine (R-PIA, 100 nM), respectively, significantly attenuated inhibition of evoked [(3)H]-ACh release induced by muscarinic M(2) receptor activation with oxotremorine (10 microM). Attenuation of the inhibitory effect of oxotremorine (10 microM) by R-PIA (100 nM) was detected even in the presence of pirenzepine (1 nM) blocking M(1) autoreceptors, suggesting that suppression of M(2)-inhibiton by A(1) receptor activation is independent on muscarinic M(1) receptor activity. Conversely, the negative crosstalk between M(1) and M(2) autoreceptors seems to involve endogenous adenosine tonically activating A(1) receptors. This was suggested, since attenuation of the inhibitory effect of oxotremorine (10 microM) by McN-A-343 (3 microM) was suppressed by the A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (2.5 nM), and by reducing extracellular adenosine with adenosine deaminase (0.5 U/mL) or with the adenosine transport blocker, S-(p-nitrobenzyl)-6-thioinosine (NBTI, 10 microM). The results suggest that the negative crosstalk between muscarinic M(1) and M(2) autoreceptors involves endogenous adenosine outflow via NBTI-sensitive (es) nucleoside transport system channelling to the activation of presynaptic inhibitory A(1) receptors at the rat motor endplate.

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.

Synergism between A2A-adenosine receptor activation and vasoactive intestinal peptide to facilitate [3H]-acetylcholine release from the rat motor nerve terminals

Abstract The effect of vasoactive intestinal peptide (VIP) on evoked [ 3 H]-acetylcholine ([ 3 H]-ACh) release from motor nerve terminals, and its interaction with presynaptic facilitatory A 2A -adenosine receptors was investigated in the rat phrenic nerve-hemidiaphragms. Facilitation of [ 3 H]-ACh release by VIP (100 nM) only becomes apparent when high frequency (50 Hz) or long lasting pulses (1 ms) were delivered to the phrenic nerve; VIP excitation was prevented by removal of endogenous adenosine tonus, with adenosine deaminase (2.5 units/ml) or with the A 2A -receptor antagonist, 3,7-dimethyl-1-propargyl xanthine, (10 μM). Pretreatment with the selective A 2A -receptor agonist, CGS 21680C (2 nM), potentiated the neurofacilitatory action of VIP (100 nM). The results suggest that tonic A 2A -receptors activation by endogenous adenosine is required to trigger the facilitatory action of VIP on evoked [ 3 H]-ACh release from motor nerve endings.

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.

Neuromuscular paralysis by the basic phospholipase A2 subunit of crotoxin from Crotalus durissus terrificus snake venom needs its acid chaperone to concurrently inhibit acetylcholine release and produce muscle blockage

Background and purpose: Crotoxin (CTX), a heterodimeric phospholipase A2 (PLA2) neurotoxin from Crotalus durissus terrificus snake venom, promotes irreversible blockade of neuromuscular transmission. Indirect electrophysiological evidence suggests that CTX exerts a primary inhibitory action on transmitter exocytosis, yet contribution of a postsynaptic action of the toxin resulting from nicotinic receptor desensitization cannot be excluded. Here, we examined the blocking effect of CTX on nerve‐evoked transmitter release measured directly using radioisotope neurochemistry and video microscopy with the FM4‐64 fluorescent dye. Experimental approach: Experiments were conducted using mice phrenic‐diaphragm preparations. Real‐time fluorescence video microscopy and liquid scintillation spectrometry techniques were used to detect transmitter exocytosis and nerve‐evoked [3H]‐acetylcholine ([3H]ACh) release, respectively. Nerve‐evoked myographic recordings were also carried out for comparison purposes. Key results: Both CTX (5 &mgr;g/mL) and its basic PLA2 subunit (CB, 20 &mgr;g/mL) had biphasic effects on nerve‐evoked transmitter exocytosis characterized by a transient initial facilitation followed by a sustained decay. CTX and CB reduced nerve‐evoked [3H]ACh release by 60% and 69%, respectively, but only the heterodimer, CTX, decreased the amplitude of nerve‐evoked muscle twitches. Conclusion and implications: Data show that CTX exerts a presynaptic inhibitory action on ACh release that is highly dependent on its intrinsic PLA2 activity. Given the high safety margin of the neuromuscular transmission, one may argue that the presynaptic block caused by the toxin is not enough to produce muscle paralysis unless a concurrent postsynaptic inhibitory action is also exerted by the CTX heterodimer. HighlightsCrotoxin (CTX) is the paralysing venom from Crotalus durissus terrificus rattlesnake.CTX heterodimer and its monomeric PLA2 subunit decrease nerve‐evoked ACh release.In contrast to CTX, the isolated PLA2 subunit is unable to cause muscle paralysis.Coupling of PLA2 subunit to its chaperone CA is required for neuromuscular paralysis.