IJUP08 - Universidade do Porto

Impairment of retrograde signalling via adenosine in toxininduced
Myasthenia gravis: cross-talk with muscarinic
autoreceptors
Diogo Trigo 1 , Ana Sá-e-Sousa 1 , Tiago Morais 1 , Maria Alexandrina Timóteo 1 , Teresa
Magalhães-Cardoso 1 , Laura Oliveira 1 & PauloCorreia-de-Sá 1
1 Laboratório de Farmacologia e Neurobiologia, Unidade Multidisciplinar de Investigação
Biomédica (UMIB), Instituto de Ciências Biomédicas de Abel Salazar – Universidade do Porto
(ICBAS-UP), Portugal.
While adenosine acts predominantly as an inhibitory signal (via A1 receptors) under resting conditions,
amplification of neuromuscular transmission depends on facilitation of acetylcholine (ACh) release via
muscarinic M1 autoreceptors [1,2]. Upon increasing the stimulation frequency, predominant activation of
adenosine A2A receptors counteracts the M1 positive feedback mechanism causing a shift on muscarinic
neuromodulation towards the activation of inhibitory muscarinic M2 autoreceptors [2]. These complex
receptor-receptor interactions, involving distinct second messengers and effectors [3], may offer the potential
for regulating neurotransmitter exocytosis and, hence, minimization of transmission deficits observed in
myasthenic patients. This prompted us to evaluate the role of adenosine and muscarinic autoreceptors in a rat
model of toxin-induced Myasthenia gravis (TIMG).
Wistar rats (70-100 g) were injected once every 48h with saline (controls) or α-bungarotoxin (α-BTX, an
irreversible muscle-type α1 nicotinic receptor antagonist) (TIMG-model) for a period up to 6 weeks [4].
Dosage of α-BTX was adjusted by monitoring myasthenic symptoms. The procedures for measuring evoked
[ 3 H]-ACh release and diaphragm contractile responses were described previously. [ 3 H]-ACh release was
evoked by phrenic nerve stimulation with either 5 Hz-trains (750 pulses) or 50 Hz-bursts (5 bursts of 150
pulses, 20-s interburst interval). Fatigue tests were carried out using high frequency (50 Hz) intermittent (17
pulses per sec, during 3 minutes) nerve stimulation.
In contrast with that observed in control animals, inactivation of endogenous adenosine with adenosine
deaminase (ADA, 2.5 U/ml) and blockade of A1 receptors with 1,3-dipropyl-8-cyclopentyl xanthine
(DPCPX, 10 nM) failed to increase [ 3 H]-ACh release evoked by 5 Hz-trains. ADA (2.5 U/ml) decreased (-
18±6%, n=6) evoked [ 3 H]-ACh release when the phrenic nerve of TIMG animals was stimulated with 50 Hzbursts,
but this effect was significantly (P