Negative crosstalk between M 1 and M 2 muscarinic autoreceptors involves endogenous adenosine activating A 1 receptors at the rat motor endplate

Abstract

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 [ 3H]-choline (2.5 μCi/ml). Selective activation of muscarinic M 1 and adenosine A 1 receptors with 4-( N-[3-clorophenyl]-carbamoyloxy)-2-butyryltrimethylammonium (McN-A-343, 3 μM) and R- N 6 -phenylisopropyladenosine (R-PIA, 100 nM), respectively, significantly attenuated inhibition of evoked [ 3H]-ACh release induced by muscarinic M 2 receptor activation with oxotremorine (10 μM). Attenuation of the inhibitory effect of oxotremorine (10 μM) 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 μM) by McN-A-343 (3 μM) 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 μM). 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.