Presynaptic A1 inhibitory/A2A facilitatory adenosine receptor activation balance depends on motor nerve stimulation paradigm at the rat hemidiaphragm
Authors: Correia-de-Sa P. , Timoteo M. A. , Ribeiro J. A. 
Published: December 2017

1. Adenosine modulates acetylcholine (ACh) release from the rat motor nerve terminals. Tonic activation of presynaptic A1 inhibitory and/or A2A facilitatory adenosine receptors is regulated by the concentration of the nucleoside at the synapse. The parameters (frequency, duration of pulses, train length) of nerve stimulation determine the amount of transmitter and/or modulator released, and have long been proposed as important features of synaptic control. This prompted us to investigate which was the prevailing response to adenosine on evoked [3H]-ACh release from rat phrenic nerve hemidiaphragm preparations in different stimulation conditions. 2. With low-frequency, short-duration pulses (5 Hz, 40 microseconds in duration), the adenosine inhibitory tonus (approximately 30%) predominates. The magnitude of the adenosine tonic inhibition was dependent on the number of pulses (250–750) delivered in each stimulation train, e.g., the facilitatory effect of adenosine deaminase (ADA, 0.5 U/ml) and the inhibitory effect of the adenosine uptake blocker S-(p-nitrobenzyl)-6-thioinosine (NBTI, 5 microM) reached significance only when > 250 pulses were applied. Facilitation was only observed with high concentrations of either exogenous adenosine (> 100 microM) or NBTI (> 10 microM). 3. When the stimulation pulse duration was increased to 1 ms (5 Hz, 750 pulses), endogenously generated adenosine consistently facilitated evoked [3H]-ACh release. In these conditions, ADA (0.5 U/ml) decreased evoked [3H]-ACh release by 29 +/- 4% (mean +/- SE) (n = 3), and both NBTI (3-30 microM) and adenosine (10–500 microM), which had biphasic effects with pulses of 40 microseconds, facilitated transmitter release. 4. When high-frequency “trains” (50 Hz, 40 microseconds, 500 pulses) were applied, both ADA (0.5 U/ml) and NBTI (5 microM) failed to modify evoked [3H]-ACh release. To bypass putative feedforward inhibition of ecto-5'-nucleotidase induced by released ATP, which might reduce adenosine formation during high-frequency trains, experiments containing a series of five high-frequency “bursts” (50 Hz, 40 microseconds, 100 pulses) with variable interburst intervals (5–20 s) were performed. In such conditions, the prevailing tonic response to adenosine turned out to be facilitatory, because ADA (0.5 U/ml) inhibited and NBTI (5 microM) facilitated evoked [3H]-ACh release. The magnitude of the inhibitory effect of ADA (0.5 U/ml) ranged from -9 +/- 6% (n = 4) to -54 +/- 8% (n = 5) as the interburst interval changed from 5 to 20 s, respectively. 5. Prolongation of individual pulses from 40 microseconds to 1 ms (5 Hz frequency) or increasing the frequency of stimulation (1–50 Hz, 40 microseconds) did not significantly change the excitatory effect of the A2A receptor agonist 2-[4-(2-p-carboxyethyl)phenylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680C). In contrast, the inhibitory effect of the A1 receptor agonist R-N6-phenylisopropyladenosine was significantly attenuated in both stimulation conditions. 6. In conclusion, the results suggest that high-intensity, high-frequency motor nerve stimulation critically influences endogenous adenosine formation and the A1/A2A receptor activation balance, i.e., it potentiates the tonic adenosine A2A-receptor-mediated facilitation of ACh release, whereas activation of the inhibitory A1 receptors becomes less effective. A model is proposed that attempts to further elucidate adenosine's involvement in synaptic transmission adaptation.