Oliveira_2004_J.Physiol_560_157

Motor nerve terminals possess multiple voltage-sensitive calcium channels operating acetylcholine (ACh) release. In this study, we investigated whether facilitation of neuromuscular transmission by adenosine generated during neuronal firing was operated by Ca(2+) influx via 'prevalent' P-type or via the recruitment of 'silent' L-type channels. The release of [(3)H]ACh from rat phrenic nerve endings decreased upon increasing the stimulation frequency of the trains (750 pulses) from 5 Hz (83 +/- 4 x 10(3) disintegrations per minute per gram (d.p.m. g(-1)); n = 11) to 50 Hz (30 +/- 3 x 10(3) d.p.m. g(-1); n = 5). The P-type Ca(2+) channel blocker, omega-agatoxin IVA (100 nm) reduced (by 40 +/- 10%; n = 6) the release of [(3)H]ACh evoked by 50-Hz trains, while nifedipine (1 microM, an L-type blocker) was inactive. Tetanic depression was overcome (88 +/- 6 x 10(3) d.p.m. g(-1); n = 12) by stimulating the phrenic nerve with 50-Hz bursts (five bursts of 150 pulses, 20 s interburst interval). In these conditions, omega-agatoxin IVA (100 nM) failed to affect transmitter release, but nifedipine (1 microM) decreased [(3)H]ACh release by 21 +/- 7% (n = 4). Inactivation of endogenous adenosine with adenosine deaminase (ADA, 0.5 U ml(-1)) reduced (by 54 +/- 8%, n = 5) the release of [(3)H]ACh evoked with 50-Hz bursts. This effect was opposite to the excitatory actions of adenosine (0.5 mm), S-(p-nitrobenzyl)-6-thioinosine (5 microM, an adenosine uptake blocker) and CGS 21680C (3 nM, a selective A(2A) receptor agonist); as the A(1) receptor agonist R-N(6)-phenylisopropyl adenosine (R-PIA, 300 nM) failed to affect the release of [(3)H]ACh, the results indicate that adenosine generated during 50-Hz bursts exerts an A(2A)-receptor-mediated tonus. The effects of ADA (0.5 U ml(-1)) and CGS 21680C (3 nm) were prevented by nifedipine (1 microM). Blocking tonic A(2A) receptor activation, with ADA (0.5 U ml(-1)) or 3,7-dimethyl-1-propargyl xanthine (10 microM, an A(2A) antagonist), recovered omega-agatoxin IVA (100 nM) inhibition and caused the loss of function of nifedipine (1 microM). Data indicate that, in addition to the predominant P-type Ca(2+) current triggering ACh release during brief tetanic trains, motoneurones possess L-type channels that may be recruited to facilitate transmitter release during high-frequency bursts. The fine-tuning control of Ca(2+) influx through P- or L-type channels is likely to be mediated by endogenous adenosine. Therefore, tonic activation of presynaptic A(2A) receptors operating Ca(2+) influx via L-type channels may contribute to overcome tetanic depression during neuronal firing.