On the adenosine receptor and adenosine inactivation at the rat diaphragm neuromuscular junction

1. The effects of adenosine and adenosine analogues 2-chloroadenosine (CADO), L-N6-phenylisopropyladenosine (L-PIA), D-N6-phenylisopropyladenosine (D-PIA), N6-cyclohexyladenosine (CHA) and 5'-N-ethylcarboxamide adenosine (NECA) on evoked endplate potentials (e.p.ps) and on twitch tension were investigated in innervated diaphragms of the rat. 2. Adenosine and its analogues decreased, in a concentration-dependent manner, the amplitude of both the e.p.ps and the twitch responses evoked by nerve stimulation. The order of potency in decreasing the twitch tension was CHA, L-PIA, NECA greater than D-PIA greater than CADO greater than adenosine. L-PIA was about 8 times more potent than D-PIA. Neither adenosine nor the adenosine analogues affected the twitch responses of directly stimulated tubocurarine-paralysed muscles. 3. 8-Phenyltheophylline (8-PT), theophylline and isobutylmethylxanthine (IBMX), in concentrations virtually devoid of effect on neuromuscular transmission, antagonized the inhibitory effect of 2-chloroadenosine. The order of potency of the alkylxanthines as antagonists of the adenosine receptor at the rat diaphragm neuromuscular junction was 8-PT greater than IBMX greater than theophylline. The antagonism by these xanthines was shown to be competitive, the pA2 value for 8-PT being 7.16. In concentrations slightly higher than those used to test its ability to antagonize the adenosine receptor, IBMX and 8-PT increased the amplitude of e.p.ps without modifying their decay phase or the resting membrane potential of the muscle fibre. 4. The adenosine uptake inhibitor, nitrobenzylthioinosine (NBI) and the adenosine deaminase inhibitor, erythro-9(2-hydroxy-3-nonyl)adenine (EHNA), in concentrations virtually devoid of effect on neuromuscular transmission, potentiated the inhibitory effect of adenosine at the rat diaphragm neuromuscular junction. The potentiation factors were about 2.6 for NBI (5 microM), 2.2 for EHNA (25 microM) and 4.6 for the combination of NBI (5 microM) and EHNA (25 microM). 5. It is concluded that both uptake and deamination contribute to the inactivation of adenosine at the rat diaphragm neuromuscular junction and that in this preparation the inhibitory effect of adenosine on transmission is mediated by a xanthine-sensitive adenosine receptor with an agonist profile which does not fit the criteria for its classification either as an A1 or A2-adenosine receptor.