Evidence for both adenosine A1 and A2A receptors activating single vagal sensory C-fibres in guinea pig lungs

Abstract

We addressed the hypothesis that single vagal afferent C-fibres can be stimulated via either the adenosine A1 or A2A receptor subtypes. The effect of adenosine on the nerve terminals of vagal sensory nerve subtypes was evaluated in an ex vivo perfused guinea pig lung preparation using extracellular recording techniques. Adenosine (10 microm) consistently evoked action potential discharge in lung C-fibre terminals arising from the nodose ganglia, but failed to evoke action potential discharge in most jugular ganglion C-fibres. Adenosine also failed to activate stretch-sensitive nodose A-fibres in the lungs. The selective A1 antagonist DPCPX (0.1 microm) or the selective A2A antagonist SCH 58261 (0.1 microm) partially inhibited the nodose C-fibre activation by adenosine, and the combination of both antagonists almost completely inhibited the response. The adenosine-induced action potential discharge in nodose C-fibres was mimicked by either the selective A1 agonist CCPA (1 microm) or the selective A2A agonist CGS 21680 (1 microm). Single cell PCR techniques revealed that adenosine A1 and A2A receptor mRNA was expressed in individual nodose neurons retrogradely labelled from the lungs. The gramicidin-perforated patch clamp technique on neurons retrogradely labelled from the lungs was employed to study the functional consequence of adenosine receptor agonists directly on neuronal membrane properties. Both the selective A1 agonist CCPA (1 microm) and the selective A2A agonist CGS 21680 (1 microm) depolarized the airway-specific, capsaicin-sensitive, nodose neurons to action potential threshold. The data support the hypothesis that adenosine selectively depolarizes vagal nodose C-fibre terminals in the lungs to action potential threshold, by stimulation of both adenosine A1 and A2A receptor subtypes located in the neuronal membrane.

Figure 1. Concentration–response curve of adenosine in vagal nodose C-fibres in the guinea pig lungs

Nodose C-fibres responded to adenosine in a concentration-dependent manner (n = 5–12), whereas the jugular C-fibres did not respond (6 of 9) or poorly responded (3 of 9) to 10 μm adenosine. The potencies should be interpreted in light of the fact that the agonists were perfused for 20 s and were unlikely to have reached equilibrium conditions. Inset, representative trace of an extracellular recording of action potential shows discharge from a nodose C-fibre responding to 10 μm adenosine.

Figure 2. Concentration–response curves of the selective A₁ receptor agonist CCPA (n = 4) and the selective A_2A receptor agonist CGS 21680 (n = 5) for action potential discharge in vagal bronchopulmonary nodose C-fibres in guinea pigs

Each point represents the mean ± s.e.m.

Figure 3

A, RT-PCR of adenosine A₁ and A_2A receptors in nodose and jugular ganglia. Three distinct bands (227 bp, 467 bp and 265 bp) represent A₁, A_2A and β-actin (positive control), respectively. Omission of reverse transcriptase or the template resulted in no amplification product (not shown). B, single cell RT-PCR of adenosine A₁ and A_2A receptors in individual nodose ganglion neurons retogradely labelled from the lungs. N1–N6 represent six individual lung-specific nodose neurons. In this experiment, A₁ receptor expression was detected in 2 of 3 neurons, whereas A_2A receptor was expressed in 1 of 3 neurons. Omission of the reverse transcriptase (RT-, n = 5) or using a sample of the bath solution as the template (B, n = 6) resulted in no amplification products (negative controls). NOD = total RNA from multiple nodose neurons (positive control).

Figure 4. Representative action potential discharge evoked by adenosine (10 μm), the selective A₁ receptor agonist CCPA (0.1 μm) and the selective A_2A receptor agonist CGS 21680 (0.1 μm) in gramicidin-perforated patch clamp recordings in current clamp mode of lung-labelled capsaicin-sensitive nodose neurons

Filled bars under the tracings are the duration for the perfusion of each agonist.