Mutual occlusion of P2X ATP receptors and nicotinic receptors on sympathetic neurons of the guinea-pig

Abstract

1. The interaction of ion channels activated by nicotinic receptor agonists with ion channels gated by extracellular ATP (i.e. P2X receptors) was studied on sympathetic neurons acutely dissociated from coeliac ganglia of the guinea-pig. Patch clamp methods were used to measure the inward current generated through these non-selective cationic channels under voltage clamp. 2. At the whole cell level, the specific nicotinic receptor agonists nicotine (5-100 microM) or cytisine (50-75 microM) and the P2X receptor agonists ATP (0.1-7 microM) or alpha,beta-methylene ATP (6 microM) were examined separately and in the presence of the other receptor activator. When a nicotinic and P2X receptor agonist were applied together, mutually occlusive effects were generally observed. This occurred even with concentrations of agonists that in themselves generated little to no inward current. 3. The occlusive effects of nicotinic agonists on ATP-gated currents were blocked by the nicotinic receptor/ion channel blocker hexamethonium (150 microM). The occlusive effects of ATP analogues on inward currents generated by nicotinic agonists were blocked by the P2X receptor antagonist suramin (100 microM). 4. Mutual occlusion of the effects of nicotinic agonists and ATP analogues were also observed when currents through single channels were studied in excised (outside-out) patches. 5. The results suggest that nicotinic receptors and P2X ATP receptors do not act independently in these sympathetic neurons.

Figure 1. Inhibition by nicotine (5 μm) of currents generated by ATP (6 μm)

In this experiment, after a rough concentration-response curve was constructed for nicotine (A), a concentration of 5 μm nicotine was chosen to examine the interaction with 6 μm ATP (B). Note the complete occlusion of the inward current produced by 6 μm ATP in the presence of nicotine. The effect was fully reversible (C). Traces A, B and C were recorded continuously from the same cell. In all figures, drugs were applied by rapid fast-flow superfusion during the period represented by the bar. The cell was held at -110 mV in the whole cell recording mode.

Figure 5. The mutually occlusive effects of ATP (6 μm) and cytisine (60 μm) at the single channel level

Note that shortly after application of ATP, multiple ATP-gated channels are opened and saturation of the amplifier is observed. After the cessation of superfusion with ATP, discrete openings characteristic of ATP are observed as the extracellular concentration of ATP declines. In B, application of cytisine opens nicotinic receptor-gated ion channels in the same patch. As above, saturation of the patch clamp amplifier occurred with the opening of two or more nicotinic receptors. In C, the combination of ATP (6 μm) + cytisine (60 μm) reveals mutual occlusion of the discrete channel openings. The effect was reversible (data not shown). The excised patch was made from the same cell as in Fig. 5 using the same concentrations of agonists and the same holding potential (-110 mV). For further details, see text.

Figure 6. The occlusion of ATP-gated channel openings (6 μm ATP, A) by nicotine (5 μm, B)

The effect is reversible (C). An excised (outside-out) patch was held at -110 mV.

Figure 2. The P2X ATP receptor agonist α,β-methylene ATP (6μm) inhibits nicotinic receptor activation

Note that a concentration of α,β-methylene ATP that produces no significant inward current in this experiment, reduced the inward current produced by 5 μm nicotine by greater than 50%. The cell was held at -110 mV in the whole cell recording mode.

Figure 3. Prevention of occlusion by antagonists of nicotinic receptors (A) and ATP receptors (B)

In A, note that the nicotinic receptor/channel blocker hexamethonium (150 μm) prevents the occlusive effect of nicotine on ATP-gated inward currents. In B, the ATP-receptor antagonist suramin (100 μm) prevents the occlusive effect of ATP on nicotinic receptors. Cells were held at -110 mV in the whole cell recording mode.

Figure 4. Lack of additivity of ATP (6 μm) and the nicotinic receptor agonist cytisine (60 μm)

The cell was held at -110 mV in the whole cell recording mode (from the same recording as Fig. 4). In 4 of 5 experiments using these concentrations of cytisine and ATP, co-application of cytisine and ATP reduced the response of ATP to 59 ± 11% of control (mean ± 1 s.e.m., range 41-90%). In the remaining experiment, the combined effects were less than that of sum of the two independent effects. In 2 of 3 experiments using α,β-methylene ATP (6 μm) and cytisine (60 μm), cytisine reduced the response of α,β-methylene ATP (6 μm) to 75% and 74% of control. In the remaining experiment, the effects of α,β-methylene ATP (6 μm) and cytisine were less than the sum of the two independent effects.