Inhibition of mechanical activation of guinea-pig airway afferent neurons by amiloride analogues

Abstract

1. The aim of this study was to investigate a role for Epithelial Sodium Channels (ENaCs) in the mechanical activation of low-threshold vagal afferent nerve terminals in the guinea-pig trachea/bronchus. 2. Using extracellular single-unit recording techniques, we found that the ENaC blocker amiloride, and its analogues dimethylamiloride and benzamil caused a reduction in the mechanical activation of guinea-pig airway afferent fibres. 3. Amiloride and it analogues also reduced the sensitivity of afferent fibres to electrical stimulation such that greater stimulation voltages were required to induce action potentials from their peripheral terminals within the trachea/bronchus. 4. The relative potencies of these compounds for inhibiting electrical excitability of afferent nerves were similar to that observed for inhibition of mechanical stimulation (dimethylamiloride approximately benzamil > amiloride). This rank order of potency is incompatible with the known rank order of potency for blockade of ENaCs (benzamil > amiloride >> dimethylamiloride). 5. As voltage-gated sodium channels play an important role in determining the electrical excitability of neurons, we used whole-cell patch recordings of nodose neuron cell bodies to investigate the possibility that amiloride analogues caused blockade of these channels. At the concentration required to inhibit mechanical activation of vagal nodose afferent fibres (100 microM), benzamil caused significant inhibition of voltage-gated sodium currents in neuronal cell bodies acutely isolated from guinea-pig nodose ganglia. 6. Combined, our findings suggest that amiloride and its analogues did not selectively block mechanotransduction in airway afferent neurons, but rather they reduced neuronal excitability, possibly by inhibiting voltage-gated sodium currents.

Figure 1

Inhibition of mechanical and electrical activation of guinea-pig airway afferent neurons by amiloride and is analogues. (A) Typical trace from an experiment showing mechanically-evoked action potentials recorded prior to (control, upper trace) and following perfusion of the trachea with benzamil (100 μM, lower trace). Traces are from single unit recordings, the horizontal line is background noise and vertical lines represent the arrival of an action potential at the recording electrode. (B) Concentration-dependence for inhibition of mechanical activation by amiloride (n=4), dimethylamiloride (n=4) and benzamil (n=4). (C) Concentration-dependence for inhibition of electrical excitability by amiloride (n=4), dimethylamiloride (n=4) and benzamil (n=4).

Figure 2

Inhibition by benzamil of voltage-gated sodium currents in guinea-pig acutely isolated nodose ganglion neurons. (A and B) Typical traces from whole cell patch-clamp recording experiments showing sodium currents that were elicited from (A) a holding potential of −50 mV and (B) −100 mV prior to (control) and following application of benzamil (100 μM for 4 min. (C) Magnitude of peak sodium currents that were elicited from a holding potential of −50 mV and −100 mV prior to (n=3) or following (n=3) a 4 min application of 100 μM benzamil. ^*P<0.05.

Figure 3

Inhibition of mechanical and electrical activation of guinea-pig airway afferent neurons by TTX. (A) Typical trace from an extracellular recording experiment showing mechanically-evoked action potentials recorded prior to (control, upper trace) and following perfusion of the trachea with TTX (30 nM, lower trace). Traces are from single unit recordings, the horizontal line is background noise and vertical lines represent the arrival of an action potential at the recording electrode. (B) Concentration-dependence for inhibition of mechanical activation by TTX (n=7). (C) Concentration-dependence for inhibition of electrical excitability by TTX (n=7 for 10 nM, n=4 for 30 nM (maximum electrical stimulation (100 volts) evoked action potentials in only four of seven preparations that were exposed to 30 nM TTX).