Characterization of acid signaling in rat vagal pulmonary sensory neurons

Abstract

Local tissue acidosis frequently occurs in airway inflammatory and ischemic conditions. The effect of physiological/pathophysiological-relevant low pH (7.0-5.5) on isolated rat vagal pulmonary sensory neurons was investigated using whole cell perforated patch-clamp recordings. In voltage-clamp recordings, vagal pulmonary sensory neurons exhibited distinct pH sensitivities and different phenotypes of inward current in responding to acidic challenge. The current evoked by lowering the pH of extracellular solution to 7.0 consisted of only a transient, rapidly inactivating component with small amplitude. The amplitude of this transient current increased when the proton concentration was elevated. In addition, a slow, sustained inward current began to emerge when pH was reduced to <6.5. The current-voltage curve indicated that the transient component of acid-evoked current was carried predominantly by Na+. This transient component was dose-dependently inhibited by amiloride, a common blocker of acid-sensing ion channels (ASICs), whereas the sustained component was significantly attenuated by capsazepine, a selective antagonist of transient receptor potential vanilloid receptor subtype-1 (TRPV1). The two components of acid-evoked current also displayed distinct recovery kinetics from desensitization. Furthermore, in current-clamp recordings, transient extracellular acidification depolarized the membrane potential and generated action potentials in these isolated neurons. In summary, our results have demonstrated that low pH can stimulate rat vagal pulmonary sensory neurons through the activation of both ASICs and TRPV1. The relative roles of these two current species depend on the range of pH and vary between neurons.

Fig. 1

Representative acid-evoked whole cell inward currents in rat vagal pulmonary sensory neurons. Low pH with increasing proton concentrations were applied for 6 s (as indicated by the horizontal bars) to four different jugular ganglion neurons. The cell capacitances for the neurons in A, B, C, and D were 24.2, 23.1, 15.6, and 11.1 pF, respectively. Note distinct pH sensitivities and different phenotypes of inward currents in response to acidic challenges.

Fig. 2

Acid-evoked transient inward currents at different membrane potentials in rat vagal pulmonary sensory neurons. A: representative records illustrating pH 6.5-evoked transient currents in a jugular neuron (23.2 pF) voltage clamped at the membrane potentials indicated in the figure. These membrane voltages were held for 90 s before the acid application to ensure any voltage-gated conductance were inactivated. V_m, membrane potential. B: current-voltage relationship of low pH (6.5–5.5; 6 s) -evoked transient currents from eight sensory neurons; the reversal potential was estimated at ∼65.4 mV. I, peak inward current evoked by acid application. Data represent means ± SE.

Fig. 3

Effect of amiloride on the acid-evoked transient inward currents in rat vagal pulmonary sensory neurons. A: representative records illustrating pH 6.5-evoked transient currents in the absence and presence of a 2-min pretreatment of increasing concentration of amiloride in a jugular neuron (36.5 pF). B: group data showing the inhibitory effect of amiloride on low pH (6.5–5.5; 2–6 s) -evoked transient currents from seven sensory neurons; Chapman fit: EC₅₀, 32.6 μM.

Fig. 4

Effect of capsazepine on the acid- and capsaicin-evoked currents in rat vagal pulmonary sensory neurons. A, B and C: representative records illustrating the effect of capsazepine (CZP; 10 μM; 2 min) pretreatment on pH 5.5 (4–6 s) -evoked inward currents in nodose (29 pF), jugular (15.6 pF), and nodose (18.6 pF) neurons, respectively. D: representative records illustrating the effect of CZP (10 μM; 2 min) on capsaicin (Cap; 1 μM, 2 s) -evoked currents in a jugular neuron (15.9 pF). E and F: group data showing the effect of CZP on the inward currents evoked by low pH (6.5–5.5; 4–6 s) and capsaicin (1 μM; 2–4 s), respectively. * P < 0.05 as compared with the corresponding control. Data represent means ± SE.

Fig. 5

Effects of amiloride and capsazepine on the acid-evoked currents in the same rat vagal pulmonary sensory neurons. A: representative records illustrating the effects of amiloride (100 μM; 2 min) and capsazepine (CZP; 10 μM, 2 min) on pH 5.5 (6 s)-evoked inward currents in a jugular neuron (23.1 pF). B: group data showing the effects of 2-min pretreatment with amiloride (100 μM) and CZP (10 μM) on both transient and sustained components evoked by low pH (6.5–5.5; 6 s). * P < 0.05 as compared with the corresponding control. Data represent means ± SE.

Fig. 6

Different recovery kinetics of acid- and capsaicin-evoked currents in rat vagal pulmonary sensory neurons. A and C: inward currents evoked by repeated application of low pH (5.5; 6 s) and capsaicin (Cap; 1 μM, 3 s), respectively, at different intervals in the same pulmonary sensory neuron (nodose; 24.7 pF). B and D: group data showing the different recovery kinetics of acid- (6.5–5.5; 6 s) and capsaicin- (1 μM; 2–4 s) evoked currents in these sensory neurons, respectively. * P < 0.05 as compared with the corresponding control (Con). Data are means ± SE.

Fig. 7

Changes in membrane potential (current-clamp) and inward current (voltage-clamp) in response to different stimuli in rat vagal pulmonary sensory neurons. A and B: acid- (pH 7.0–5.5; 6 s) and capsaicin- (Cap; 1 μM, 6 s), respectively, evoked changes in membrane potential (V_m; upper traces; current clamp) and inward current (lower traces; voltage clamp). C: action potentials (upper trace) evoked by a 50 pA current injection (198-ms duration; lower trace). A, B and C were recorded from a single nodose neuron (23.3 pF). D: action potentials (upper traces) and inward currents (lower traces) evoked by pH 5.5 (6 s) challenge at different intervals in a jugular neuron (30.1 pF). Insets, the action potentials triggered by various stimuli are shown on a large scale.