Regulation of acid signaling in rat pulmonary sensory neurons by protease-activated receptor-2

Abstract

Airway acidification has been consistently observed in airway inflammatory conditions and is known to cause cardiorespiratory symptoms that are, at least in part, mediated through the activation of bronchopulmonary C fibers and the subsequent reflexes. Protease-activated receptor-2 (PAR(2)) is expressed in a variety of cells in the lung and airways and is believed to play a role in airway inflammation and hyperresponsiveness. This study was carried out to investigate the effect of PAR(2) activation on the acid signaling in rat bronchopulmonary C-fiber sensory neurons. Our RT-PCR results revealed the expression of mRNAs for transient receptor potential vanilloid receptor 1 (TRPV1) and four functional acid-sensing ion channel (ASIC) subunits 1a, 1b, 2a, and 3 in these sensory neurons. Preincubation of SLIGRL-NH(2), a specific PAR(2)-activating peptide, markedly enhanced the Ca(2+) transient evoked by extracellular acidification. Pretreatment with PAR(2) agonists significantly potentiated both acid-evoked ASIC- and TRPV1-like whole cell inward currents. Activation of PAR(2) also potentiated the excitability of these neurons to acid, but not electrical stimulation. In addition, the potentiation of acid-evoked responses was not prevented by inhibiting either PLC or PKC nor was mimicked by activation of PKC. In conclusion, activation of PAR(2) modulates the acid signaling in pulmonary sensory neurons, and the interaction may play a role in the pathogenesis of airway inflammatory conditions, where airway acidification and PAR(2) activation can occur simultaneously.

Fig. 1.

Detection of the presence of acid-sensing ion channels (ASICs) and transient receptor potential vanilloid receptor 1 (TRPV1) mRNAs in rat pulmonary sensory neurons using RT-PCR. Cytoplasm of 20 individual pulmonary nodose neurons was collected into single PCR tubes. One-step RT-PCR and the nested PCR were carried out to detect the presence of the transcripts for TRPV1 and ASIC subunits 1a, 1b, 2a, and 3. M, DNA molecular weight marker.

Fig. 2.

Potentiation of acid-evoked Ca²⁺ transient by protease-activated receptor-2-activating peptide (PAR₂-AP) in rat pulmonary sensory neurons. A: Ca²⁺ transients evoked by extracellular acidification (pH 5.5, 40 s) before and after pretreatment with PAR₂-AP (SLIGRL-NH₂; 100 μM, 5 min). The neuronal sensitivity to capsaicin (Cap; 0.5 μM, 40 s) was tested at the end of experimental run. B: group data showing the enhancement of acid-evoked Ca²⁺ transient by PAR₂-AP pretreatment. *Significantly different from the control response before PAR₂-AP (P < 0.05, n = 26).

Fig. 3.

Effect of PAR₂-AP on the acid-evoked ASIC-like inward current in rat pulmonary sensory neurons. A–C: inward currents evoked by acid (pH 7.0, 6.5, 6.0, and 5.5; 6 s each) before, immediately after pretreatment with PAR₂-AP (100 μM, 2 min), and after 5-min washout, respectively. In all panels, Current 1 is defined as the peak of transient ASIC-like inward current, and Current 2 is measured as the amplitude of sustained inward current when the acid challenge was terminated. D: group data showing that both the peak (Current 1) and sustained (Current 2) components of acid-evoked current were significantly enhanced after PAR₂-AP pretreatment. *Significantly different from the corresponding control (P < 0.05, n = 17).

Fig. 4.

Pretreatment with 2-furoyl-LIGRLO-NH₂ mimicked the effect of PAR₂-AP. A: inward current evoked by pH 5.5 (6 s) before and after pretreatment with 2-furoyl-LIGRLO-NH₂ (100 μM, 2 min), a selective PAR₂ agonist. The acid-evoked current was evaluated by the amplitudes of its peak (Current 1) and sustained (Current 2) components. B: group data showing that both the peak and sustained components of ASIC-like current were significantly increased after 2-furoyl-LIGRLO-NH₂. *Significantly different from the corresponding control (P < 0.05, n = 7).

Fig. 5.

Potentiation of both acid-evoked ASIC-like and TRPV1-like currents by PAR₂-AP in rat pulmonary sensory neurons. A: acid (pH 5.5, 18 s)-evoked ASIC-like (first peak) and TRPV1-like (second peak) inward currents at control, after pretreatment with amiloride (ASIC inhibitor; 300 μM, 2 min), amiloride + PAR₂-AP (300 μM and 100 μM, respectively; 2 min), and PAR₂-AP (100 μM, 2 min) alone, and after 10-min washout. B and C: group data showing the peak amplitudes of pH 5.5-evoked ASIC-like and TRPV1-like inward currents, respectively, after different pretreatments as shown in A. *Significantly different from the control response (P < 0.05, n = 10). †Significant difference between pretreatments with amiloride + PAR₂-AP and amiloride alone (P < 0.05, n = 10).

Fig. 6.

Pretreatment with PAR₂-AP increased the excitability of rat pulmonary sensory neurons to acid stimulation. A: acid (pH 5.5, 6 s)-evoked changes in membrane potential (V_m) at control, after pretreatment with PAR₂-AP (100 μM, 2 min), and after 5-min washout. V_m-1 and V_m-2 refer to the values of membrane potential at the first peak and at the termination of acid challenge, respectively. B: effect of PAR₂-AP on the pH 5.5-evoked membrane potential changes. C: effect of PAR₂-AP on the number of pH 5.5-evoked action potentials. *Significantly different from the corresponding control (P < 0.05, n = 9).

Fig. 7.

Inhibition of PLC and PKC did not prevent the potentiating effect of PAR₂-AP. A: acid (pH 5.5, 6 s)-evoked inward current at control, after pretreatment with U73122 (a PLC inhibitor; 1 μM, 4 min) + PAR₂-AP (100 μM, 2 min), and after 5-min washout. B: group data showing that PAR₂-AP potentiated both the acid-evoked transient and sustained inward currents in the presence of U73122. *Significantly different from the corresponding control (P < 0.05, n = 7). C: acid (pH 5.5, 6 s)-evoked inward current at control, after pretreatment with chelerythrine (a PKC inhibitor; 10 μM, 4 min) + PAR₂-AP (100 μM, 2 min), and after 5-min washout. D: group data showing that PAR₂-AP potentiated both the acid-evoked transient and sustained inward currents in the presence of chelerythrine. *Significantly different from the corresponding control (P < 0.05, n = 6).

Fig. 8.

Lack of potentiating effect of phorbol 12-myristate 13-acetate (PMA) on the acid-evoked inward current in rat pulmonary sensory neurons. A: pretreatment with PMA (0.1 μM, 2 min), a PKC activator, did not affect the acid (pH 5.5, 6 s)-evoked ASIC-like inward current, whereas the latter was markedly potentiated by PAR₂-AP pretreatment (100 μM, 2 min) in the same neuron. B: pretreatment with PMA (0.1 μM, 2 min) failed to potentiate either ASIC-like or TRPV1-like inward currents evoked by a prolonged acid application (pH 5.5, 22 s). A and B were recorded from 2 different neurons. C: group data showing that PMA did not significantly affect the acid-evoked inward current (n = 12). The acid-evoked whole cell responses were evaluated as the amplitudes of transient peak current (Current 1) and sustained current at the termination of acid challenge (Current 2), regardless of the phenotypes of inward currents as shown in A and B.