Thrombin and trypsin directly activate vagal C-fibres in mouse lung via protease-activated receptor-1

Abstract

The nature of protease-activated receptors (PARs) capable of activating respiratory vagal C-fibres in the mouse was investigated. Infusing thrombin or trypsin via the trachea strongly activated vagal lung C-fibres with action potential discharge, recorded with the extracellular electrode positioned in the vagal sensory ganglion. The intensity of activation was similar to that observed with the TRPV1 agonist, capsaicin. This was mimicked by the PAR1-activating peptide TFLLR-NH(2), whereas the PAR2-activating peptide SLIGRL-NH(2) was without effect. Patch clamp recording on cell bodies of capsaicin-sensitive neurons retrogradely labelled from the lungs revealed that TFLLR-NH(2) consistently evokes a large inward current. RT-PCR revealed all four PARs were expressed in the vagal ganglia. However, when RT-PCR was carried out on individual neurons retrogradely labelled from the lungs it was noted that TRPV1-positive neurons (presumed C-fibre neurons) expressed PAR1 and PAR3, whereas PAR2 and PAR4 were rarely expressed. The C-fibres in mouse lungs isolated from PAR1(-/-) animals responded normally to capsaicin, but failed to respond to trypsin, thrombin, or TFLLR-NH(2). These data show that the PAR most relevant for evoking action potential discharge in vagal C-fibres in mouse lungs is PAR1, and that this is a direct neuronal effect.

Figure 1. Activation of PAR1-evoked action potential discharge in mouse nodose/jugular ganglion C-fibres

Single-fibre recordings in the isolated perfused lung preparation illustrating that thrombin (A; 10 μg ml⁻¹) and trypsin (B; 10 μg ml⁻¹) delivered to the mouse lung elicit action potential discharge in C-fibres of the vagal ganglia. The PAR2-activating peptide SLIGRL-NH₂ (10 μm, n= 9; 100 μm, n= 6) did not elicit action potential discharge in these fibres (C); however, activation of the PAR1 receptor with its activating peptide TFLLR-NH₂ (D; 10 μm) did. Arrow and bar indicate start and duration (0.5 min), respectively, of the perfusion of drugs into the trachea. The action potential discharge is quantified in Table 2.

Figure 2. Inward currents elicited by activation of PAR1 in a capsaicin-sensitive mouse lung-specific jugular/nodose ganglion neuron

Whole-cell patch clamp recording in a perforated patch configuration showing that TFLLR-NH₂ (PAR1-activating peptide) elicited an inward current. Holding potential was −60 mV. Membrane capacitance was 15.8 pF. Drugs were delivered at the noted concentration via superfusion solution (bar). The response to 10 μm TFLLR-NH₂ is represented by 6 additional experiments with capsaicin (1 μm)-sensitive neurons in which the current density averaged −29.8 ± 13.2 pA pF⁻¹. TFLLR-NH₂ (10 μm) did not evoke an inward current in capsaicin-insensitive neurons (0/7 neurons studied).

Figure 3. Lung-specific, TRPV1+ mouse jugular/nodose ganglion neurons express PAR1 and PAR3 mRNA

A, conventional RT-PCR showing that PAR1–4 mRNA are expressed in a mouse jugular nodose ganglion complex. ‘blank’ indicates lane was not loaded with PCR product. –RT indicates RNA control of β-actin. B, single-cell RT-PCR showing the distribution of PAR1, PAR2, PAR3 and PAR4 mRNA in individual TRPV1+ lung-specific cells isolated from the mouse jugular/nodose ganglion complex. ‘bath’ indicates RT-PCR processed with the solution in which cells were perfused during cell picking. –RT indicates RNA control of β-actin.