Voltage-gated sodium channels in nociceptive versus non-nociceptive nodose vagal sensory neurons innervating guinea pig lungs

Abstract

Lung vagal sensory fibres are broadly categorized as C fibres (nociceptors) and A fibres (non-nociceptive; rapidly and slowly adapting low-threshold stretch receptors). These afferent fibre types differ in degree of myelination, conduction velocity, neuropeptide content, sensitivity to chemical and mechanical stimuli, as well as evoked reflex responses. Recent studies in nociceptive fibres of the somatosensory system indicated that the tetrodotoxin-resistant (TTX-R) voltage-gated sodium channels (VGSC) are preferentially expressed in the nociceptive fibres of the somatosensory system (dorsal root ganglia). Whereas TTX-R sodium currents have been documented in lung vagal sensory nerves fibres, a rigorous comparison of their expression in nociceptive versus non-nociceptive vagal sensory neurons has not been carried out. Using multiple approaches including patch clamp electrophysiology, immunohistochemistry, and single-cell gene expression analysis in the guinea pig, we obtained data supporting the hypothesis that the TTX-R sodium currents are similarly distributed between nodose ganglion A-fibres and C-fibres innervating the lung. Moreover, mRNA and immunoreactivity for the TTX-R VGSC molecules Na(V)1.8 and Na(V)1.9 were present in nearly all neurons. We conclude that contrary to findings in the somatosensory neurons, TTX-R VGSCs are not preferentially expressed in the nociceptive C-fibre population innervating the lungs.

Figure 1. Voltage-gated sodium currents in lung-specific nodose ganglion neurons

A, pharmacological method for isolating TTX-R I_Na. Families of currents evoked by 30 ms voltage steps from −90 mV to 30 mV in 5 mV increments from a holding potential of −120 mV. Inset: voltage protocol. Total I_Na was recorded in a reduced sodium solution (upper traces). Only 20 ms is depicted for clarity. TTX-R I_Na was recorded in the presence of 0.3 μm TTX (middle traces). Digital subtraction of the TTX-R I_Na from the total I_Na revealed the TTX-S component (lower traces). B, activation and steady-state inactivation curves for TTX-S I_Na (n = 33 and 29, respectively) and TTX-R I_Na (n = 39 and 31, respectively). Circles represent TTX-S I_Na. Squares represent TTX-R I_Na. Filled symbols, activation. Open symbols, steady-state inactivation. Continuous and dotted lines represent Boltzmann fits for TTX-S I_Na and TTX-R I_Na, respectively. G is conductance. C, prepulse inactivation method for isolating TTX-R I_Na. In a separate neuron, total I_Na was recorded in a reduced sodium solution using the identical voltage protocol in A (upper traces). Middle traces: TTX-R-like I_Na was generated using a prepulse inactivation step to −55 mV for 500 ms followed by a series of 30 ms steps from −90 mV to 30 mV in 5 mV increments (inset). Digital subtraction of the TTX-R I_Na from the total I_Na revealed the TTX-S component (lower traces). D, activation curves for TTX-S I_Na (n = 23) and TTX-R I_Na (n = 23). •, TTX-S-like I_Na. ▪, TTX-R-like I_Na. ▴, recordings in zero-concentration sodium (n = 4). Data are means ±s.e.m.

Figure 2. TTX-R I_Na recorded from intact neurons in the nodose ganglion

A, representative traces from a lung-specific non-nociceptive neuron showing a family of TTX-R I_Na evoked using a prepulse inactivation protocol (inset; see Fig. 1 legend). B, activation curves fitted using a Boltzmann function (n = 5). G is conductance. C, action potential elicited with a suprathreshold current step in the presence of TTX (1 μm). Inset depicts an expanded time scale. Data are means ±s.e.m.

Figure 3. Three distinct phenotypes of nodose fibres innervate airways and lungs of guinea pigs

Histograms of conduction velocities were obtained from recordings in isolated innervated trachea or lung preparation (see methods in Riccio et al. 1996b; Undem et al. 2004). Nodose ganglion fibres innervating the intrapulmonary compartment segregate into two populations. Upper panel: the faster conducting group (n = 114) has a conduction velocity centred around 13.6 ± 0.7 m s⁻¹, which is classified as Aβ. In general, this population possesses low-threshold mechanosensitivity and is often attributed as stretch receptors. Note the different frequency scale used. Lower panel: the slower conducting group (n = 149) has a conduction velocity centred around 0.7 ± 0.01 m s⁻¹, which falls in the C fibre range. Hallmarks of this group include sensitivity to a variety of chemicals, including capsaicin, but relative insensitivity to mechanical stimulation. Middle panel: nodose nerves innervating the extrapulmonary airways (larynx, trachea and main bronchi) have an intermediate conduction velocity centred around 5.1 ± 0.3 m s⁻¹, which is classified as Aδ (n = 190). This population is not activated by capsaicin, but is highly sensitive to punctate mechanical stimulation, which evokes cough in intact animals. Histograms in upper, middle, and lower panels were binned at 0.1, 1, and 2 m s⁻¹, respectively. Data are means ±s.e.m.

Figure 4. TTX-R I_Na in three distinct phenotypes of nodose ganglion neurons innervating the lungs and airways

TTX-R I_Na as a percentage of total I_Na was evaluated in lung-specific capsaicin-sensitive (n = 9), lung-specific capsaicin-insensitive (n = 14), and trachea-specific capsaicin-insensitive (n = 13) neurons. Cap is capsaicin. Data are means ±s.e.m.

Figure 5. Voltage-gated α subunit mRNA expression in guinea pig tissues

Quantitative real-time RT-PCR was used to determine expression of 9 sodium channel α subunits in a variety of guinea pig tissues. Tissues studied were nodose ganglia (n = 3), jugular ganglia (n = 3), dorsal root ganglia (n = 3), brain tissue (n = 2), heart (n = 3), and skeletal muscle (n = 2). Note that relative amounts of mRNA within a given tissue type are approximate as the relative efficiencies of the RT step for each of the 9 Na_Vs is unknown. Data are means ±s.e.m.

Figure 6. Coexpression of Na_V1.7, Na_V1.8 and Na_V1.9 α subunits in lung-specific nodose neurons

Single-cell RT-PCR was used to evaluate coincident expression of α subunit of Na_V1.7, Na_V1.8 and Na_V1.9 in TRPV1-positive (presumed capsaicin-sensitive) and TRPV1-negative (presumed capsaicin-insensitive) lung-specific nodose ganglion neurons. Examples of TRPV1-negative (A) and TRPV1-positive (B) neurons are depicted. C, no signals were detected in bath controls. D, non-neuronal material surrounding the cells was also evaluated. Message for β actin was routinely detected. PCR products were run on 1.5% agarose gels. Left-most lane represents a 50 bp ladder. Predicted sizes for TRPV1, β-actin, Na_V1.7, Na_V1.8 and Na_V1.9 were 284 bp, 132 bp, 158 bp, 123 bp and 117 bp, respectively.

Figure 7. Coexpression of Na_V1.8-like and Na_V1.9-like I_Na in a majority of lung-labelled nodose ganglion neurons

A, TTX-R I_Na evoked with 100 ms voltage steps from −90 mV to 30 mV in 5 mV increments in the presence of 0.3 μm TTX. Holding potential was −120 mV. Fluoride was the primary anion in the pipette solution in this set of experiments. Inset: voltage protocol. B, a 500 ms prepulse to −40 mV prior to voltage steps protocol in A yielded the Na_V1.8-like current. Inset: voltage protocol. C, digital subtraction of the Na_V1.8-like current from the total TTX-R I_Na revealed the Na_V1.9-like current. D, activation curves for Na_V1.8 (•; n = 17) and Na_V1.9 (▪; n = 13). Data were fitted with Boltzmann function (line). E, comparison of Na_V1.8 and Na_V1.9 current density among capsaicin-sensitive (hatched bars; n = 5) and -insensitive (open bars; n = 13) lung-specific nodose ganglion neurons. *Statistically significant comparison (P < 0.05). Data are means ±s.e.m.

Figure 8. Immunoreactivity for Na_V1.7, Na_V1.8, and Na_V1.9 found in virtually all cells in nodose ganglion

A, representative low magnification (top) and high magnification (bottom) photomicrograph showing Na_V1.7-like immunoreactivity using 1 : 1500 primary antibody concentration. Immunoreactivity was visualized using the chromagen DAG/horseradish peroxidase reaction. Mayer's haematoxylin was used to counterstain the tissues. B, low magnification (top) and high magnification (bottom) photomicrographs showing Na_V1.8-like immunoreactivity (1 : 3000). C, low magnification (top) and high magnification (bottom) photomicrographs showing Na_V1.9-like immunoreactivity (1 : 1500). D, rabbit IgG isotype control (2.5 μg ml⁻¹). Top and bottom photomicrographs are at low and high magnification, respectively. Bars indicate 300 μm and 100 μm for low magnification and high magnification photomicrographs.