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.
- acid-sensing ion channels
- transient receptor potential vanilloid receptor subtype-1
- low pH
pulmonary interstitial acidosis develops when the production of CO2 is exceedingly high and/or the elimination of CO2 from the lungs is hindered. Such changes can occur in both physiological (e.g., exercise) and pathophysiological (e.g., chronic obstructive pulmonary disease) conditions (6, 43). Excessive production of lactic acid also occurs commonly during anaerobic metabolism such as during tissue ischemia or hypoxia. A recent study from our laboratory demonstrated that pulmonary C fibers were activated by a bolus intravenous injection of lactic acid that lowered the pH in pulmonary venous blood to ∼7.1 (17). The fact that similar responses of C fibers were also evoked when the same molar concentration of other types of acids (formic acid, glycolic acid, etc.) with almost identical pKa (∼3.8) as lactic acid were injected suggests a major role of H+ in this action. However, we could not draw a more definitive conclusion because acid solution is also known to induce local release of potent autacoids that can activate these afferents (14).
Furthermore, local tissue acidosis is known to occur in a number of pathophysiological conditions, such as inflammation, ischemia, and carcinogenesis, in which extracellular pH (pHo) can decrease by more than two pH units (4, 30, 42). A decrease of pHo is known to activate acid-sensing ion channels (ASICs) in both peripheral sensory and central neurons (2, 21, 39, 42). ASICs, a newly described class of ligand-gated channels, have been suggested to play important roles in various physiological/pathophysiological conditions, from sensory transmission (such as nociception, mechanosensation, and taste) to ischemia, retinal function, and learning memory (7, 21, 25, 34, 45). In addition, it has been demonstrated that protons can also directly activate transient receptor potential vanilloid receptor subtype-1 (TRPV1; see Refs. 9 and 19). The contributions of ASICs and/or TRPV1 to acid signaling have been recently demonstrated in the nociceptors (2, 5, 10, 13, 28, 33, 36, 44). However, little is known about the relative roles of these two types of ion channels in the response to different levels of acidity in vagal pulmonary sensory neurons.
In light of the existing knowledge described above and the information that is currently lacking, this study was carried out to characterize the acid-signaling properties in rat vagal pulmonary sensory neurons by using whole cell perforated patch-clamp recordings.
MATERIALS AND METHODS
Labeling vagal pulmonary sensory neurons.
Cell bodies of vagal sensory nerves arising from airways and lungs reside in nodose and intracranial jugular ganglia. These sensory neurons were identified by retrograde labeling from the lungs by using the fluorescent tracer, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) as described previously (15). Young adult Sprague-Dawley rats (∼160 g) were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg) and intubated with a polyethylene catheter (PE-150) with its tip positioned in the trachea above the thoracic inlet. DiI was initially sonicated and dissolved in ethanol, diluted in saline (1% ethanol vol/vol), and then instilled into the lungs (0.2 mg/ml; 0.2 ml × 2) with the animal's head tilted up at ∼30°.
Isolation and culture of nodose and jugular ganglion neurons.
After 7–10 days, an interval previously determined to be sufficient for DiI to diffuse to the cell body, the rats were anesthetized with halothane inhalation and decapitated. The head was immediately immersed in ice-cold Hanks' balanced salt solution. Nodose and jugular ganglia were extracted under a dissecting microscope and placed in ice-cold Dulbecco’s minimal essential medium (DMEM/F-12) solution. Each ganglion was desheathed, cut into ∼10 pieces, placed in 0.125% type IV collagenase, and incubated for 1 h in 5% CO2 in air at 37°C. The ganglion suspension was centrifuged (150 g, 5 min) and supernatant aspirated. The cell pellet was resuspended in 0.05% trypsin in Hanks' balanced salt solution for 5 min and centrifuged (150 g, 5 min); the pellet was then resuspended in a modified DMEM/F-12 solution [DMEM/F-12 supplemented with 10% (vol/vol) heat-inactivated FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 100 μM MEM nonessential amino acids] and gently triturated with a small-bore fire-polished Pasteur pipette. The dispersed cell suspension was centrifuged (500 g, 8 min) through a layer of 15% BSA to separate the cells from the myelin debris. The pellets were resuspended in the modified DMEM/F-12 solution supplemented with 50 ng/ml 2.5S nerve growth factor, plated on poly-l-lysine-coated glass coverslips, and then incubated overnight (5% CO2 in air at 37°C).
Whole cell perforated patch-clamp recordings were carried out as described previously (15). Briefly, the coverslip containing the attached cells was placed in the center of a small recording chamber (0.2 ml) that was perfused by gravity feed (VC-6 perfusion valve controller; Warner Instruments, Hamden, CT) with standard extracellular solution (ECS). The chemical stimulants were applied by a pressure-driven drug delivery system (ALA-VM8; ALA Scientific Instruments, Westbury, NY), with its tip positioned to ensure that the cell was fully within the stream of the injectate. Recordings were made in the whole cell perforated-patch configuration (50 μg/ml gramicidin) using Axopatch 200B/pCLAMP 9.0 (Axon Instruments, Union City, CA). The data were acquired at 5 kHz and filtered at 2 kHz. The series resistance was usually in the range of 4–8 MΩ and was not compensated. The resting membrane potential was held at −70 mV except where otherwise indicated. The experiments were performed at room temperature (∼22°C).
Solutions and chemicals.
Standard ECS contained (in mM): 136 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 0.33 NaH2PO4, 10 glucose, and 10 HEPES, at pH 7.4. For solutions with pH ≤6.0, MES was used instead of HEPES for more reliable pH buffering. The intracellular solution contained (in mM): 92 potassium gluconate, 40 KCl, 8 NaCl, 1 CaCl2, 0.5 MgCl2, 10 EGTA, and 10 HEPES, at pH 7.2.
DiI was purchased from Molecular Probes (Eugene, OR). DMEM/F-12, trypsin, and nerve growth factor were obtained from Invitrogen (Carlsbad, CA). All other chemicals were obtained from Sigma Chemical (St. Louis, MO). Stock solution of capsaicin (1 mM) was prepared in a vehicle of 10% Tween 80, 10% ethanol, and 80% ECS. Stock solutions of amiloride and capsazepine were prepared in DMSO at concentrations of 500 and 20 mM, respectively. The solutions of these chemicals at desired concentrations were prepared daily by dilution with ECS. No detectable effect of the vehicles of these chemical agents was found in our preliminary experiments.
Data were analyzed by a one-way ANOVA. When the ANOVA showed a significant interaction, pairwise comparisons were made with a post hoc analysis (Fisher's least significant difference). Results were considered significant at P < 0.05. Data are means ± SE.
A total of 93 pulmonary sensory neurons isolated from nodose and jugular ganglia of 23 rats were studied. The whole cell capacitances of these neurons were in the range of 11.1–42.1 pF (average: 22.5 ± 0.8 pF; n = 93); the majority of them (83 out of 93) were small in size (capacitance ≤30 pF). The average resting membrane potential of these neurons was −64.4 ± 1.3 mV (range: −50 to −82 mV; n = 33).
Characteristics of acid-evoked inward current in rat vagal pulmonary sensory neurons.
At the holding potential of −70 mV, a rapid drop of pHo from 7.4 evoked a transient, rapidly inactivating inward current (Fig. 1) that could be further differentiated based on activation and inactivation kinetics into a fast type (e.g., Fig. 1B) and a slow type (e.g., Fig. 1C). In some of the neurons, the transient inward current was followed by a sustained component that did not desensitize during the presence of the acidic stimulus (e.g., Fig. 1, A and B), whereas a small subset of neurons showed only a sustained current upon the acid challenge without a transient component (e.g., Fig. 1D).
As shown in Table 1, rat vagal pulmonary sensory neurons exhibited distinct pH sensitivities. A mild pHo drop to 7.0 activated 45.2% of these sensory neurons, and pH 6.5, 6.0, and 5.5 were able to activate 83.1, 91.5, and 92.5% of these neurons, respectively. In response to acidic challenge, pulmonary sensory neurons also showed different phenotypes of inward currents. The current evoked by pH 7.0 consisted of only the transient component with small amplitude (e.g., Fig. 1, B and C). The amplitude of this transient current increased when the proton concentration increased (Table 1). Interestingly, the transient current apparently reached the maximum amplitude even at pH 6.5 in some neurons (e.g., Fig. 1A). In contrast, the sustained current increased its amplitude in a larger percentage of neurons when a lower pHo was applied (e.g., Fig. 1, A and B and Table 1). Our data did not indicate a close association between the cell size of these sensory neurons and their pH sensitivities (Table 1).
In 45 of these neurons, we also tested their responses to capsaicin. Rapid application of capsaicin (1 μM; 2–6 s) evoked an inward current in 31 (68.9%) neurons, the average amplitude of which was 1,183.4 ± 250.2 pA. The average whole cell capacitance of these capsaicin-sensitive neurons was 20.9 ± 1.0 pF.
Na+ is the charge-carrying ion of the transient component of acid-evoked current in rat vagal pulmonary sensory neurons.
The current-voltage (I-V) curve was constructed by plotting the peak amplitude of the transient component of the current evoked by a pHo drop from 7.4 to 6.5 (or 6.0 to 5.5) at different membrane potentials. Neurons were voltage clamped initially at −80 mV and then changed at a step of +20 mV every 5 min. At each of these holding potentials, the current activated by the same low-pH challenge applied for 6 s was recorded.
As shown in Fig. 2, the transient component of acid-evoked current in these pulmonary sensory neurons had a linear I-V relationship with a reversal potential at ∼65.4 mV (n = 8), which is close to the theoretical Na+ equilibrium potential (71.6 mV with extra- and intracellular solutions containing 136 and 8 mM Na+, respectively), indicating that the transient current evoked by low pH was selective for Na+.
Effects of amiloride and capsazepine on acid-evoked current in rat vagal pulmonary sensory neurons.
To determine the contribution of ASICs to acid-evoked currents, we applied amiloride, a known blocker of ASICs. As shown in Fig. 3, 2-min pretreatment with amiloride dose dependently attenuated the transient current evoked by low pH with EC50 at 32.6 μM. High dose of amiloride (1 mM) significantly inhibited the peak transient current to a mean of 34.1 ± 8.9% of the control responses (Fig. 3B; n = 7). The effect of amiloride was reversible after 10-min washout in all seven neurons tested (e.g., Fig. 3A).
We also examined the relative contribution of TRPV1, another channel gated by protons (9, 19), to the acid-evoked current in these pulmonary sensory neurons. Pretreatment with capsazepine (10 μM; 2 min), a specific antagonist of TRPV1, significantly attenuated the sustained (e.g., Fig. 4, A, B, and E), but not the transient (e.g., Fig. 4, A, C, and E), component of the current evoked by low pH. Consistent with what we demonstrated previously (15), this dose of capsazepine was able to completely abolish the capsaicin-evoked inward current in these sensory neurons (Fig. 4, D and F). Both acid- and capsaicin-evoked currents were reversible 20–30 min after the termination of capsazepine treatment (Fig. 4).
In 14 pulmonary sensory neurons, we examined the effects of amiloride and capsazepine in the same sensory neurons. As shown in Fig. 5, the transient component of acid-evoked current was primarily sensitive to amiloride, whereas the sustained component was mainly sensitive to capsazepine. There seemed no apparent interactions between these two chemicals when applied to the neurons simultaneously (Fig. 5).
Recovery time course of acid-evoked current in rat vagal pulmonary sensory neurons.
To test the recovery from acid-induced desensitization, neurons were exposed to a low-pH stimulus (6.5–5.5) for 6 s, a duration sufficient to completely desensitize the transient component of acid-evoked current. Neurons were then bathed in the standard ECS (pH 7.4) for progressively shortening intervals before they were again challenged with the same low-pH solution. The amplitudes of both components of the subsequent acid-evoked current were then compared with those of their corresponding controls. Our results showed that the transient current required 10–30 s for a complete recovery (n = 7), whereas at least 60–120 s had to elapse before the sustained current fully recovered from desensitization (n = 4; Fig. 6, A and B).
To compare the recovery time course between acid- and capsaicin-evoked currents, in a group of five neurons, we challenged cells with capsaicin with a protocol similar to that described for low pH. Short application (2–4 s) of 1 μM capsaicin evoked a comparable or a slightly smaller inward current in the same neurons, whereas the full recovery time for capsaicin (6–8 min) was much longer compared with that for low pH (Fig. 6, C and D).
Effect of acid on the excitability of rat vagal pulmonary sensory neurons.
This series of studies was carried out to test whether depolarization caused by transient extracellular acidification was sufficient to trigger action potentials in these isolated neurons. As shown in Fig. 7A, pH 7.0 failed to evoke a detectable inward current in this nodose ganglion neuron, but it induced a clear depolarization of the membrane potential (resting membrane potential: −71 mV). When the pHo was dropped to 6.5, action potentials were generated. In this particular neuron, with pHo decreasing from 6.5 to 6.0 and 5.5, the amplitudes of both the transient and sustained components of acid-evoked current in voltage-clamp recordings increased correspondingly. Similarly, in current-clamp mode, the temporal pattern of membrane depolarization also exhibited the transient and sustained components, coinciding with those displayed in the voltage-clamp mode. The pattern of the action potentials evoked by capsaicin (e.g., Fig. 7B) or current injection (e.g., Fig. 7C) in the same neurons differed from that by low pH (n = 5). Moreover, when ≥10 s elapsed, the depolarization/action potentials evoked by acidic challenge in current-clamp recordings were almost fully recovered from desensitization in all four neurons tested (e.g., Fig. 7D).
Our results showed that physiological/pathophysiological-relevant low pH (7.0–5.5) activated rat vagal pulmonary sensory neurons. In voltage-clamp recordings, these sensory neurons exhibited distinct pH sensitivities and different phenotypes of inward currents in response to low-pH challenge. About one-half of these neurons responded to pH 7.0 with a small, transient current. The amplitude of this transient current increased when the proton concentration was elevated. This transient current was predominantly carried by Na+ and was dose dependently blocked by amiloride, indicating that it was mediated by ASICs. In addition, a slow, sustained inward current after the transient component began to emerge when pH ≤6.5 was applied. This sustained current was attenuated significantly by capsazepine, indicating that it was mediated primarily through TRPV1. In current-clamp recordings, transient extracellular acidification depolarized the membrane potential and generated action potentials in these sensory neurons. Furthermore, the inward currents and depolarization/action potentials mediated by these two different acid-activated channels displayed distinct activation, inactivation, and recovery properties.
ASICs, a novel class of ligand-gated cation channels activated by protons, belong to the amiloride-sensitive degenerin/epithelial Na+ channel superfamily (3, 12, 21, 39, 41). So far, four genes encoding six ASIC subunits have been cloned. Four of these subunits can form functional homomultimeric channels (45). The pH of half-maximal activation (pH0.5) of these channels differs as follows: ASIC1a, pH0.5 = 6.2 (39); ASIC1b, a splice variant of ASIC1a, pH0.5 = 5.9 (10); ASIC2a, pH0.5 = 4.4 (29, 40); and ASIC3, pH0.5 = 6.5 (34, 38). Neither ASIC2b nor ASIC4 can form functional homomeric channels (1, 26). Whereas ASIC1a, ASIC1b, and ASIC2a display transient activation characteristics, ASIC3 responds to acid stimuli biphasically, with a quick desensitizing and a late sustained current (16). ASICs have been shown to be expressed throughout the neurons of mammalian central and peripheral nervous systems (2, 21, 42). Like other ligand-gated ion channels, functional ASICs can be formed by homomultimers and heteromultimers (5, 39). Different homomeric and heteromeric ASICs are known to have distinct pH sensitivity, ion selectivity, and channel kinetics (16). In dorsal root ganglion neurons, where many subtypes are expressed, native ASICs are believed to be heteromultimeric (2, 5). Our results have demonstrated for the first time that the acid-evoked inward currents in these pulmonary sensory neurons exhibited the properties of ASIC1 (e.g., Fig. 1B)-, ASIC2 (e.g., Fig. 1C)-, and ASIC3 (e.g., Fig. 1, A and B)-like responses. However, we do not have more definitive evidence of their expressions in these neurons. The relative narrow physiological/pathophysiological range of low pH (7.0–5.5) applied in this study further limited our ability to measure the pH0.5 in these native neurons. Nevertheless, the reversal potential of the transient component, estimated from the I-V relationship (Fig. 2), was close to the theoretical Na+ equilibrium potential. This Na+ selectivity, together with amiloride sensitivity (Figs. 3 and 5), and rapid recovery from desensitization (Fig. 6) have provided the critical evidence of the activation of ASICs in these vagal pulmonary sensory neurons.
In this study, a slow, sustained inward current began to emerge when pHo was lowered to ≤6.5, and the response did not inactivate during the presence of the acidic stimulus (e.g., Fig. 1, A, B, and D). The percentage of the neurons exhibiting this current and the amplitude of the current increased when the pHo was reduced further; for example, when pH 5.5 (4–6 s) was applied, ∼62% of neurons showed the sustained current (Table 1). Coincidently, a similar percentage of neurons (∼69%) responded to capsaicin (1 μM; 2–6 s) challenge. In contrast to its effect on the acid-evoked transient current, amiloride did not significantly affect the sustained current in these sensory neurons (Fig. 5). Instead, the latter was markedly attenuated by capsazepine (Figs. 4 and 5), a selective TRPV1 antagonist, indicating that it was mainly mediated by the activation of TRPV1 in these sensory neurons. Indeed, it has been known that, in addition to ASICs, protons can also directly activate TRPV1 (9, 19). The contribution of TRPV1 to the acid signaling has been extensively studied recently in the cutaneous nociceptive neurons (9, 35, 36, 37), where it has been shown that TRPV1 contributes to a major part of acid-induced nociception, especially the generation of the persistent pain under more severe acidification (8, 36).
Our results have shown that the ASIC-mediated transient component of acid-evoked inward current recovered completely from desensitization within 30 s, whereas >60 s were required for the TRPV1-mediated sustained component to reach a full recovery (Fig. 6, A and B). It is noteworthy that the latter was still significantly shorter than that for the capsaicin-evoked current (at least 6 min; Fig. 6, C and D), which was known to be solely mediated by TRPV1 (e.g., Fig. 4, D and F). This difference in the recovery time course between acid- and capsaicin-evoked TRPV1 responses may be, in part, the result of the difference in the amplitudes of the peak currents evoked by these two chemical stimuli (Fig. 6). An alternative explanation could be that this difference was the result of the different binding sites and different activation and inactivation kinetics of these two chemicals on this ion channel. It has been known that capsaicin binds to an intracellular site to activate TRPV1 (8), whereas proton has been suggested to interact with an extracellular site(s) on the channel complex (35). Indeed, several candidate sites located within putative extracellular loops of TRPV1 have been identified for such interactions, with two glutamate residuals E648 and E600 of particular interest (8, 18). Our study has also shown that low pH can cause the depolarization of membrane potential and generation of action potentials in these pulmonary sensory neurons. Interestingly, the acid-evoked action potentials always fired at the transient (peak) but not the sustained (plateau) phase of the depolarization (e.g., Fig. 7, A and D). A similar observation has also been reported in acid-induced activation of dorsal root ganglion neurons (27). It is believed that the tetrodotoxin-resistant Na+ channels that are expressed in a distinctly higher percentage in capsaicin-sensitive pulmonary neurons (22) play a critical role in determining the activating properties of these neurons (23, 27).
The present study has shown that pH 5.5 stimulated the majority of pulmonary sensory neurons (92.5%) through the activation of ASICs and/or TRPV1, which is in agreement with the observation made by Kollarik and Undem (20). These investigators recorded the extracellular action potential in an isolated airway nerve preparation and demonstrated that the activation of guinea pig airway afferents by low pH was mediated by both slowly (TRPV1 dependent) and rapidly (TRPV1 independent) inactivating mechanisms. However, our results did not indicate a significant difference between pulmonary sensory neurons isolated from nodose and jugular ganglia in responding to acid challenge (data not shown); nor did our data show a significant difference in cell size between the groups of neurons with different pH sensitivities (Table 1). Whether the differences in animal species (rat vs. guinea pig) and experimental approaches contribute to the discrepancy between these two studies is not known.
It should be noted that our data of acid-sensing properties in the present study were obtained from isolated pulmonary sensory neurons; whether there is a difference in the sensitivity to low pH between the sensory terminal and its neuronal soma remains to be determined. However, it has been recognized that airway acidification induces cough and bronchoconstriction in humans and laboratory animals, which has been suggested to result mainly from the release of neuropeptides from the acid-evoked activation of bronchopulmonary C fibers (20, 31). Indeed, the majority of the vagal pulmonary afferent nerves are nonmyelinated (C) fibers. Stimulation of these C-fiber afferents is known to elicit a number of reflex responses mediated through the central and/or autonomic nervous systems, including bronchoconstriction, mucus hypersecretion, cough, dyspneic sensation, and bronchial vasodilatation (11, 24). In addition, sensory neuropeptides such as tachykinins (e.g., substance P, neurokinin A) and calcitonin gene-related peptide are synthesized in the cell bodies of these C fibers and released locally from the sensory terminals upon stimulation. These peptides are known to act on a number of effector cells (e.g., airway smooth muscles, cholinergic ganglia, inflammatory cells, mucous glands) and produce potent local effects such as bronchoconstriction, extravasation of macromolecules, and edema of airway mucosa in various species, including humans (24, 32).
In summary, our results demonstrate that low pH can activate the majority of rat vagal pulmonary sensory neurons. The pH sensitivity and pattern of responses vary between neurons; and the acid signaling in these neurons is mediated through the activation of ASICs and TRPV1. Because the pH levels used in this study are well within the range of those reported for various pathophysiological conditions, such as tissue inflammation and ischemia (4, 30), these findings may provide the information for elucidating the acid-signaling mechanisms in various airway diseases in which extracellular acidification is known to occur.
This study was supported by National Heart, Lung, and Blood Institute Grants HL-58686 and HL-67379, and the Kentucky Lung Cancer Research Program. Q. Gu is a Parker B. Francis Fellow in Pulmonary Research.
We thank Michelle E. Wiggers and Robert F. Morton for technical assistance.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2006 the American Physiological Society