Avian intrapulmonary chemoreceptors (IPC) are vagal respiratory afferents that are inhibited by high lung Pco 2 and excited by low lung Pco 2. Previous work suggests that increased CO2 inhibits IPC by acidifying intracellular pH (pHi) and that pHi is determined by a kinetic balance between the rate of intracellular carbonic anhydrase-catalyzed CO2 hydration/dehydration and transmembrane extrusion of acids and/or bases by various exchangers. Here, the role of amiloride-sensitive Na+/H+ exchange (NHE) in the IPC CO2 response was tested by recording single-unit action potentials from IPC in anesthetized ducks, Anas platyrhynchos. For each of the IPC tested, blockade of the NHE using dimethyl amiloride (DMA) elicited a marked (>50%) dose-dependent decrease in mean IPC discharge (P < 0.05), suggesting that NHE is important for pHi regulation and CO2 transduction in IPC. In addition, activation of the NHE using 12-O-tetradecanoylphorbol 13-acetate stimulated six of the seven IPC tested, although the overall effect was not statistically significantly (P = 0.07). Taken together, these findings suggest that CO2 transduction in IPC is dependent on transmembrane NHE although it is likely to be much slower than carbonic anhydrase-catalyzed hydration-dehydration of CO2.
- carbon dioxide chemosensitivity
- intracellular pH regulation
- respiratory control
birds have intrapulmonary chemoreceptors (IPC) that monitor lung Pco 2 and exert reflex effects on the pattern of breathing (9, 15, 16, 36, 37, 43, 45). These IPC have afferent axons in the vagus nerves, cell bodies in the nodose ganglia, and sensory endings in the parabronchial tissue of the lungs (9,25, 34). The action potential discharge of IPCs responds to both rapidly changing (i.e., phasic) and sustained (or tonic) levels of intrapulmonary Pco 2, thereby encoding information about the temporal relationships between ventilation, perfusion, and metabolism (4, 9, 15, 16, 19, 24, 34). IPC sensory feedback helps terminate inspiration by sensing CO2washout from the lung, helps maintain arterial homeostasis in response to moderate inspired hypercapnia (35, 37, 45), and helps adjust breathing to metabolic demands (4, 9, 19, 24, 50).
IPC are unusual respiratory chemoreceptors because their action potential discharge rate is inversely proportional to intrapulmonary Pco 2. Low Pco 2stimulates IPC firing, and high Pco 2 inhibits firing (9, 15, 16); thus the IPC response is backward compared with that of traditional respiratory chemoreceptors like the carotid bodies (18, 29), many presumptive central chemoreceptors (40), and snail pneumostome ganglia chemoreceptors (13, 14). Many presumptive chemoreceptor neurons located in the mammalian medullary raphe, however, have also been shown to be inhibited by high Pco 2(41, 54), as are reptilian IPC, mammalian pulmonary stretch receptors, and mammalian laryngeal CO2chemoreceptors (10, 32, 39, 44). Thus the inverse CO2 response (discharge rate inhibited by high CO2) may be a common variant of respiratory chemosensitivity, just somewhat overlooked.
IPC, like some other chemoreceptors that are excited by decreasing Pco 2 levels, are critically dependent on carbonic anhydrase (CA) activity for CO2 transduction (11, 27, 38, 43, 44). Pharmacological inhibition of intracellular CA with acetazolamide (but not extracellular CA with benzolamide) abolishes avian IPC response to CO2 and causes sustained high-frequency action potential discharge (27,43). Under normal conditions, steady high-frequency discharge occurs only if IPC are exposed to hypocapnia, suggesting that CA inhibition mimics the effect of constant low Pco 2 and/or alkalinity. In contrast, when CA inhibitors are given to traditional respiratory chemoreceptors, the receptors remain normally responsive to tonic Pco 2 levels, but their response to phasic Pco 2 changes is attenuated, and their steady discharge rate is increased as if they were seeing increased Pco 2 or acidity (11, 13, 29). Therefore, rapid CO2 hydration/dehydration is somehow critical for CO2 chemotransduction in IPC but not in traditional respiratory chemoreceptors. This suggests that IPC do not sense CO2 directly but respond instead to one of the products of its hydration reaction (CO2 + H2O ⇌ H+ + HCO ). Additionally, the CO2hydration/dehydration equilibrium does not appear to be the predominant determinant of intracellular pH (pHi) in IPC because even with complete CA inhibition the uncatalyzed CO2hydration/dehydration equilibrium should still occur, although more slowly. The critical dependence of IPC on fast intracellular CA catalytic rates suggests that IPC chemotransduction reflects a dynamic coupling of the kinetics of rapid CO2 hydration/dehydration to other acid-base regulatory mechanisms.
Our proposed model of CO2 chemoreception in IPC is shown in Fig. 1 (27, 47). In the model, CO2 is thought to diffuse freely across IPC sensory endings, but H+ and HCO do not. When Pco 2 increases, CO2 is rapidly hydrated to H+ and HCO by CA (43) located in the intracellular space (27). When Pco 2 decreases, the reaction reverses. Critical dependence of IPC chemotransduction on a high CA catalysis rate occurs because pHi at a given Pco 2 is dependent on a dynamic balance between rapid kinetic rates of H+ and HCO production by CA-catalyzed CO2 hydration/dehydration, and the slower kinetic rates of H+ and/or HCO transmembrane exchange. Upsetting this dynamic balance is hypothesized to change the steady-state pHi at a given Pco 2 and alter discharge rate. For example, inhibiting CA with acetazolamide should slow H+production rate from CO2 hydration and make the IPC relatively alkalotic at a given Pco 2. However, extrusion of intracellular H+ by Na+/H+ exchangers (NHE) may also be depressed by acetazolamide, due to limitation of substrate (H+) and allosteric inhibition by intracellular alkalosis (2), resulting in a less pronounced alkalosis. If CA inhibition has greater inhibitory effects on CO2 hydration rate than on H+ extrusion rate, the model suggests that pHiand IPC discharge should increase with acetazolamide, as is seen experimentally. If acetazolamide has greater inhibitory effects on H+ extrusion rate than on CO2 hydration rate, the model suggests that pHi and IPC discharge should decrease with acetazolamide, which is not seen experimentally. As a final link, alkaline pHi may stimulate IPC membrane excitability and acidic pHi may depress membrane excitability through as yet unidentified pH-sensitive ion channels (an area of future study).
We began testing the model by blocking HCO /Cl− transmembrane exchange with DIDS and found that it modestly stimulated IPC discharge at all CO2 levels (47). This is consistent with model predictions because DIDS should cause intracellular HCO accumulation and alkalosis, thereby exciting IPC. However, the small observed response suggests that DIDS-sensitive HCO /Cl− exchange is not the main transmembrane acid/base regulating mechanism in IPC. In the present study, the model was tested again. Here, the effect of the Na+/H+ antiporter (NHE) on IPC chemotransduction was tested. In vivo single-cell IPC responses to CO2 were determined before and after administration of the NHE blocker dimethyl amiloride (DMA) and before and after administration of the NHE enhancer 12-O-tetradecanoylphorbol 13-acetate (TPA). We hypothesized that slowing or stopping any essential mechanism for H+ extrusion should cause intracellular acidosis and depress IPC discharge.
All experiments were performed under a protocol approved by the Institutional Animal Care and Use Committee at Northern Arizona University in accordance with The American Physiological Society's “Guiding Principles for Research Involving Animals and Human Beings” (1). Sixteen mallard ducks, Anas platyrhynchos, aged 4–6 mo, body mass 1.0–1.4 kg, of either sex, were studied. Ducks were anesthetized to a deep surgical level (i.e., absence of withdrawal response to a strong toe pinch) by intravenous infusion of 35–40 mg/kg pentobarbital sodium. A polyethylene catheter was implanted in the left brachial vein for administration of supplemental dosages of pentobarbital sodium (3.5–5.0 mg/kg iv, as needed) and for administration of experimental drugs. Another polyethylene catheter was implanted in the brachial artery for monitoring arterial blood pressure and acid-base status. A thermistor probe was inserted 7–10 cm into the colon, and body temperature was regulated at 40 ± 1°C using a circulating hot water pad and hot water bottles.
The thorax was opened with a midsternal incision, and the lungs were independently unidirectionally ventilated (8) through Foley catheters (7 Fr) inserted into the right and left primary bronchi. The inflatable cuffs of the catheters were positioned to occlude the ostia of the medioventral secondary bronchi. Each lung was then ventilated with 600 ml/min of air supplemented with 3% CO2. Gases were mixed and delivered by two Cameron Instruments GF-1 electronic mass flow controllers. A loose umbilical tape ligature was placed around the left pulmonary artery and fashioned into a snare. The snare could be tightened to occlude blood flow through the left lung and redirect all blood flow to the right lung, or loosened to allow blood flow through both lungs. The thorax was then draped in plastic wrap to conserve body heat and moisture.
The left vagus nerve was exposed in the neck, raised slightly on a black plastic dissecting stage, and bathed in a mineral oil pool. Single-unit recordings were made by dissecting fine filaments from the vagus nerve and placing them on a monopolar platinum-iridium electrode. Electrical activity in the filaments was referenced to an indifferent Ag-AgCl electrode on the nerve sheath through a high-impedance differential probe (Grass HIP). Action potentials were amplified with a Grass P511K AC preamplifier and AM-5 audio amplifier. Only single-unit recordings were accepted for study, and single units were identified by the constant amplitude and shape of their action potentials using a Haer slope/height window discriminator, which generated a digital pulse with each occurrence of a single-unit action potential. Interspike intervals and the overall discharge rates of IPC action potentials were timed using the digital pulses from the window discriminator and a Compupro S-100 8085/8088 microcomputer sampling at 14,500 Hz. Analog action potential signals were band pass-filtered between 100 and 3,000 Hz, visualized on an oscilloscope, and recorded on a Vetter 4 channel VHS tape system (using pulse code modulation). IPCs were identified by their nearly immediate response to step changes in ventilatory gas Pco 2.
Normal IPC Response to CO2
We tested left vagal filaments for IPC activity while stepping left lung inspired CO2 between 0 and 5% (in air) at 11-s intervals. When an IPC was identified, we tightened the left pulmonary artery snare to shunt all pulmonary blood flow to the right lung, which provided gas exchange for the animal. In the absence of left lung blood flow, left lung Pco 2 and therefore the intrapulmonary Pco 2 stimulus delivered to the IPC were equal to the left lung inspired Pco 2.
The left lung inspired CO2 step was then turned off, left lung inspired CO2 was adjusted to a steady 1%, and the IPC discharge rate was allowed to stabilize (usually occurring in 1 or 2 min). Steady-state IPC discharge was then recorded on tape and computer. Inspired CO2 was increased by 1%, the receptor was allowed to stabilize, and steady-state discharge was again recorded. This was repeated until steady-state IPC responses to 1, 2, 3, 4, 5, 6, and 7% had been recorded. Duplicate recordings of IPC discharge were then made by returning to one or more CO2levels and checking the discharge rates for reproducibility (±10%). IPC with nonreproducible responses to steady CO2 were not studied further. After establishing a normal IPC response to Pco 2, left lung blood flow was restored by releasing the pulmonary artery snare.
IPC Response to CO2 After Blockade and Activation of the NHE
After a normal IPC response to Pco 2 in air was measured, the effects of blockade or activation of the NHE on the IPC response to Pco 2 were examined. DMA was used the to selectively block the NHE, and TPA was used to activate the NHE. TPA is an activator of protein kinase C (PKC), which has been shown to stimulate Na+/H+ antiport activity in some cells (5, 6, 21, 22, 48). Both DMA and TPA were obtained from Sigma Chemical. DMA was dissolved in distilled water and prepared fresh on the day of the experiment; TPA was initially dissolved in DMSO (1.2 μl/ml), brought to its final concentration by adding distilled water, and stored at −20°C. A vehicle solution using the same concentration of DMSO was used in two experiments before injection of TPA as a control for nonspecific effects on IPC discharge.
Eight animals were given DMA intravenously, and their IPC responses were retested using the same Pco 2 stimuli as during control conditions. DMA dosages studied were 1 μmol/kg (n = 7 IPC), 3 μmol/kg (n = 8 IPC), and 8 μmol/kg (n = 5 IPC). The IPC response to the smallest dose was studied first (3 μmol/kg was the smallest dose in one case), and then additional (cumulative) dosages were given and studied. IPC usually responded to DMA injections rapidly, and within 5–10 min, their discharge rates were generally stable. Left lung blood flow was then redirected to the right lung with the pulmonary artery snare, and IPC responses to steady levels of Pco 2 were recorded as described above. The pulmonary artery snare was then loosened, and the procedure was repeated for higher cumulative DMA dosages. Two-way ANOVA was used to test for main effects of CO2 level (treatment 1, 7 levels) and DMA dosage (treatment 2, 4 levels) on IPC discharge.
Seven animals were given TPA intravenously, and their IPC responses were retested using the same Pco 2 stimuli as during control conditions. One or two dosages of TPA ranging from 14 to 60 nmol/kg were used in each animal studied. IPC usually responded to TPA injections within 1–3 min, after which they maintained a stable discharge rate. Left lung blood flow was then redirected to the right lung with the pulmonary artery snare, and IPC responses to steady levels of Pco 2 were recorded as described above. Two-way ANOVA was used to test for main effects of CO2 level (treatment 1, 7 levels) and TPA dosage (treatment 2, 3 levels) on IPC discharge rate.
Logarithmic regressions of the form were used to quantify the individual IPC stimulus-response relationships under each treatment condition (27), wheref IPC is the IPC discharge rate (s−1), A is the determined slope parameter, loge Pco 2is the natural logarithm of intrapulmonary Pco 2(Torr), and B is the determined intercept parameter. Mean and SEs for the slope and intercept parameters were determined for the various treatment conditions and compared with the normal condition using t-tests, for which the criterion level ofP < 0.05 was considered significant.
Blood Acid-Base Analysis
Blood buffer curves were measured before and 30–60 min after administration of 8 μmol/kg DMA in three animals to test for possible metabolic acid-base disturbances in the extracellular fluid. Arterial Pco 2 and pH were analyzed on a Cameron Instruments blood gas analyzer using modified Radiometer electrodes. Blood was sampled into 1-ml heparinized glass syringes from the brachial artery catheter while animals were ventilated with inspired CO2 values ranging from 1 to 7%. The blood-buffer relationships before and 30–90 min after DMA injection were quantified by regression of arterial pH on log10 Pco 2. Slopes and intercepts of the regression equations obtained pre- and post-DMA were compared using t-tests, and P < 0.05 was considered significant.
Effects of DMA on IPC Response to CO2
In each of the animals tested, blockade of the NHE by intravenous injection of DMA depressed IPC discharge in a dose-dependent manner at each level of Pco 2. Figure2 shows an example of this dose-dependent relationship for one IPC studied at Pco 2 levels of 5.4, 16.2, and 27.0 Torr. As expected, before administration of DMA (i.e., control), IPC discharge decreased in response to increasing Pco 2. After administration of DMA, IPC discharge was reduced at each level of Pco 2, with larger DMA dosages producing a greater reduction in the discharge rate. Although most IPC still responded to increased Pco 2 with decreasing discharge rate after DMA administration, some IPC were silenced by the higher dosages of DMA regardless of the level of Pco 2 (not shown).
The mean CO2 responses measured after administration of DMA at dosages of 1, 3, and 8 μmol/kg are shown in Fig.3. IPC discharge rate was depressed in a dose-dependent manner from normal (control). Two-way ANOVA revealed significant effects of CO2 on IPC discharge (P < 0.0001), with significant inhibition of IPC discharge frequency being observed at each DMA dosage examined (1 μmol/kg, P < 0.003; 3 μmol/kg, P< 0.0001; 8 μmol/kg, P < 0.0001). In addition, there was a significant interaction between the effects of DMA and CO2 (P < 0.05). Further, the average magnitude of the slopes and intercepts of the logarithmic stimulus-response regression equations were also smaller after administration of DMA (Table 1).
Effects of TPA on IPC Response to CO2
In six of the seven animals tested, activation of the NHE by intravenous injection of TPA produced a small increase in IPC discharge at some or all levels of Pco 2. Figure4 shows an example of one IPC studied before and after administration of 14 nmol/kg TPA at Pco 2 levels of 16.2, 21.6, and 27.0 Torr. Both before (control) and after administration of TPA, IPC discharge decreased in response to increasing Pco 2. After administration of TPA, in this example, an increase in IPC discharge at each level of Pco 2 was seen. In general, in the six animals in which TPA increased IPC discharge, the increase in discharge rate ranged from approximately 2 to 40%, with discharge increasing to a greater extent at lower levels of Pco 2.
The mean CO2 responses measured after administration of TPA showed a trend for increased IPC discharge rate relative to control, especially at lower levels of Pco 2. The mean IPC responses, however, were not significantly different from normal (P > 0.18). Likewise, the average slopes and intercepts of the logarithmic stimulus-response regression equations were not significantly altered by TPA (Table2) but trended toward increased responsiveness to CO2 (P > 0.06). In contrast to the small stimulatory effect elicited by TPA, IPC discharge was unchanged or slightly reduced at each level of Pco 2 examined in response to injection of vehicle solution containing DMSO.
Figure 5 shows the blood-buffer relationships before and 30–90 min after administration of 8 μmol/kg DMA. The blood pH response to changing arterial Pco 2 was not significantly changed by DMA treatment, indicating no significant metabolic alterations in extracellular pH over this time period. Parameters of the blood-buffer regression equations, pH = (slope) log10 Pco 2 + intercept, for the curves shown in Fig. 5 are provided in Table3.
Critique of the Hypothesized “Balance” Between Catalyzed CO2 Hydration-Dehydration Rates and Transmembrane Acid-Base Exchanger Rates
Our model of IPC CO2 chemotransduction suggests that H+ production or loss by CO2hydration/dehydration is balanced by transmembrane acid-base exchange. However, it is clear that the relative rates of CO2hydration/dehydration (even uncatalyzed) far exceed those possible for antiport acid-base exchange (20). The hypothesized “balance” then is actually a kinetic mismatch, which is probably critical for CO2 chemotransduction in IPC. If the rates of H+ production and H+ extrusion were exactly matched in magnitude at all Pco 2 values, pHi would probably change little with Pco 2. Thus a kinetic mismatch between H+ production and extrusion is required to generate a pHi signal for transduction of the Pco 2 stimulus. Our mathematical modeling of this relationship indicates that a CO2hydration/dehydration rate that is 100- to 1,000-fold faster than the acid-base transmembrane exchange rate gives phasic and tonic IPC responses to Pco 2 that closely simulate experimental observations (26).
Modulation of Activity of the NHE
In the current experiments, DMA (a selective blocker of the NHE) strongly inhibited, in a dose-dependent manner, action potential discharge in all IPC studied and reduced the slope and intercept of the IPC stimulus-response relationships to Pco 2. In addition, a specific effect of DMA on IPC CO2 sensitivity was suggested by the significant interaction between DMA and CO2 on IPC discharge rate in the two-way ANOVA.
In several IPC, the highest dosages of DMA completely inhibited discharge, but on average IPC showed an attenuated response to CO2 after NHE blockade. The largest attenuation of discharge rates occurred when IPC were exposed to the lowest levels of Pco 2, hence the significantly reduced slope and intercept of the IPC stimulus-response lines (see Fig. 3), and the significant CO2-DMA interaction in the ANOVA. One possible explanation for this finding is that DMA inhibition of NHE produced the greatest relative acidotic shift in pHi when Pco 2 levels were lowest. Other CO2chemoreceptor cells have NHEs that are inhibited by extracellular acidosis (14, 40, 42), and hypercapnia-induced extracellular acidosis may similarly inhibit NHEs in IPC. If so, IPC may be less sensitive to DMA inhibition in hypercapnia if their NHEs are already depressed by extracellular acidosis, and IPC may be more sensitive to DMA inhibition in hypocapnia because their NHEs may be operating more freely due to extracellular alkalosis. Another possible explanation for the larger inhibitory effect at low Pco 2 is that the pHi change is larger for equimolar changes in H+ concentration under alkalotic conditions compared with acidotic conditions due to the logarithmic nature of the pH relationship. For example, [H+] increasing from 35 to 40 nM (a relative intracellular alkalosis) causes a ΔpH of −0.058, but [H+] increasing from 75 to 80 nM (a relative intracellular acidosis) causes a ΔpH = −0.028. Thus, even if DMA inhibited H+ transport by the same amount during hypercapnia and hypocapnia, the relative effect on pHiwould be greater in hypocapnia due to its associated alkalosis.
It should be noted, however, that chronic blockade of NHE via administration of amiloride for many hours or days can cause metabolic acidosis due to inhibitory effects on NHE in renal tubular epithelia (28). Because extracellular metabolic acidosis would clearly complicate the interpretation of the DMA-induced effects on IPC, our experimental design focused on the acute effects of DMA that started within seconds after intravenous DMA infusion and reached a steady state after 5–10 min. Further, no changes were observed in systemic blood-buffer relationships, and no evidence of metabolic acidosis or base deficit was detected even at 90 min (i.e., the maximum duration of our experiments) after DMA infusion (see Fig. 5). This supports the conclusion that the large changes in IPC response produced by DMA were not due to changes in extracellular pH but rather to direct effects of DMA on acid extrusion in IPC sensory endings.
It should also be noted that in our present investigation, DMA was used rather than its parent compound amiloride. DMA is a potent and specific blocker of NHE (31, 51), whereas amiloride is a less specific blocker (23, 31, 33). Further, the concentration of amiloride that is required to inhibit NHE activity has also been reported to inhibit epithelial Na+ channels, Na+/Ca2+ exchange, Na+/K+-ATPase, and a number of kinases (6, 12, 31, 46, 49, 51). Because DMA has not been demonstrated to exert similar nonspecific (i.e., “non-NHE” related) effects, we believe that the DMA-induced effects on IPC discharge observed in the present study were mediated by blockade of NHE. The data from the present study, however, provide no insight into which of the five isoforms of the NHE identified (reviewed by Ref.40) were present in IPC or to the contribution of any of these isoforms to our current findings because DMA is an effective inhibitor of most NHE isoforms.
Phorbol esters are activators of PKC and stimulate amiloride-sensitive NHE and intracellular alkalization in many cells, including vascular smooth muscle, rat renal epithelial cells, rat parotid gland, HL-60 cultured cells, human neutrophils, rat smooth muscle, chicken skeletal muscle, Swiss 3T3 cultured cells, A431 cultured human fibroblasts, and others (21-23). We hypothesized that the phorbol ester TPA would stimulate NHE in IPC and cause intracellular alkalization and increased IPC discharge rate. The increases in mean IPC response to CO2 induced by TPA were not significant (0.6 ≤ P≤ 0.18), suggesting that TPA is not a powerful stimulant of NHE for the average IPC. However, no IPC were inhibited by TPA and several were markedly stimulated, which also suggests a wide variation in individual IPC responses.
The variability of the TPA effects on IPC discharge rate has several possible explanations. PKC may not be as important for regulating NHE in IPC as it is in some other cells. Alternatively, the NHE in IPC may already operate in a stimulated state due to high endogenous PKC activity, and further stimulation by TPA may be limited. It is also possible that TPA does stimulate IPC, but the sample size and individual IPC variability in the present experiment produced inadequate statistical power to prevent a type II error.
It should also be noted that in our current experiments, TPA was initially dissolved in DMSO. It has previously been suggested that 0.2% DMSO solvent (a concentration higher than that used in our study) may produce a small stimulation of H+ efflux (5). We believe, however, that the small increase in IPC discharge observed in our current study was a specific effect of TPA and not DMSO (i.e., vehicle) because vehicle injection was ineffective in increasing IPC discharge. Thus it is unlikely that DMSO (vehicle) was the primary source of increased discharge rate in avian IPC.
Comparison to Other CO2 Chemoreceptors
IPC differ in several ways from traditional CO2-stimulated respiratory chemoreceptors in the mammalian brain stem, mammalian and avian carotid body, and pulmonate ganglion ofHelix aspersa. For example, traditional chemoreceptors increase excitability as Pco 2 increases, but IPC decrease excitability as Pco 2 increases (14, 18, 29, 40-42). Traditional chemoreceptors continue to respond to Pco 2 after CA inhibition by acetazolamide, and they are mildly stimulated because CA inhibition causes an extracellular acidosis (11, 13, 29). In contrast IPC do not respond to Pco 2 after CA inhibition by acetazolamide, and their discharge is strongly stimulated, perhaps because CA inhibition results in intracellular alkalosis (27,43).
IPC are also similar in some ways to traditional CO2-stimulated respiratory chemoreceptors. For example, pHi rather than CO2 per se seems to be the sensed physiological variable in both traditional chemoreceptors and IPC (3, 17, 27, 52, 53). Traditional chemoreceptors in the mammalian medulla, carotid body, and in Helix aspersa use NHE as an important acid-base regulator. Furthermore, these traditional chemoreceptors show attenuated pHi recovery during a normal hypercapnic stimulus because their NHEs are inhibited by extracellular acidosis (7, 14, 40, 42). Recently, inhibition of the NHE-3 isoform with the selective blocker S8218 has been shown to increase the mammalian central CO2 ventilatory response and lower the apneic threshold, suggesting an important function of NHE-3 in mammalian CO2 sensing (30). Avian IPC also appear to rely on NHEs for their inverse response to CO2stimuli as demonstrated in the current investigation. DMA, an amiloride analog, caused profound inhibition of IPC CO2chemotransduction; however, it was not possible to determine the specific NHE isoform from our results. DMA also caused a smaller absolute reduction in IPC discharge rate when IPC were transducing high Pco 2 compared with low Pco 2 (see Fig. 2). Perhaps NHE in IPC, like NHE in traditional respiratory chemoreceptors, are inhibited by the hypercapnia-induced extracellular acidosis, and therefore the effect of further inhibition of the NHE by DMA is smallest at high Pco 2.
Respiratory CO2 chemosensitivity is important for pH and metabolic homeostasis in active air-breathing animals, and CO2 chemosensitivity seems to have evolved many times in different forms (14). These forms include the intrapulmonary chemoreceptors studied here, brain stem and peripheral arterial chemoreceptors of vertebrates, nasal and upper airway chemoreceptors in reptiles, amphibians, and mammals, and pneumostome ganglia chemoreceptors in pulmonate snails. Experimental evidence suggests that inverse CO2 sensitivity involves differences in CO2 signal transduction that set IPC apart from traditional CO2-stimulated respiratory chemoreceptors although some similarities also exist. These differences may be a product of evolutionary pressures to produce a rapid, positive response to decreasing intrapulmonary CO2 levels.
We thank L. Hempleman for technical assistance, and students K. Wallace, C. King, and P. Morgan for help with blood gas analysis.
This work was supported by National Science Foundation Grant 9723783 and National Institutes of Health Grant GM-56931.
Address for reprint requests and other correspondence: S. C. Hempleman, Dept. of Biological Sciences, Box 5640, Northern Arizona Univ., Flagstaff, AZ 86011-5640 (E-mail:).
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.
First published February 20, 2003;10.1152/ajpregu.00519.2002
- Copyright © 2003 the American Physiological Society