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Am J Physiol Regul Integr Comp Physiol 284: R1551-R1559, 2003. First published February 20, 2003; doi:10.1152/ajpregu.00519.2002
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Vol. 284, Issue 6, R1551-R1559, June 2003

CO2 transduction in avian intrapulmonary chemoreceptors is critically dependent on transmembrane Na+/H+ exchange

Steven C. Hempleman1, Thomas P. Adamson2, Rowin S. Begay1, and Irene C. Solomon3

1 Department of Biological Sciences, Northern Arizona University, Flagstaff, Arizona 86011-5640; 2 Division of Neurobiology, Physiology and Behavior, University of California, Davis, California 95616; and 3 Department of Physiology and Biophysics, State University of New York, Stony Brook, New York 11794-8661


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Avian intrapulmonary chemoreceptors (IPC) are vagal respiratory afferents that are inhibited by high lung PCO2 and excited by low lung PCO2. 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; neuron; acid; base


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

BIRDS HAVE INTRAPULMONARY chemoreceptors (IPC) that monitor lung PCO2 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 PCO2, 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 CO2 washout 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 PCO2. Low PCO2 stimulates IPC firing, and high PCO2 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 PCO2 (41, 54), as are reptilian IPC, mammalian pulmonary stretch receptors, and mammalian laryngeal CO2 chemoreceptors (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 PCO2 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 PCO2 and/or alkalinity. In contrast, when CA inhibitors are given to traditional respiratory chemoreceptors, the receptors remain normally responsive to tonic PCO2 levels, but their response to phasic PCO2 changes is attenuated, and their steady discharge rate is increased as if they were seeing increased PCO2 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 right-left-harpoons  H+ + HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>). Additionally, the CO2 hydration/dehydration equilibrium does not appear to be the predominant determinant of intracellular pH (pHi) in IPC because even with complete CA inhibition the uncatalyzed CO2 hydration/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<UP><SUB>3</SUB><SUP>−</SUP></UP> do not. When PCO2 increases, CO2 is rapidly hydrated to H+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> by CA (43) located in the intracellular space (27). When PCO2 decreases, the reaction reverses. Critical dependence of IPC chemotransduction on a high CA catalysis rate occurs because pHi at a given PCO2 is dependent on a dynamic balance between rapid kinetic rates of H+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> production by CA-catalyzed CO2 hydration/dehydration, and the slower kinetic rates of H+ and/or HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transmembrane exchange. Upsetting this dynamic balance is hypothesized to change the steady-state pHi at a given PCO2 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 PCO2. 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 pHi and 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).


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  		Fig. 1.   Conceptual model of CO2 transduction in avian intrapulmonary chemoreceptors (IPC). The role of Na+/H+ exchange in IPC chemotransduction was examined in the present investigation. See text for additional details.

We began testing the model by blocking HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/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<UP><SUB>3</SUB><SUP>−</SUP></UP> accumulation and alkalosis, thereby exciting IPC. However, the small observed response suggests that DIDS-sensitive HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

General

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 PCO2.

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 PCO2 and therefore the intrapulmonary PCO2 stimulus delivered to the IPC were equal to the left lung inspired PCO2.

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 CO2 levels 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 PCO2, 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 PCO2 in air was measured, the effects of blockade or activation of the NHE on the IPC response to PCO2 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 PCO2 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 PCO2 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 PCO2 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 PCO2 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.

Regression Analysis

Logarithmic regressions of the form
f<SUB>IPC</SUB><IT>=A</IT> log<SUB><IT>e</IT></SUB> (P<SC>co</SC><SUB>2</SUB>)<IT>+B</IT>
were used to quantify the individual IPC stimulus-response relationships under each treatment condition (27), where fIPC is the IPC discharge rate (s-1), A is the determined slope parameter, loge PCO2 is the natural logarithm of intrapulmonary PCO2 (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 of P < 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 PCO2 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 PCO2. Slopes and intercepts of the regression equations obtained pre- and post-DMA were compared using t-tests, and P < 0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 PCO2. Figure 2 shows an example of this dose-dependent relationship for one IPC studied at PCO2 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 PCO2. After administration of DMA, IPC discharge was reduced at each level of PCO2, with larger DMA dosages producing a greater reduction in the discharge rate. Although most IPC still responded to increased PCO2 with decreasing discharge rate after DMA administration, some IPC were silenced by the higher dosages of DMA regardless of the level of PCO2 (not shown).


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  		Fig. 2.   Examples of IPC action potential (AP) discharge in response to changes in inspired CO2 before and after intravenous administration of dimethyl amiloride (DMA). Responses from a single IPC are shown for 3 of the levels of CO2 tested under control conditions and after increasing dosages of DMA (as indicated). DMA reduced IPC discharge rate at each CO2 level studied in a dose-dependent manner. PCO2, inspired PCO2. TTL, digital pulses from window discriminator.

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).


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  		Fig. 3.   Average IPC discharge frequencies in response to changes in inspired CO2 before and after intravenous administration of DMA. The effects of both CO2 and DMA on IPC discharge frequency were significant (P < 0.05). Further, a significant interaction between the effects of CO2 and DMA were observed (P < 0.05). Values are means ± SE.


                              
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  		Table 1.   Effects of DMA on logarithmic stimulus-response regression equations

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 PCO2. Figure 4 shows an example of one IPC studied before and after administration of 14 nmol/kg TPA at PCO2 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 PCO2. After administration of TPA, in this example, an increase in IPC discharge at each level of PCO2 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 PCO2.


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  		Fig. 4.   Example of IPC action potential discharge in response to changes in inspired CO2 before and after intravenous administration of 12-O-tetradecanoylphorbol 13-acetate (TPA). Responses from a single IPC are shown for 3 of the levels of CO2 tested under control conditions and after TPA (14 nmol/kg). TPA enhanced IPC discharge rate at each CO2 level studied, with the greatest effects being seen at the lower levels of PCO2.

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 PCO2. 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 (Table 2) 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 PCO2 examined in response to injection of vehicle solution containing DMSO.

                              
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  		Table 2.   Effects of TPA on logarithmic stimulus-response regression equations

Blood-Buffer Curves

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 PCO2 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 PCO2 + intercept, for the curves shown in Fig. 5 are provided in Table 3.


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  		Fig. 5.   Blood-buffer relationship before and after administration of DMA. No significant difference was observed in the relationship between arterial pH and changing arterial PCO2 between control conditions and 30-90 min after administration of DMA (8 µmol/kg). Values are means ± SE. CI, confidence intervals.


                              
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  		Table 3.   Effects of DMA on blood buffer regression equations


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 CO2 hydration/dehydration is balanced by transmembrane acid-base exchange. However, it is clear that the relative rates of CO2 hydration/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 PCO2 values, pHi would probably change little with PCO2. Thus a kinetic mismatch between H+ production and extrusion is required to generate a pHi signal for transduction of the PCO2 stimulus. Our mathematical modeling of this relationship indicates that a CO2 hydration/dehydration rate that is 100- to 1,000-fold faster than the acid-base transmembrane exchange rate gives phasic and tonic IPC responses to PCO2 that closely simulate experimental observations (26).

Modulation of Activity of the NHE

DMA. 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 PCO2. 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 PCO2, 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 PCO2 levels were lowest. Other CO2 chemoreceptor 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 PCO2 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 Delta pH of -0.058, but [H+] increasing from 75 to 80 nM (a relative intracellular acidosis) causes a Delta pH = -0.028. Thus, even if DMA inhibited H+ transport by the same amount during hypercapnia and hypocapnia, the relative effect on pHi would 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.

TPA. 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 of Helix aspersa. For example, traditional chemoreceptors increase excitability as PCO2 increases, but IPC decrease excitability as PCO2 increases (14, 18, 29, 40-42). Traditional chemoreceptors continue to respond to PCO2 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 PCO2 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 CO2 stimuli as demonstrated in the current investigation. DMA, an amiloride analog, caused profound inhibition of IPC CO2 chemotransduction; 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 PCO2 compared with low PCO2 (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 PCO2.

Perspectives

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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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: Steven.Hempleman{at}nau.edu).

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

Received 27 August 2002; accepted in final form 18 February 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   American Physiological Society. Guiding principles for research involving animals and human beings. Am J Physiol Regul Integr Comp Physiol 283: R281-R283, 2002[Free Full Text].

2.   Aronson, PS, Nee J, and Suhm MA. Modifier role of internal H+ in activating the Na+-H+ exchanger in renal microvillus membrane vesicles. Nature 299: 161-163, 1982[Medline].

3.   Bebout, DE, and Hempleman SC. Chronic hypercapnia resets CO2 sensitivity of avian intrapulmonary chemoreceptors. Am J Physiol Regul Integr Comp Physiol 276: R317-R322, 1999[Abstract/Free Full Text].

4.   Berger, PJ, Tallman RD, Jr, and Kunz AL. Discharge of intrapulmonary chemoreceptors and its modulation by rapid FICO2 changes in decerebrate ducks. Respir Physiol 42: 123-130, 1980[ISI][Medline].

5.   Besterman, JM, and Cuatrecasas P. Phorbol esters rapidly stimulate amiloride-sensitive Na+/H+ exchange in a human leukemic cell line. J Cell Biol 99: 340-343, 1984[Abstract/Free Full Text].

6.   Besterman, JM, May WS, Jr, LeVine H, III, Cragoe EJ, Jr, and Cuatrecasas P. Amiloride inhibits phorbol ester-stimulated Na+/H+ exchange and protein kinase C. An amiloride analog selectively inhibits Na+/H+ exchange. J Biol Chem 260: 1155-1159, 1985[Abstract/Free Full Text].

7.   Buckler, KJ, Vaughan-Jones RD, Peers C, and Nye PCG Intracellular pH and its regulation in isolated type I carotid body cells of the neonatal rat. J Physiol 436: 107-129, 1991[Abstract/Free Full Text].

8.   Burger, RE, and Lorenz FW. Artificial respiration in birds by unidirectional gas flow. Poult Sci 39: 236-237, 1960[ISI].

9.   Burger, RE, Osborne JL, and Banzett RB. Intrapulmonary chemoreceptors in Gallus domesticus: adequate stimulus and functional localization. Respir Physiol 22: 87-97, 1974[ISI][Medline].

10.   Coates, EL, Knuth SL, and Bartlett D, Jr. Laryngeal CO2 receptors: influence of systemic PCO2 and carbonic anhydrase inhibition. Respir Physiol 104: 53-61, 1996[ISI][Medline].

11.   Coates, EL, Li AH, and Nattie EE. Acetazolamide on the ventral medulla of the cat increases phrenic output and delays the ventilatory response to CO2. J Physiol 441: 433-451, 1991[Abstract/Free Full Text].

12.   Cuthbert, AW, and Fanelli GM. Effects of some pyrazinecarboxamides on sodium transport in frog skin. Br J Pharmacol 63: 139-149, 1978[ISI][Medline].

13.   Erlichman, JS, Coates EL, and Leiter JC. Carbonic anhydrase and CO2 chemoreception in the pulmonate snail Helix aspersa. Respir Physiol 98: 27-41, 1994[ISI][Medline].

14.   Erlichman, JS, and Leiter JC. Comparative aspects of central CO2 chemoreception. Respir Physiol 110: 177-185, 1997[ISI][Medline].

15.   Fedde, MR, Gatz RN, Slama H, and Scheid P. Intrapulmonary CO2 receptors in the duck. I. Stimulus specificity. Respir Physiol 22: 99-114, 1974[ISI][Medline].

16.   Fedde, MR, and Peterson DF. Intrapulmonary receptor response to changes in airway-gas composition in Gallus domesticus. J Physiol 209: 609-625, 1970[Abstract/Free Full Text].

17.   Filosa, JA, Dean JB, and Putnam RW. Role of intracellular and extracellular pH in the chemosensitive response of rat locus coeruleus neurones. J Physiol 541: 493-509, 2002[Abstract/Free Full Text].

18.   Fitzgerald, RS, Shirahata M, and Lahiri S. Amiloride and carotid body chemoreception of hypercapnia and hypoxia. Respir Physiol 81: 337-347, 1990[ISI][Medline].

19.   Furilla, RA, and Bernstein MH. Intrapulmonary CO2-rise time and ventilation in ducks. J Appl Physiol 79: 1397-1404, 1995[Abstract/Free Full Text].

20.   Geers, C, and Gros G. Carbon dioxide transport and carbonic anhydrase in blood and muscle. Physiol Rev 80: 681-715, 2000[Abstract/Free Full Text].

21.   Green, J, and Muallem S. A common mechanism for activation of the Na+/H+ exchanger by different types of stimuli. FASEB J 3: 2408-2414, 1989[Abstract].

22.   Grinstein, S, Cohen S, Goetz JD, and Rothstein A. Osmotic and phorbol ester-induced activation of Na+/H+ exchange: possible role of protein phosphorylation in lymphocyte volume regulation. J Cell Biol 101: 269-276, 1985[Abstract/Free Full Text].

23.   Grinstein, S, Rotin D, and Mason MJ. Na+/H+ exchange and growth factor-induced cytosolic pH changes. Role in cellular proliferation. Biochim Biophys Acta 988: 73-97, 1989[Medline].

24.   Hempleman, SC, and Bebout DE. Increased venous PCO2 enhances dynamic responses of avian intrapulmonary chemoreceptors. Am J Physiol Regul Integr Comp Physiol 266: R15-R19, 1994[Abstract/Free Full Text].

25.   Hempleman, SC, and Burger RE. Receptive fields of intrapulmonary chemoreceptors in the Pekin duck. Respir Physiol 57: 317-330, 1984[ISI][Medline].

26.   Hempleman, SC, and Posner RG. A mathematical model of the intracellular pH response of intrapulmonary chemoreceptors to CO2. FASEB J 16: A813, 2002.

27.   Hempleman, SC, Rodriguez TA, Bhagat YA, and Begay RS. Benzolamide, acetazolamide, and signal transduction in avian intrapulmonary chemoreceptors. Am J Physiol Regul Integr Comp Physiol 279: R1988-R1995, 2000[Abstract/Free Full Text].

28.   Horie, S, Moe O, Tejedor A, and Alpern RJ. Preincubation in acid medium increases Na/H antiporter activity in cultured renal proximal tubule cells. Proc Natl Acad Sci USA 87: 4742-4745, 1990[Abstract/Free Full Text].

29.   Iturriaga, R, Lahiri S, and Mokashi A. Carbonic anhydrase and chemoreception in the cat carotid body. Am J Physiol Cell Physiol 261: C565-C573, 1991[Abstract/Free Full Text].

30.   Kiwull-Schone, H, Wiemann M, Frede S, Bingmann D, Wirth KJ, Heinelt U, Lang HJ, and Kiwull P. A novel inhibitor of the Na+/H+ exchanger type 3 activates the central respiratory CO2 response and lowers the apneic threshold. Am J Respir Crit Care Med 164: 1303-1311, 2001[Abstract/Free Full Text].

31.   Kleyman, TR, and Cragoe EJ, Jr. Amiloride and its analogs as tools in the study of ion transport. J Membr Biol 105: 1-21, 1988[ISI][Medline].

32.   Lai, CJ, and Kou YR. Inhibitory effect of inhaled wood smoke on the discharge of pulmonary stretch receptors in rats. J Appl Physiol 84: 1138-1143, 1998[Abstract/Free Full Text].

33.   Mahnensmith, RL, and Aronson PS. The plasma membrane sodium-hydrogen exchanger and its role in physiological and pathophysiological processes. Circ Res 56: 773-788, 1985[Abstract/Free Full Text].

34.   Nye, PC, and Burger RE. Chicken intrapulmonary chemoreceptors: discharge at static levels of intrapulmonary carbon dioxide and their location. Respir Physiol 33: 299-322, 1978[ISI][Medline].

35.   Osborne, JL, and Mitchell GS. Regulation of arterial PCO2 during inhalation of CO2 in chickens. Respir Physiol 31: 357-364, 1977[ISI][Medline].

36.   Peterson, DF, and Fedde MR. Receptors sensitive to carbon dioxide in lungs of chicken. Science 162: 1499-1501, 1968[Abstract/Free Full Text].

37.   Powell, F. Respiration. In: Sturkie's Avian Physiology (5th ed.), edited by Whittow G.. San Diego, CA: Academic, 2000.

38.   Powell, FL, Fedde MR, Gratz RK, and Scheid P. Role of avian intrapulmonary chemoreceptors in the ventilatory response to inhaled CO2: effects of acetazolamide in the duck. In: Respiratory Function in Birds, Adult and Embryonic, edited by Piiper J.. Berlin: Springer, 1978.

39.   Powell, FL, Milsom WK, and Mitchell GS. Effects of intrapulmonary CO2 and airway pressure on pulmonary vagal afferent activity in the alligator. Respir Physiol 74: 285-298, 1988[ISI][Medline].

40.   Putnam, RW. Intracellular pH regulation of neurons in chemosensitive and nonchemosensitive areas of brain slices. Respir Physiol 129: 37-56, 2001[ISI][Medline].

41.   Richerson, GB, Wang W, Tiwari J, and Bradley SR. Chemosensitivity of serotonergic neurons in the rostral ventral medulla. Respir Physiol 129: 175-189, 2001[ISI][Medline].

42.   Ritucci, NA, Chambers-Kersh L, Dean JB, and Putnam RW. Intracellular pH regulation in neurons from chemosensitive and nonchemosensitive areas of the medulla. Am J Physiol Regul Integr Comp Physiol 275: R1152-R1163, 1998[Abstract/Free Full Text].

43.   Scheid, P, Gratz RK, Powell FL, and Fedde MR. Ventilation response to CO2 in birds. II. Contribution by intrapulmonary CO2 receptors Respir Physiol 35: 361-372, 1978[ISI][Medline].

44.   Scheid, P, Kuhlmann WD, and Fedde MR. Intrapulmonary receptors in the Tegu lizard. II. Functional characteristics and localization. Respir Physiol 29: 49-62, 1977[ISI][Medline].

45.   Scheid, P, and Piiper J. Control of breathing in birds. In: Handbook of Physiology. The Respiratory System. Bethesda, MD: Am. Physiol. Soc, 1986, p. 815-832.

46.   Schellenberg, GD, Anderson L, and Swanson PD. Inhibition of Na+-Ca2+ exchange in rat brain by amiloride. Mol Pharmacol 24: 251-258, 1983[Abstract].

47.   Shoemaker, JM, and Hempleman SC. Avian intrapulmonary chemoreceptor discharge rate is increased by anion exchange blocker DIDS. Respir Physiol 128: 195-204, 2001[ISI][Medline].

48.   Siffert, W, and Scheid P. A phorbol ester and 1-oleoyl-2-acetylglycerol induce Na+/H+ exchange in human platelets. Biochem Biophys Res Commun 141: 13-19, 1986[ISI][Medline].

49.   Soltoff, SP, and Mandel LJ. Amiloride directly inhibits the Na,K-ATPase activity of rabbit kidney proximal tubules. Science 220: 957-958, 1983[Abstract/Free Full Text].

50.   Tallman, RD, Jr, and Grodins FS. Intrapulmonary CO2 receptor discharge at different levels of venous PCO2. J Appl Physiol 53: 1386-1391, 1982[Abstract/Free Full Text].

51.   Vigne, P, Frelin C, Cragoe EJ, Jr, and Lazdunski M. Structure-activity relationships of amiloride and certain of its analogues in relation to the blockade of the Na+/H+ exchange system. Mol Pharmacol 25: 131-136, 1984[Abstract].

52.   Vivoni, M, Castro MI, Lojo L, and Furilla RA. Airway ammonia negates the normal ventilatory response to airway CO2 in garter snakes. Respir Physiol 127: 13-22, 2001[ISI][Medline].

53.   Wang, W, Bradley SR, and Richerson GB. Quantification of the response of rat medullary raphe neurones to independent changes in pHo and PCO2. J Physiol 540: 951-970, 2002[Abstract/Free Full Text].

54.   Wang, W, Tiwari JK, Bradley SR, Zaykin RV, and Richerson GB. Acidosis-stimulated neurons of the medullary raphe are serotonergic. J Neurophysiol 85: 2224-2235, 2001[Abstract/Free Full Text].

Am J Physiol Regul Integr Comp Physiol 284(6):R1551-R1559
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