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Calcium and avian intrapulmonary chemoreceptor response to CO₂

S. C. Hempleman,¹ S. X. Egan,^1, J. Q. Pilarski,¹ T. P. Adamson,² and I. C. Solomon³

¹Department of Biological Sciences, Northern Arizona University, Flagstaff, Arizona; ²Division of Neurobiology, Physiology and Behavior, University of California at Davis, Davis, California; and ³Department of Physiology and Biophysics, State University of New York at Stony Brook, Stony Brook, New York

Submitted 24 January 2006 ; accepted in final form 31 July 2006

	ABSTRACT

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Intrapulmonary chemoreceptors (IPC) are highly responsive respiratory chemoreceptors that innervate the lungs of birds and diapsid reptiles. IPC are stimulated by low levels of lung PCO₂, inhibited by high levels of lung PCO₂, and their vagal afferents serve as a sensory limb for reflex adjustments of breathing depth and rate. Most IPC exhibit both phasic and tonic sensitivity to CO₂, and spike frequency adaptation (SFA) contributes to their phasic CO₂ responsiveness. To test whether CO₂ responsiveness and SFA in IPC is modulated by a Ca²⁺-linked mechanism, we quantified the role of transmembrane Ca²⁺ fluxes and Ca²⁺-related channels on single-unit IPC function in response to phasic changes in inspired PCO₂. We found that 1) broad-spectrum blockade of Ca²⁺ channels using cadmium or cobalt and blockade of L-type Ca²⁺ channels using nifedipine increased IPC discharge; 2) activation of L-type Ca²⁺ channels using BAY K 8644 reduced IPC discharge; 3) blockade of Ca²⁺-activated potassium channels using charybdotoxin (antagonist of large-conductance Ca²⁺-dependent K⁺ channel) increased IPC discharge, but neither charybdotoxin nor apamin affected SFA; and 4) blockade of chloride channels, including Ca²⁺-activated chloride channels, with niflumic acid decreased IPC discharge at low PCO₂ and increased IPC discharge at high PCO₂, resulting in a net attenuation of the IPC CO₂ response. We conclude that Ca²⁺ influx through L-type Ca²⁺ channels has an inhibitory effect on IPC afferent discharge and CO₂ sensitivity, that spike frequency adaptation is not due to apamin- or charybdotoxin-sensitive Ca²⁺-activated K⁺ channels in IPC, and that chloride channels blocked by niflumic acid help modulate IPC CO₂ responses.

signal transduction; intracellular pH; spike frequency adaptation; lung; vagus

Reflex studies and extracellular single-unit recordings of IPC action potentials indicate that IPC are stimulated by low levels of inspired PCO₂, inhibited by high levels of inspired PCO₂, and are most active at low to normal PCO₂ levels (7, 9, 11). IPC spike discharge responds rapidly to intrapulmonary CO₂ fluctuations (21), producing rapid reflex changes in breathing (4, 23, 28). IPC are phasically active during the breathing cycle (19), and most show spike-frequency adaptation (SFA) in response to step changes in PCO₂ (5, 21). Thus the kinetic responsiveness of IPC spike discharge is well suited for dynamic within-breath monitoring of lung CO₂ (11).

Numerous observations suggest that IPC sense intracellular pH (alkaline excites, acid inhibits), rather than molecular CO₂ directly. Normal IPC function, for example, requires intracellular carbonic anhydrase (12, 25), which catalyzes the reaction CO₂ + H₂O H⁺ + HCO₃^–; thus the actual IPC stimulus appears to be the products of CO₂ hydration. IPC signal transduction involves Na⁺/H⁺ antiports [since blockade with dimethyl amiloride depresses IPC discharge (8)], and HCO₃^–/Cl^– antiports [since blockade with DIDS excites IPC discharge (29)]. These antiport exchangers function in intracellular H⁺ and HCO₃^– balance, respectively. IPC CO₂ response is also dependent on H⁺-sensitive imidazole groups on proteins [since blockade with diethylpyrocarbonate nearly abolishes IPC CO₂ sensitivity (24)]. Finally, chronic hypercapnia resets IPC CO₂ sensitivity to higher levels, but the underlying H⁺ sensitivity remains the same (2). Consistent with these observations are the results of mathematical modeling, which indicate that intracellular pH in IPC at any given PCO₂ probably reflects a dynamic coupling between the rates of CO₂ hydration-dehydration and the rates of transmembrane H⁺ and HCO₃^– transport (11).

As dynamic chemoreceptors, most IPC exhibit significant SFA in response to phasic CO₂ stimuli. SFA enhances the IPC ability to respond to the large and abrupt within-breath PCO₂ changes that occur in the avian lung and to provide phasic feedback for ventilatory control (11). Although the cellular mechanism responsible for the SFA in IPC is unknown, we hypothesized that IPC SFA may be due to feedback inhibition of cellular excitability by Ca²⁺-activated K⁺ (K_Ca) channels, as occurs in many other neurons (13). Since K_Ca channels have been linked to CO₂ chemotransduction in brain stem chemoreceptor neurons and carotid bodies (22, 26, 32), it seemed likely that calcium or calcium-activated ion channels might also be involved in IPC chemotransduction. To test this, we manipulated transmembrane Ca²⁺ fluxes and/or ion channels involved with Ca²⁺ while recording IPC discharge responses to dynamic (i.e., phasic) changes in inspired CO₂. Three series of experiments were conducted, representing the progression of our inquiry from general to specific.

In series I, we quantified the effects of the inorganic, nonspecific calcium channel blockers cadmium chloride (CdCl₂) and cobalt chloride (CoCl₂) on IPC discharge in vivo. We found that cadmium (especially) and cobalt excited IPC, suggesting that Ca²⁺ influx into IPC had inhibitory effects that were reduced when cadmium and cobalt were present. However, because both cadmium and cobalt are nonspecific Ca²⁺ channel blockers, with many pathophysiological and toxicological side effects, these results were suggestive but not definitive evidence for Ca²⁺ involvement in IPC responses. Therefore in series II and III, we used much more specific antagonists and agonists of Ca²⁺-related ion channels to further test the hypothesis.

In series II, we quantified the effects of the specific L-type Ca²⁺ channel blocker nifedipine and the specific L-type Ca²⁺ channel agonist BAY K 8644 on IPC discharge in vivo. L-type Ca²⁺ channels have been found to contribute to CO₂ sensitivity in other respiratory chemoreceptor cells (11, 26). Based on our series I results, we hypothesized that IPC might also possess L-type Ca²⁺ channels, and if so nifedipine should increase IPC excitability and BAY K 8644 should decrease IPC excitability.

In series III, to explore the inhibitory effect of Ca²⁺ influx on IPC CO₂ response, we quantified the effects of the K_Ca channel blockers charybdotoxin [CBTX, antagonist of large-conductance Ca²⁺-dependent K⁺ (BK_Ca) channels] and apamin [antagonist of small-conductance K_Ca (sK_Ca) channels] and the chloride channel blocker niflumic acid [which also antagonizes Ca²⁺-activated Cl^– (Cl_Ca) channels] on IPC discharge in vivo. We hypothesized that, if any of these channels contributed to IPC function, changes in IPC discharge and phasic responses to CO₂ step stimuli should occur with antagonist treatment. For example, if CBTX or apamin reduced SFA in IPC, it would indicate that this adaptation is due to K_Ca channel activation.

	METHODS

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General.   Experiments were 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). Thirty-nine mallard ducks, Anas platyrhynchos, body mass 1.0–1.4 kg, of either sex, were studied. Animals were anesthetized by intravenous infusion of 35–40 mg/kg pentobarbital sodium (dissolved in 10% ethanol), and a polyethylene cannula was implanted in the left brachial vein for administration of supplemental dosages of pentobarbital sodium (3–5 mg/kg iv, as needed to maintain a deep level of surgical anesthesia, i.e., absence of withdrawal response to a strong toe pinch) and experimental drugs. A thermistor probe was inserted into the colon, and body temperature was regulated at 40 ± 1°C using a circulating hot water pad and hot water bottles. Electrocardiogram was monitored using a Grass P511K AC preamplifier coupled to a Grass AM5 audio amplifier and Hitachi analog oscilloscope.

The interclavicular air sac was opened through a skin incision, and a pediatric cuffed endotracheal tube was inserted into the trachea. During subsequent surgery, animals were unidirectionally ventilated with a continuous 1 l/min stream of humidified air supplemented with 3% CO₂, which entered through the endotracheal tube and exited via the interclavicular air sac opening. Gases from tanks were mixed and delivered by a Cameron Instruments GF-1 electronic mass flow controller. Unidirectional ventilation and deep surgical anesthesia precluded any breathing movements.

The left vagus nerve was exposed in the neck, freed from surrounding tissue, and draped over a small plastic platform submerged in light mineral oil. Single-unit recordings were made from fine filaments dissected from the exposed vagus nerve and placed on a 35-gauge monopolar platinum-iridium electrode ("fiber-picking" technique). Electrical activity in the filaments was referenced to an Ag-AgCl electrode on the nerve sheath through a differential high-impedance probe (Grass HIP). Signals were amplified 10,000-fold with a Grass P511K AC preamplifier. IPCs were identified by their nearly immediate response to step changes in ventilatory gas PCO₂ and their inverse sensitivity to PCO₂. Single-unit IPCs were identified by the constant amplitude and shape of their action potential waveforms using a Haer slope/height window discriminator. The window discriminator also generated a digital pulse (5 V, 10 µs) with each occurrence of the discriminated action potential. Interspike intervals and discharge rates were measured by timing the digital pulses from the window discriminator using a microcomputer sampling at 14,500 Hz (timing accuracy ±69 µs). Analog action potential signals were band-pass filtered between 100 and 3,000 Hz, notch-filtered at 60 Hz, visualized on an analog Hitachi oscilloscope, auditioned with a Grass AM-5 audio amplifier, and recorded on a Vetter four-channel VHS tape system using pulse-code modulation.

Measuring IPC response to CO₂.   Experiments were conducted using a repeated-measures design. We first monitored the normal IPC responses to CO₂ during a 15- to 20-min control period to ensure that the IPC discharge was stable (e.g., Fig. 1A). We then administered test drug(s) listed in series I–III below and measured the IPC response again. Vagal filaments were tested for CO₂-linked action potential activity by stepping inspired CO₂ between 0 and 6% with an 11-s cycle period, in a continuous unidirectional air flow of 2 l/min. When an IPC was identified, single-unit spike discharge responses were recorded on tape and computer during 10 complete CO₂-step stimulus cycles (300–2,000 action potentials). Digital timing pulses triggering the analog CO₂ step stimulus were logged simultaneously with times of action potential occurrence by computer (20). These recordings were analyzed to produce cycle-triggered stimulus histograms of IPC discharge responses to the stimulus cycle. Consistency and signal strength of the IPC response to the repetitive stimulus were quantified using a two-way ANOVA (20). IPC with significant (P < 0.05) predrug discharge entrainment to the CO₂ step stimuli were accepted for further analysis (10, 20).

View larger version (11K): [in this window] [in a new window]   Fig. 1. A: time-stable reproducibility of intrapulmonary chemoreceptors (IPC) discharge rate (means ± SE, n = 15 IPC) during repetitive CO2 step stimuli (two measurements made 15–20 min apart for each IPC). Trace with solid symbols (control measurement) shows average discharge rates measured at beginning of the control period. Trace with open symbols (repeat control measurement) shows average discharge rates measured from the same IPC 15–20 min later, but still during control period. Between control and repeat control measurements, animals received intravenous injections of 3–5 mg/kg pentobarbital in 10% ethanol (vol. 0.05 ml), flushed with 1–3 ml of saline, which also did not affect IPC discharge. B: vehicle control experiment showing lack of effect of intravenous infusion of 1 ml of 10% ethanol on IPC discharge response to CO2 step stimuli (n = 2 IPC). Solid symbols, control; open symbols, after ethanol infusion.

Series II: effects of L-type calcium channel antagonist and agonist on IPC discharge.   Nifedipine (N-7634, Sigma RBI, St. Louis, MO) was dissolved in 95% ethanol and diluted 1:100 with normal saline immediately before use. S-(–)-BAY K 8644 (B-133, Sigma RBI) was dissolved in 10% ethanol and then diluted 1:10 with normal saline immediately before use. After quantifying an IPC's normal response to CO₂ steps (see above), either 1 mg/kg nifedipine (n = 7) or 2 mg/kg BAY K 8644 (n = 6) were slowly infused over a period of 1–2 min, via the brachial vein cannula, while IPC discharge and CO₂ stimulus steps were recorded. IPC discharge was allowed to stabilize for 5 min, reaching a new steady state, and then IPC discharge response to CO₂ steps was again measured.

Series III: effects of K_Ca and Cl_Ca channel blockers.   CBTX (C-7802, Sigma RBI) and apamin (A1289, Sigma RBI) were dissolved in water, aliquoted, and frozen before use. Niflumic acid (N-0630, Sigma-RBI) was dissolved in saline immediately before use. After an IPC's normal response to CO₂ steps was quantified (see above), either 1–9 nmol/kg CBTX (n = 5), 100 nmol/kg apamin (n = 5), or 100 µmol/kg niflumic acid (n = 4) were slowly infused over a period of 1–2 min, via the brachial vein cannula, while IPC discharge and CO₂ stimulus steps were recorded. IPC discharge was allowed to stabilize for 5 min, reaching a new steady state, and then IPC discharge response to CO₂ steps was again measured.

Statistical analysis drug effects on cycle triggered stimulus histograms.   IPC were studied before and after drug treatment using a two-way repeated-measures ANOVA. To assess IPC function, each IPC was studied while responding to an 11-s CO₂-stimulus cycle that stepped between high (6%) CO₂ (from 0 to 5.5 s) and low (0%) CO₂ (from 5.5 to 11 s). Time within each stimulus cycle was divided into 43 time bins, each with 0.256-s duration. IPC discharge rate in each time bin was calculated as the total number of action potential occurrences in that time bin (summed over all stimulus cycles), divided by the width of each time bin (0.256 s), divided by the number of stimulus cycles given. These discharge rates produced separate cycle-triggered histograms of discharge frequency for each IPC under control and drug-treated conditions.

Treatment effects of drugs and CO₂ on IPC discharge frequency were quantified using a two-way repeated-measures ANOVA. Main effects were time ("", mapping to the CO₂ stimulus as indicated above), drug ("", control or treatment), and IPC identifier ("", to quantify inter-IPC variation). Interaction terms time * drug (), time * IPC (), and drug * IPC () completed the model. Tests were run in JMP-IN version 4.0 (SAS, Cary, SC) on a PC. The statistical model was:

(1)

where Y_i,j,k is the observed discharge frequency for IPC (k), with drug (j), at time (i); µ... is the grand mean IPC discharge over all IPC, drugs, and times; and _i,j,k is the residual error associated with Y_i,j,k, which was the three-way interaction term ()_i,j,k in this repeated-measures model. Type I error rate of = 0.05 was used for all tests. Post hoc multiple comparisons were made with Tukey's honestly significant difference test in JMP-IN version 4 that controlled for the overall type I error rate ( = 0.05).

Statistical interpretation: modulation of IPC discharge and/or modulation of IPC CO₂ sensitivity.   Two statistical effects on IPC discharge were important in the present analysis: the "main effect of the drug treatment relative to control"; and the "interaction effect" of the drug treatment with CO₂-time relative to control. The "main effect" of the drug measured whether the drug significantly modulates mean IPC discharge rate compared with control (discharge frequency), but it does not specifically quantify whether the drug affects the CO₂ sensitivity of IPC discharge. The "interaction effect" of the drug with CO₂-time specifically measures whether the drug significantly modifies the IPC CO₂ sensitivity (discharge frequency/PCO₂). For example, cadmium treatment was found to be significant as a main effect (see RESULTS), meaning that the mean IPC discharge rate (averaged over all CO₂ stimulus times) was significantly different during cadmium treatment compared with control. (Interpretation: cadmium significantly modulates IPC discharge.) There was also a significant interaction between cadmium and CO₂-time (see RESULTS), which indicates that the magnitude of the cadmium effect on IPC discharge rate was significantly different during different parts of the CO₂-time stimulus cycle (cadmium had a greater stimulatory effect at high CO₂ than at low CO₂, Fig. 2). [Interpretation: cadmium significantly changes IPC CO₂ sensitivity (discharge frequency/PCO₂).]

View larger version (29K): [in this window] [in a new window]   Fig. 2. Series I: cycle-triggered averages of IPC discharge rate (means ± SE) during repetitive CO2 step stimuli. A: before and after infusion of 100 µmol/kg CdCl2 (n = 5). B: before and after infusion of 1 mmol/kg CoCl2 (n = 7). Paired differences (means ± SE) comparing effects of drug infusion with control are shown for cadmium (C) and for cobalt (D).

	RESULTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

Vehicle control: ethanol.   Figure 1B shows that intravenous injection of 1 ml of 10% ethanol in saline (vehicle control) in two different IPC had no effect on discharge response to CO₂ (P > 0.6). This dosage of ethanol was 10-fold greater than the 1 ml of 1% ethanol used as a vehicle for nifedipine and BAY K 8644 in series II. Ten percent ethanol (volume 0.2–0.4 ml) was also used as a vehicle for pentobarbital anesthesia given in Fig. 1A between the control and repeat control measurements, also with no effect on IPC CO₂ response (P > 0.7).

Series I: effects of CdCl₂ and CoCl₂ on IPC discharge.   The overall statistical fits of the repeated-measures ANOVA model (Eq. 1) to CdCl₂ and CoCl₂ data were significant (CdCl₂: P < 0.0001, R² = 0.877; CoCl₂: P < 0.0001, R² = 0.849). Figures 2, 7, and 8 show that both cadmium and cobalt increased the mean IPC discharge averaged over the entire stimulus cycle. The ANOVA model revealed significant main effects of CdCl₂ and CoCl₂ compared with controls (P < 0.0001 for each), indicating that both CdCl₂ and CoCl₂ modulated (increased) mean IPC discharge rate, by 50 and 30%, respectively (see Fig. 7). ANOVA showed significant interaction between CdCl₂ * CO₂-time (P < 0.05), indicating CdCl₂ reduced IPC CO₂ sensitivity by 30% (see Fig. 8). No significant CoCl₂ * CO₂-time interaction was seen (P > 0.9), indicating that CoCl₂ did not significantly alter IPC CO₂ sensitivity.

View larger version (57K): [in this window] [in a new window]   Fig. 3. Effect of cadmium on IPC CO2 response. Three-dimensional plot of action potential discharge rates of one IPC responding to 25 repetitive 11-s cycles of a time-linked CO2 step stimulus. CO2 level was 6% (inhibitory) from 2.5 to 8 s and 0% (excitatory) the rest of the time. Cycles 1–19 show the normal (control) response of this IPC. At arrow mark in cycle 20, CdCl2 (100 µmol/kg) was infused, causing IPC discharge rate to increase and stop responding to the low-CO2 stimulus.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Series II: cycle-triggered averages of IPC discharge rate (means ± SE) during repetitive CO2 step stimuli. A: before and after infusion of 1 mg/kg nifedipine (n = 7). B: before and after infusion of 2 mg/kg BAY K 8644 (n = 6). Paired differences in IPC discharge rate (means ± SE) between drug infusion and control are shown for nifedipine (C) and for BAY K 8644 (D). E: comparison of paired differences for both drugs, which illustrates their nearly symmetrical effects.

View larger version (23K): [in this window] [in a new window]   Fig. 5. Series III: cycle-triggered averages of IPC discharge rate (means ± SE) during repetitive CO2 step stimuli. A: before and after infusion of 9 nmol/kg charybdotoxin (CBTX; n = 5). B: before and after infusion of 100 nmol/kg apamin (n = 5). Paired differences in IPC discharge rate (means ± SE) between drug infusion and control are shown for CBTX (C) and for apamin (D). E: comparison of paired differences for both drugs.

View larger version (17K): [in this window] [in a new window]   Fig. 6. Series III: cycle-triggered averages of IPC discharge rate (means ± SE) during repetitive CO2 step stimuli. A: before and after infusion of 100 µmol/kg niflumic acid (n = 4). B: paired differences in IPC discharge rate (means ± SE) between niflumic acid and control.

View larger version (14K): [in this window] [in a new window]   Fig. 7. Percentage changes in mean (±SE) IPC discharge rates produced by indicated drug treatments relative to control (i.e., "main effect" of drug in ANOVA). Mean discharge rates were calculated as the average discharge rate over the entire CO2 stimulus cycle.

View larger version (13K): [in this window] [in a new window]   Fig. 8. Bars show effect of indicated drug treatments on the magnitudes of CO2-induced IPC discharge oscillations relative to control. Pretreatment controls are represented by solid bars, with normalized discharge rates ranging from 0% (minimum) to 100% (maximum). Drug treatments are represented by shaded bars, with minimum and maximum discharge rates expressed as a percentage of control. Dotted lines trace the changes in minimum, mean, and maximal discharge rates with drug treatment. Nonparallel dotted lines for some drug treatments (e.g., niflumic acid) reflect the interaction between the drug and the CO2 stimulus (i.e., a change in IPC CO2 sensitivity with the drug). Error bars indicate SEs of minima and maxima.

Although both CdCl₂ and CoCl₂ elevated IPC discharge, the effects of these blockers were not identical. With infusion of CoCl₂, the increase in IPC discharge was seen during both the high- and low-CO₂ portions of the CO₂ stimulus cycle (Fig. 2B), in contrast to the effects of CdCl₂, which were only apparent at high CO₂. The difference in IPC discharge frequency between control and CoCl₂ treatment is summarized in Fig. 2D and was smaller than the effect of CdCl₂.

Series II: effects of nifedipine and BAY K 8644 on IPC discharge.   The overall statistical fits of the repeated-measures ANOVA model (Eq. 1) to nifedipine and BAY K 8644 data were significant (nifedipine: P < 0.0001, R² = 0.952; BAY K 8644: P < 0.0001, R² = 0.915). Figures 4, 7, and 8 show that blockade of the L-type Ca²⁺ channels with nifedipine and activation of the L-type Ca²⁺ channels with BAY K 8644 exerted symmetrically opposing effects on IPC discharge. ANOVA indicated significant (P < 0.0001) main effects of nifedipine and BAY K 8644, indicating that both drugs modulated mean IPC discharge rate (nifedipine increased it by 50%, BAY K 8644 decreased it by 30%, see Fig. 7). ANOVA also indicated significant (P < 0.001) interaction between nifedipine * CO₂-time and BAY K 8644 * CO₂-time, indicating that both drugs changed IPC CO₂ sensitivity (nifedipine increased it by 40%, BAY K 8644 decreased it by 50%, see Fig. 8).

IPC discharge was increased at low CO₂ following infusion of nifedipine and decreased at low CO₂ following infusion of BAY K 8644. The average cycle-triggered stimulus histograms of IPC discharge before and after 1 mg/kg nifedipine infusion and before and after 2 mg/kg BAY K 8644 infusion are shown in Fig. 4, A and B. Following infusion of nifedipine, IPC discharge was increased at low CO₂, but remained unchanged at high CO₂ during the CO₂ stimulus cycle. In addition, the relative amount of SFA was unaffected by nifedipine. The paired difference in IPC discharge frequency between control and nifedipine treatment is summarized in Fig. 4, C and E. In contrast to these effects, following infusion of BAY K 8644, IPC discharge was decreased at low CO₂ and slightly elevated at high CO₂ (i.e., BAY K 8644 exerted a slight excitatory effect at high CO₂). BAY K 8644 also decreased SFA relative to control. The difference in IPC discharge frequency between control and BAY K 8644 treatment is summarized in Fig. 4, D and E. For direct comparison, the effects of nifedipine infusion and BAY K 8644 infusion on the paired changes in IPC discharge (drug-control) during the CO₂ stimulus cycle are presented in Fig. 4E, emphasizing the nearly symmetrical opposing effects on IPC discharge. Infusions of nifedipine and BAY K 8644 were well tolerated by the animals, and the drugs produced stable changes in IPC response.

Series III: effects of CBTX, apamin, and niflumic acid on IPC discharge.   The overall statistical fits of the repeated-measures ANOVA model (Eq. 1) to CBTX, apamin, and niflumic acid data were each significant (CBTX: P < 0.0001, R² = 0.983; apamin: P < 0.0001, R² = 0.978; niflumic acid: P < 0.0001, R² = 0.951). Blockade of BK_Ca, sK_Ca, and Cl_Ca channels with CBTX, apamin, and niflumic acid, respectively, differentially affected IPC discharge (Fig. 5, K_Ca; Fig. 6, Cl_Ca; also see Figs. 7 and 8). ANOVA showed significant main effects of CBTX (P < 0.0001) and niflumic acid (P = 0.0003), but no main effect of apamin (P = 0.17). The main effect results indicate that both CBTX and niflumic acid modulated mean IPC discharge rate (an 18% increase with CBTX, and a 10% decrease with niflumate), but apamin did not modulate mean IPC discharge rate. Interaction was seen for apamin * CO₂-time (P = 0.03) and niflumate * CO₂-time (P = 0.0008), indicating that apamin and niflumate changed IPC CO₂ sensitivity (apamin slightly increased it, niflumate decreased it 30%). There was no interaction between CBTX * CO₂-time, indicating that CBTX did not alter IPC CO₂ sensitivity (P = 0.88).

The effect of CBTX was generally excitatory, while that of apamin was nearly imperceptible. Neither CBTX nor apamin significantly reduced SFA in IPC. The average cycle-triggered stimulus histograms of IPC discharge before and after 1–9 nmol/kg CBTX infusion and before and after 100 nmol/kg apamin infusion are shown in Fig. 5, A and B. The difference in IPC discharge frequency between control and CBTX treatment is summarized in Fig. 5, C and E. Also illustrated in Fig. 5E is the direct comparison of the changes in IPC discharge during the CO₂ stimulus cycle observed for CBTX and apamin, emphasizing that CBTX had generally excitatory effects and that apamin's effect was usually indistinguishable from control. Infusions of CBTX and apamin were both well tolerated by the animals.

In contrast to the effects of blockade of BK_Ca and sK_Ca channels, infusion of niflumic acid increased IPC discharge at high CO₂ and decreased it at low CO₂, thereby reducing the overall phasic response of IPC to CO₂. The average cycle-triggered stimulus histograms of IPC discharge before and after 100 µmol/kg niflumic acid infusion is shown in Fig. 6A. The difference in IPC discharge frequency between control and niflumic acid treatment is summarized in Fig. 6B.

Summary data.   The magnitudes of the drug-induced changes in overall mean (±SE) IPC discharge for the different experimental series are summarized in Fig. 7. Figure 8 shows the average (±SE) maximum and minimum IPC discharge rate responses to step CO₂ stimuli to illustrate any interaction of drug treatment with the CO₂ response. Cadmium, cobalt, nifedipine, and BAY K 8644 treatments modulated mean IPC discharge by greater than ±20% (shown by the magnitude of the "main effect" of the drug, Fig. 7). Cadmium, nifedipine, BAY K 8644, and niflumic acid treatments produced greater than ±20% change in IPC CO₂ sensitivity (shown by the magnitude of the "interaction effect" between drug and CO₂ level, Fig. 8).

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
This study shows that influx of calcium through membrane ion channels modulates IPC response to phasic CO₂ stimulation. In series I, IPC excitability increased in the presence of nonspecific Ca²⁺ channel blockers, but series I was complicated by the toxicity and nonspecificity of the heavy metal blockers CdCl₂ and CoCl₂ (see below). Therefore series II experiments further tested this observation using specific, relatively nontoxic dihydropyridine agents targeting L-type Ca²⁺ channels. Series II revealed that Ca²⁺ influx through L-type channels inhibits IPC excitation; mean IPC discharge rate and IPC CO₂ sensitivity were augmented by L-type antagonist nifedipine and diminished by the L-type agonist BAY K 8644. Series III experiments explored the role of Ca²⁺-modulated ion channels in IPC excitability. Neither apamin (an sK_Ca channel blocker) nor CBTX (a BK_Ca channel blocker) influenced SFA in IPC, but CBTX produced a modest excitation of IPC without significantly affecting IPC CO₂ sensitivity. Niflumic acid, a blocker of Cl^– channels, including calcium-modulated Cl^– channels, significantly reduced IPC CO₂ sensitivity.

Taken together, these results reveal the physiological importance of Ca²⁺ ions for modulating IPC discharge and contributing to overall CO₂ sensitivity. Further studies are needed to determine whether Ca²⁺ ions modulate IPC sensory end organs directly or modulate some unidentified synaptic input to IPC.

Critique of systemic drug administration.   Single-unit IPC were studied in intact, anesthetized animals, and IPC discharge responses were measured before and after intravenous injection of various drugs that affect Ca²⁺-related ion channels. Because the drugs were administered systemically, we could not limit their effect to the IPC in the lung. This was especially evident in series I experiments, because cadmium produced cardiovascular collapse 10 min after infusion. We tried to avoid the complicating cardiovascular influences by recording from IPC within a few minutes of cadmium infusion. However, since IPC are sensitive to both ventilation and perfusion of the lung (11), a reduction in cardiac output (with the same level of unidirectional lung ventilation) would be expected to increase the relative importance of ventilation in determining the lung CO₂ tension and the IPC discharge response. For example, when the pulmonary artery is ligated to completely occlude blood flow to a lung, IPC discharge typically becomes higher during low-inspired CO₂ stimuli, and discharge becomes decreased more than usual during high-inspired PCO₂ stimuli (11). This pattern was not seen with cadmium: it increased IPC discharge frequency during low-inspired CO₂ but not high-inspired CO₂, so the effect of cadmium on IPC is not attributable simply to changes in lung perfusion.

Future experiments that include ligation of the pulmonary artery after infusion of calcium channel blockers or agonists might help resolve some of the uncertainty about systemic influences on IPC response. Ligation would isolate the IPC from any circulatory changes.

Critique of drug dosages.   None of these drugs or toxins had been used previously in avian IPC studies, and there were no literature references for IPC-relevant dosages. However, all had been used extensively for in vitro or in vivo studies in various other species (see discussion below), so we used those literature reports to estimate the body fluid concentrations of each drug that should produce blocking (or agonist) effects. Approximate drug dosages for ducks were then calculated on the basis of the animal's body mass, making the simplifying assumption that body mass (in kg) was approximately equal to the volume of drug distribution (in liters). We reasoned this would place trial dosages in the approximate range. We then performed pilot experiments on anesthetized ducks (not shown), giving each drug over approximately a 10-fold concentration range that bracketed average literature reports of effective in vitro and in vivo dosages. In this way, we settled on the dosage that gave a measurable drug effect on IPC discharge and was within reported range of effective values from the literature. We have no specific information on the relative permeability of these drugs to IPC sensory endings, so uncertainty about pharmacologically appropriate dosages still exists.

Series I: effects of inorganic calcium channel blockers on IPC discharge.   The divalent cations cadmium (Cd²⁺) and cobalt (Co²⁺) are typically used at micro- to millimolar concentrations to block Ca²⁺ channels in cell membranes, and usually in vitro rather than in vivo (6, 13, 15, 27, 30). In IPC, 100 µmol/kg CdCl₂ and 1 mmol/kg CoCl₂ increased mean action potential discharge rate by 50 and 30%, respectively (Figs. 2 and 7), indicating that Ca²⁺ channel block increases IPC excitability. These data suggest that, under normal conditions, Ca²⁺ flows through cell membrane channels down their electrochemical gradient and decreases IPC afferent excitability. Since the influx of Ca²⁺, a cation with a positive Nernst equilibrium potential, would by itself cause membrane depolarization, the observed Ca²⁺-dependent inhibition must occur through other means. Two candidate mechanisms we considered were activation of K_Ca or Cl_Ca channels. (series III).

Although both Cd²⁺ and Co²⁺ increased IPC discharge, Cd²⁺ was the stronger stimulant, and it exerted its effects mainly during high CO₂. The stimulatory effect of Co²⁺ was weaker, but persisted during both high CO₂ and low CO₂ (Fig. 1). The reason for the differences between the Cd²⁺ and Co²⁺ stimulation patterns was unclear but may be related to differences in Ca²⁺ channel blocking efficacy, or to other toxic mechanisms unique to the different heavy metal ions. For example, the stimulation pattern caused by Cd²⁺ was similar to the stimulation pattern caused by the carbonic anhydrase inhibitor acetazolamide (12), and we thought perhaps Cd²⁺ might inhibit carbonic anhydrase enzyme in avian IPC, as has been reported in certain tissues of some other animals, like eels (16) and estuarian crabs (31). Since we could not directly assay carbonic anhydrase activity in IPC, we tested for carbonic anhydrase inhibition using an in vitro carbonic anhydrase activity assay on duck blood (17, 24). We found no significant inhibition of avian erythrocyctic carbonic anhydrase by 100 or even 1,000 µmol/l Cd²⁺ (data not shown), so it seemed unlikely (but not impossible) that CdCl₂ affected IPC discharge by impairing carbonic anhydrase.

Critique of series I.   Cadmium and cobalt are known neurotoxins, genotoxins, and carcinogens and, therefore, relatively nonselective Ca²⁺ channel blockers. Although much of the acute neurotoxicity of cadmium and cobalt is related to their ability to interfere with Ca²⁺ channel function, their other toxic effects are numerous. In our animals, we saw stimulation of IPC discharge immediately after injection of Cd²⁺ and Co²⁺, but Cd²⁺ (not Co²⁺) also caused cardiac arrhythmia and death after 10 min. The immediate stimulation of IPC discharge by Cd²⁺ and Co²⁺ suggested that Ca²⁺ channels may be involved in IPC signal transduction, but the general toxicity made the experimental results unclear. It was also unclear whether the different toxicological profiles of Cd²⁺ and Co²⁺ and the severe cardiac depression caused by Cd²⁺ may have contributed to the different types of excitatory responses observed (Fig. 2). Because of these results and associated uncertainties about the specificity of Cd²⁺ and Co²⁺, we thought it was essential to follow up series I using much more specific and relatively nontoxic Ca²⁺ channel modulators: nifedipine and BAY K 8644 (series II).

Comparison to other studies.   There are no previous reports of the effects of Cd²⁺ or Co²⁺ or any other inorganic Ca²⁺ channel blocker on IPC function, but heavy metal ions are common probes for investigating Ca²⁺ channel function in many other chemoreceptors, especially in vitro. For example, treating neurons in rat locus ceruleus with 2 mM cobalt (or 50 µM nifedipine) blocks spiking from dendritic L-type Ca²⁺ channels that may participate in primary CO₂ sensing (6). In carotid body glomus cells, 200 µM Cd²⁺ (30) and >1 mM Co²⁺ (15) essentially abolish Ca²⁺ influx through voltage-gated Ca²⁺ channels (predominantly L-type) during hypoxic or hypercapnic stimulation. Mouse sour-taste chemoreceptors, which depend on Ca²⁺ influx through voltage-gated channels during signal transduction, are completely blocked by 500 µM Cd²⁺ (27). Interestingly, although Cd²⁺ blockade of calcium channels inhibits these other acid chemoreceptors, Cd²⁺ blockade excited IPC. We think this difference may be related to the inverse acid/CO₂ sensitivity of IPC (hypocapnia excites, hypercapnia inhibits) relative to the other chemoreceptors.

Series II: effects of L-type calcium channel antagonist and agonist on IPC discharge.   Series I results suggested that Ca²⁺ channel block may increase IPC discharge, but the inorganic blockers used were not very specific. Therefore, we investigated the effects of more specific organic probes of Ca²⁺ channels: the dihydropyridine L-type channel antagonist nifedipine and the L-type agonist BAY K 8644. In these experiments, nifedipine excited IPC discharge, as did Cd²⁺ and Co²⁺. Conversely, the Ca²⁺ channel opener BAY K 8644 caused a decrease in IPC excitability of approximately the same absolute magnitude as the increase in IPC excitability caused by the blocker nifedipine. This symmetrical action (Fig. 4E) suggests that L-type Ca²⁺ channels may be important modulators of IPC function. Interestingly, in rats, treatment of CO₂-sensitive locus ceruleus neurons with nifedipine inhibits dendritic Ca²⁺ spiking and chemoreceptor response to CO₂ (6). The reverse effect of nifedipine in IPC may reflect their opposite chemosensitivity (where hypocapnia excites, hypercapnia inhibits) compared with locus ceruleus neurons (where hypocapnia inhibits, hypercapnia excites).

The excitation of IPC discharge produced by nifedipine was quantitatively similar to that elicited by Cd²⁺ and Co²⁺ (30–50% increase in overall mean discharge, Fig. 7), suggesting that most of the Ca²⁺ influx in IPC that can be blocked by Cd²⁺ or Co²⁺ may be carried through L-type Ca²⁺ channels. Other voltage-gated Ca²⁺ channel types in IPC, however, have yet to be investigated, including fast-inactivating Ca²⁺ channels like P/Q-, N-, R-, and T-types. In carotid body glomus cells, dihydropyridine-sensitive L-type Ca²⁺ channels carry most of the transmembrane Ca²⁺ current, but other voltage-gated Ca²⁺ channels are also expressed that help shape chemoreceptor response (15, 26).

Critique of series II.   Nifedipine and BAY K 8644 were administered intravenously in 1% ethanol. Vehicle controls (Fig. 1B) showed that ethanol had no significant effect on IPC discharge. There is also considerable evidence in the literature that pentobarbital, which is the most commonly used anesthetic for IPC studies, and which is injected in a 10% ethanol vehicle, has no direct effects on IPC discharge (Fig. 1A and Ref. 11). In addition, the effects of nifedipine and BAY K 8644 on IPC discharge were opposite each other, even though both used the same dilute ethanol vehicle, supporting the interpretation that effects of these drugs were not due to the ethanol vehicle.

Series III: effects of K_Ca and Cl_Ca channel blockers.   Since the results of series I and II supported a role for Ca²⁺-dependent inhibition in IPC discharge, an effect that could be explained by calcium-linked activation of K_Ca or Cl_Ca channels, we investigated the function of BK_Ca, sK_Ca, and Cl_Ca channels in IPC discharge.

Blockade of BK_Ca channels by CBTX enhanced IPC response to the CO₂ step stimulus. This is consistent with the idea that IPC excitation normally involves Ca²⁺ influx through voltage-gated Ca²⁺ channels, which activates BK_Ca channels to provide a negative feedback limit on excitation (13). In some vertebrate CO₂ chemoreceptors, including carotid bodies (22) and cultured medullary chemoreceptors (32), K_Ca channels are inhibited by acid pH, and these channels have been implicated in CO₂ chemosensitivity. However, our IPC data are more consistent with the BK_Ca channels modulating mean IPC discharge and protecting IPC from overexcitation, rather than being the primary site of CO₂ chemosensitivity. CBTX did not significantly affect CO₂ sensitivity (i.e., the CBTX * CO₂-time interaction was nonsignificant).

In contrast to the excitatory effect of blockade of BK_Ca channels, blockade of sK_Ca channels by apamin had no significant effect on mean IPC discharge rate, and a very small effect on IPC CO₂ sensitivity, mostly at low CO₂. In designing this experiment, we thought that SFA in IPC, which in many neurons is dependent on sK_Ca channels (13), might be reduced or abolished by apamin. However, if anything, we saw slightly increased SFA with apamin, especially in those IPC that had little adaptation to begin with (Fig. 5, B, D, and E). Included within the mean responses of the five apamin-treated IPC shown in Fig. 5 were two IPC that showed 30–50% spike adaptation, and apamin also slightly increased adaptation of these IPC. We concluded that apamin-sensitive sK_Ca channels do not contribute to IPC SFA. Other (apamin-insensitive) sK_Ca channels (3), however, might contribute to IPC SFA; this requires further study.

Niflumic acid, a nonspecific blocker of chloride channels including Cl_Ca channels, reduced the amplitude of IPC discharge during the CO₂ step stimulus (Fig. 6). Niflumic acid inhibited IPC discharge at low CO₂ when IPC were discharging most rapidly, and excited IPC at high CO₂ when IPC were discharging more slowly. This brought IPC discharge closer to a median level during the CO₂ stimulus cycle and reduced the extremes in IPC discharge rate (Figs. 6 and 8).

Significant interaction observed between niflumic acid and CO₂ suggest that a Cl^– current blocked by niflumic acid contributes to overall IPC CO₂ sensitivity. The niflumate-sensitive Cl^– current appears to excite IPC during low CO₂ and inhibit IPC during high CO₂, but these results do not reveal the underlying mechanism. Two hypotheses seem testable for the future: 1) IPC may have H⁺-modulated Cl^– channels that are opened by acid (reducing excitability) and closed by alkalinity (increasing excitability), such that blockade of H⁺-modulated Cl^– channels by niflumic acid should reduce the amplitude of IPC response to CO₂; and 2) blockade of Cl^– channels by niflumic acid may reduce IPC response to CO₂ by slowing the chloride shift through Cl^– channels in the IPC cell membrane. IPC chemotransduction is known to be critically dependent on the carbonic anhydrase-catalyzed hydration-dehydration reaction: CO₂ + H₂O H⁺ + HCO₃^–. Slowing the Cl^–-HCO₃^– shift may slow this critical CO₂ hydration-dehydration reaction. For example, during high CO₂, rapid intracellular CO₂ hydration would produce H⁺ and HCO₃^–, and accumulating HCO₃^– would likely move out of the cell while extracellular Cl^– moves into the cell. During low CO₂, intracellular HCO₃^– would be rapidly converted to CO₂, and extracellular HCO₃^– would likely move into the cell in exchange for intracellular Cl^– moving out of the cell. In either case, blocking Cl^– channels and impairing the chloride shift might, by the law of mass action, slow the CO₂ hydration-dehydration reaction by causing an intracellular deficit or excess of HCO₃^–.

Critique of series III.   Repeated-measures ANOVA allows statistical detection of relatively small treatment-induced changes in IPC discharge, if the changes are consistent among IPC, just as a paired t-test offers greater detection power than an unpaired t-test. However, to determine biological significance, it is also important to consider the magnitude of the observed responses and how the responses may contribute to IPC function (Figs. 7 and 8). The IPC responses to apamin were small and relatively insignificant from a biological perspective; however, because apamin did not affect SFA, the hypothesis that sK_Ca channels underlie IPC SFA was tested and seems unlikely. The effects of CBTX and niflumic acid on IPC discharge were much larger than the apamin effects, which indicated that their biological roles in IPC excitability are probably more important. The results suggest that BK_Ca channels provide feedback inhibition of cellular excitability that may function to prevent IPC overexcitation at low PCO₂, while niflumic acid-sensitive Cl^– channels in IPC seem to contribute to CO₂ sensitivity.

Summary.   Our experiments show that transmembrane calcium influx through nifedipine and BAY K 8644-sensitive L-type Ca²⁺ channels inhibits IPC discharge, but has no effect on IPC SFA. Our experiments suggest that Ca²⁺-linked modulation of IPC discharge arises from several sources, including CBTX-sensitive K_Ca and niflumic acid-sensitive Cl^– channels, and most likely other Ca²⁺-sensitive sources that remain to be investigated. Recent reviews of mammalian central and peripheral chemoreceptors (other than IPC) also suggest a modulatory role for calcium in CO₂ chemotransduction, which is consistent with the idea that multiple intracellular sites contribute to overall CO₂ sensitivity in respiratory chemoreceptors (14, 26). Although the modulatory effects of Ca²⁺ presented here for IPC are, in many cases, opposite those seen in carotid bodies and some central chemoreceptors, it is very likely that these differences reflect the evolutionary adaptation of IPC to their function in the avian lung, i.e., their marked inverse CO₂ sensitivity (hypocapnia excites, hypercapnia inhibits) and their extremely rapid phasic responsiveness (11, 18).

	GRANTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Support for this work was provided by National Science Foundation Grant no. IBN-0217815 to S. C. Hempleman.

	ACKNOWLEDGMENTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
The authors thank Leslie Hempleman and Monica Campa for technical assistance, and Robert Bina and Dr. Del Kilgore for helpful discussions. Nifedipine and BAY K 8644 experiments were performed by Sterling X. Egan in partial fulfillment of the requirements for the Master of Science degree in Biology at Northern Arizona University.

	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.

Deceased.

	REFERENCES

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS 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. 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]
3. Bond CT, Maylie J, and Adelman JP. Small-conductance calcium-activated potassium channels. Ann NY Acad Sci 868: 370–378, 1999.[Abstract/Free Full Text]
4. 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]
5. Fedde MR. Intrapulmonary CO₂-rise time and ventilation in ducks. J Appl Physiol 79: 1395–1396, 1995.[Free Full Text]
6. Filosa JA and Putnam RW. Multiple targets of chemosensitive signaling in locus coeruleus neurons: role of K⁺ and Ca²⁺ channels. Am J Physiol Cell Physiol 284: C145–C155, 2003.[Abstract/Free Full Text]
7. Furilla RA and Bernstein MH. Intrapulmonary CO₂ rise time and ventilation in ducks. J Appl Physiol 79: 1397–1404, 1995.[Abstract/Free Full Text]
8. Hempleman SC, Adamson TP, Begay RS, and Solomon IC. CO₂ transduction in avian intrapulmonary chemoreceptors is critically dependent on transmembrane Na⁺/H⁺ exchange. Am J Physiol Regul Integr Comp Physiol 284: R1551–R1559, 2003.[Abstract/Free Full Text]
9. 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]
10. Hempleman SC, Powell FL, and Prisk GK. Avian arterial chemoreceptor responses to steps of CO₂ and O₂. Respir Physiol 90: 325–340, 1992.[CrossRef][ISI][Medline]
11. Hempleman SC and Posner RG. CO₂ transduction mechanisms in avian intrapulmonary chemoreceptors: experiments and models. Respir Physiol Neurobiol 144: 203–214, 2004.[CrossRef][ISI][Medline]
12. Hempleman SC, Rodriguez TA, Bhagat Y, 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]
13. Hille B. Ionic Channels of Excitable Membranes. Sunderland, MA: Sinauer, 1992.
14. Jiang C, Rojas A, Wang R, and Wang X. CO₂ central chemosensitivity: why are there so many sensing molecules? Respir Physiol Neurobiol 145: 115–126, 2005.[CrossRef][ISI][Medline]
15. Jiang RG and Eyzaguirre C. Calcium channels of cultured rat glomus cells in normoxia and acute hypoxia. Brain Res 1031: 56–66, 2005.[CrossRef][ISI][Medline]
16. Lionetto MG, Giordano ME, Vilella S, and Schettino T. Inhibition of eel enzymatic activities by cadmium. Aquat Toxicol (Amst) 48: 561–571, 2000.[CrossRef]
17. Maren TH. Direct measurements of the rate constants of sulfonamides with carbonic anhydrase. Mol Pharmacol 41: 419–426, 1992.[Abstract]
18. Milsom WK, Abe AS, Andrade DV, and Tattersall GJ. Evolutionary trends in airway CO₂/H⁺ chemoreception. Respir Physiol Neurobiol 144: 191–202, 2004.[CrossRef][ISI][Medline]
19. Molony V. Classification of vagal afferents firing in phase with breathing in Gallus domesticus. Respir Physiol 22: 57–76, 1974.[CrossRef][ISI][Medline]
20. Orem J and Dick T. Consistency and signal strength of respiratory neuronal activity. J Neurophysiol 50: 1098–1107, 1983.[Abstract/Free Full Text]
21. Osborne JL, Burger RE, and Stoll PJ. Dynamic responses of CO₂-sensitive avian intrapulmonary chemoreceptors. Am J Physiol Regul Integr Comp Physiol 233: R15–R22, 1977.[Abstract/Free Full Text]
22. Peers C. Effect of lowered pH on Ca²⁺-dependent K⁺ currents in type I cells from the neonatal rat carotid body. J Physiol 422: 381–395, 1990.[Abstract/Free Full Text]
23. Peterson DF and Fedde MR. Receptors sensitive to carbon dioxide in lungs of chicken. Science 162: 1499–1501, 1968.[Abstract/Free Full Text]
24. Pilarski JQ and Hempleman SC. Imidazole binding reagent diethyl pyrocarbonate (DEPC) inhibits avian intrapulmonary chemoreceptor discharge in vivo. Respir Physiol Neurobiol 150: 144–154, 2006.[CrossRef][ISI][Medline]
25. 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-Verlag, 1978.
26. Putnam RW, Filosa JA, and Ritucci NA. Cellular mechanisms involved in CO₂ and acid signaling in chemosensitive neurons. Am J Physiol Cell Physiol 287: C1493–C1526, 2004.[Abstract/Free Full Text]
27. Richter TA, Caicedo A, and Roper SD. Sour taste stimuli evoke Ca⁺⁺ and pH responses in mouse taste cells. J Physiol 547: 475–483, 2003.[Abstract/Free Full Text]
28. Scheid P and Piiper J. Control of breathing in birds. In: Handbook of Physiology. The Respiratory System. Control of Breathing. Bethesda, MD: Am. Physiol. Soc, 1986, sect. 3, vol. II, pt. 2, chapt. 26, p. 815–832.
29. Shoemaker JS and Hempleman SC. Avian intrapulmonary chemoreceptor discharge rate is increased by anion exchange blocker "DIDS". Respir Physiol 128: 195–204, 2001.[CrossRef][ISI][Medline]
30. Urena J, Fernandez-Chacon R, Benot AR, Alvarez de Toledo GA, and Lopez-Barneo J. Hypoxia induces voltage-dependent Ca⁺⁺ entry and quantal dopamine secretion in carotid body glomus cells. Proc Natl Acad Sci USA 91: 10208–10211, 1994.[Abstract/Free Full Text]
31. Vitale AM, Monserrat JM, Castilho P, and Rodriguez EM. Inhibitory effects of cadmium on carbonic anhydrase activity and ionic regulation of the estuarine crab Chasmagnathus granulata (Decapoda, Grapsidae). Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 122: 121–129, 1999.[CrossRef][Medline]
32. Wellner-Kienitz MC, Shams H, and Scheid P. Contribution of Ca⁺⁺-activated K⁺ channels to central chemosensitivity in cultivated neurons of fetal rat medulla. J Neurophysiol 79: 2885–2894, 1998.[Abstract/Free Full Text]

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