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. 2002 Jul 26;543(Pt 3):933–945. doi: 10.1113/jphysiol.2001.015750

A Possible Dual Site of Action for Carbon Monoxide-Mediated Chemoexcitation in the Rat Carotid Body

C Barbé *, F Al-Hashem *, A F Conway *, E Dubuis *, C Vandier *, P Kumar *
PMCID: PMC2290549  PMID: 12231649

Abstract

High tensions of carbon monoxide (CO), relative to oxygen, were used as a tool to investigate the mechanism of chemotransduction. In an in vitro whole organ, rat carotid body preparation, CO increased sinus nerve chemoafferent discharge in the dark, an effect that was significantly reduced (by ca 70 %) by bright white light and by the removal of extracellular Ca2+ from the superfusate or by the addition of either Ni2+ (2 mM) or methoxyverapamil (100 μM). Addition of the P2 purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (50 μM) also significantly reduced the neural response to CO. In perforated patch, whole-cell recordings of isolated rat type I cells, CO induced a depolarisation of ca 11 mV and a decrease in the amplitude of an outward current around and above the resting membrane potential. Membrane conductance between -50 and -60 mV was significantly reduced by ca 40 % by CO. These effects were not photolabile and were present also when a ‘blocking solution’ containing TEA, 4-AP, Ni2+ and zero extracellular Ca2+ was used. In conventional whole-cell recordings, CO only decreased current amplitudes above +10 mV and was without effect around the resting membrane potential. These data demonstrate a direct effect of CO upon type I cell K+ conductances and strongly suggest an effect upon a background, leak conductance that requires an intracellular mediator. The photolabile effect of CO only upon afferent neural discharge adds further evidence to a dual site of action of CO with a separate action at the afferent nerve terminal that, additionally, requires the permissive action of the neurotransmitter ATP.

Plasma membrane K+ channel conductances in type I cells of the carotid body are decreased during periods of hypoxia and hypercapnia, which can lead to cell depolarisation, voltage-gated Ca2+ entry and neurosecretion (Peers, 1997) with the postsynaptic neural discharge generated in the carotid sinus nerve providing the graded, afferent information required for cardiovascular and respiratory homeostasis (Gonzalez et al. 1994). The mechanism by which the cell senses these chemical stimuli, however, remains unresolved. A primary transducer role has been attributed to specialised, hypoxia-sensitive, plasma K+ channels (Lopez-Barneo et al. 2001), with signalling to the channel occurring perhaps through a redox-sensitive, intracellular mechanism that might not be NADPH oxidase (He et al. 2002) but may be some other associated haem protein (Riesco-Fagundo et al. 2001). This role for ion channels has, however, been questioned (Donnelly, 1997; Prabhakar & Overholt, 2000) and chemotransduction via cytochromes associated with mitochondrial respiration thus remains an attractive alternative hypothesis (Prabhakar & Overholt, 2000). Not only have inhibitors of mitochondrial oxidative phosphorylation long been known to act as powerful carotid body chemostimulants (Anichkov & Belen'kii, 1963; Mulligan et al. 1981) but less intense stimulation with graded hypoxia also reveals a unique high sensitivity to PO2 in carotid body mitochondria (Duchen & Biscoe, 1992a, b). In addition, a causal link in the type I cell between mitochondrial uncouplers (Buckler & Vaughan-Jones, 1998) and inhibitors (Wyatt & Buckler, 2000) and the inhibition of a background, leak K+ conductance has been described, with an intracellular signal mediating the interaction between mitochondrion and cell membrane. This leak current, which lacks intrinsic voltage sensitivity or time dependence and is not inhibited by TEA or 4-AP, is activated at the resting membrane potential of type I cells and is also inhibited by hypoxia (Buckler, 1997).

Another possibility has arisen from studies utilising the mitochondrial inhibitor carbon monoxide (CO), which at high partial pressures relative to oxygen, decreases carotid body O2 consumption and acts like hypoxia to increase carotid body chemoafferent neural discharge (Joels & Neil, 1962, 1968) and in a photolabile manner through an action initially presumed to be upon mitochondrial cytochrome a3 (Mills & Jobsis, 1972; Wilson et al. 1994; Lahiri et al. 1995). When coupled, however, with the finding that CO-induced dopamine release from type I cells was not photolabile (Buerk et al. 1997), this led to a recent conjecture that CO might have a non-specific action in type I cell mitochondria but a specific, CO-haem-mediated effect upon ion channel conductances in the postsynaptic nerve terminal of the carotid sinus nerve (Lahiri & Acker, 1999; Lahiri et al. 1999). The possibility, however, that there may also be a photolabile, inhibitory effect of high tensions of CO upon K+ currents in rat carotid body type I cells had not been tested directly and was one of the major objectives of the present study. In addition, the role of neurotransmitters, other than dopamine, upon CO-mediated chemodischarge had not been determined. Of these possible other transmitters, the purinoceptor agonist, ATP, seemed worthy of further examination, given the demonstrated ability of ATP-receptor antagonists to block hypoxia and CO2 sensitivity in the carotid body (Zhang et al. 2000; Al-Hashem et al. 2001).

Some of this work has been published previously in abstract form (Al-Hashem et al. 2000; Barbe et al. 2001).

METHODS

Experiments were performed in accordance with the UK Animals (Scientific Procedures) Act 1986. Adult rats (100–130 g) were anaesthetised with halothane (3–4 % in oxygen for induction and maintained at 1.5–2 %). Left and right carotid bifurcations were removed as described previously (Pepper et al. 1995), after gravity-perfusion of warmed, gassed (95 % O2-5 % CO2) bicarbonate-buffered saline through the left ventricle at a hydrostatic pressure of ca 80 mmHg, to remove blood from the carotid bodies. Animals were killed by halothane overdose and decapitation. Excised bifurcations were prepared either for single chemoafferent fibre recording from intact organ preparations or for patch-clamp recording of isolated type I cells, as described below.

Single chemoafferent fibre recording

In vitro. preparation

Each carotid bifurcation was pinned on Sylgard (Dow Corning) in a small volume (≈0.2 ml) tissue bath. A gassed (95 % O2-5 % CO2), bicarbonate-buffered saline solution (composition (mm): 125 NaCl, 3 KCl, 1.25 NaH2PO4, 5 Na2SO4, 1.3 MgSO4, 24 NaHCO2, 2.4 CaCl2, 10 glucose, pH ≈7.38) was superfused at 3 ml min−1 into the tissue bath. The temperature was continually monitored in the superfusion line immediately before entering the bath by a thermocouple (871A, Tegam Inc., Madison, OH, USA) and maintained at 36–37 °C. The excess connective tissue around the bifurcation was removed and the carotid body and its attached sinus nerve identified before sectioning the nerve at its junction with the glossopharyngeal nerve. The preparation was partially digested in a gassed enzyme solution (0.06 % collagenase (Sigma Type II), 0.02 % protease (Sigma Type IX)) for 20 min at 37 °C to facilitate the recording of neuronal activity.

Data acquisition and analysis

Extracellular recordings of afferent single fibre activity were made from the cut end of the carotid sinus nerve using glass suction electrodes and standard Neurolog modules (Digitimer Ltd, Welwyn Garden City, Hertfordshire, UK). Afferent spike activity was monitored on an oscilloscope (Gould 1604) and recorded throughout each experiment on a VHS video recorder via a DC-modified PCM digital unit (Applegarth Electronics). Chemoreceptor discharge was discriminated as activity exceeding a level which was at least 50 % of the amplitude of the baseline noise above the noise and which responded to a decrease in superfusate PO2 with a reversible increase in discharge frequency. Single fibres were discriminated on the basis of amplitude and shape. Action potentials were sampled digitally after conversion to TTL pulses via a window discriminator (Neurolog NL200) by a computer (Macintosh IIci with NB-MIO-16 DA and NB-MOI-8G DMA cards; National Instruments Co., Austin, TX, USA) running customised LabVIEW 2 (National Instruments Co.) software. TTL pulses were counted and binned into predetermined time periods.

Gas tensions and drugs

Neuronal activity was recorded under steady-state conditions of normoxic normocapnia (160 mmHg PO2; 40 mmHg PCO2, balance N2) produced by precision flow valves (Cole-Parmer Instrument Company) and calibrated against a blood gas analyser (IL 1640, Instrumentation Laboratory). Gaseous CO was added to the superfusate when required at 320 mmHg by substitution for N2.

For Ca2+-free solutions, CaCl2 was removed and 4 mm MgSO4 and 1 mm EGTA added. Methoxyverapamil (D600, 100 μM), nickel chloride (Ni2+, 2 mm) and pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS; 50 μM) were added to the saline solution as required. All drugs were obtained from Sigma except PPADS, which was supplied by Tocris.

Patch-clamp recording of isolated type I cells

Cell isolation

Carotid bodies were removed from excised bifurcations and incubated for 10 min in magnesium-free, low-Ca2+ (50 μM), bicarbonate-buffered saline solution. Collagenase (0.39 mg ml−1, Type I, Worthington) and trypsin (0.2 mg ml−1, Sigma) were then added and the solution held at 37 °C for 23 min. The enzyme solution was removed and replaced by Ham's F-12 culture medium (Sigma) supplemented with 10 % fetal bovine serum, 100 i.u. ml−1 penicillin, 100 μg ml−1 streptomycin, 80 unit l−1 insulin and 2 mm glutamine. Following trituration with fire-polished Pasteur pipettes, dispersed cells were plated onto glass coverslips and kept in a humidified incubator (5 % CO2-95 % air) at 37 °C. Cells were maintained in culture medium for 2–16 h until use.

Solutions and drugs

The standard bicarbonate-buffered extracellular saline solution contained (mm): 117 NaCl, 4.5 KCl, 23 NaHCO3, 1 MgCl2, 2.5 CaCl2 and 11 glucose and was equilibrated with 5 % CO2-95 % air (pH ≈7.4). A low external Ca2+ solution with channel blockers was made and contained (mm): 102 NaCl, 4.5 KCl, 23 NaHCO3, 1 MgCl2, 11 glucose, 2.5 NiCl2, 10 TEA and 5 4-AP. For high external K+ solutions, the same components were used but with 20 mm KCl and 86.5 mm NaCl. Solutions were equilibrated with 5 % CO2-95 % air and pH was readjusted to ≈7.4 with HCl. The NaCl concentration was adjusted to avoid osmotic effects.

The pipette filling intracellular solution used for perforated patch whole-cell recording contained (mm): 55 K2SO4, 30 KCl, 5 MgCl2, 1 EGTA, 10 glucose, 20 Hepes (pH 7.3 adjusted with NaOH). Perforated patch filling solutions also contained amphotericin B (240–360 μg ml−1). The conventional whole-cell filling solution contained (mm): 6 NaCl, 107 KCl, 2 Na2ATP, 11 Hepes, 2 EGTA, 2 MgCl2, 2 CaCl2 (pH 7.2 adjusted with KOH).

Extracellular solutions were superfused at ≈3 ml min−1 into a 200 μl bath chamber. The total change of bath solutions occurred with 800 μl of perfused solution (i.e. 4 × bath chamber solution) and the dead space was 500 μl. Thus, the new solution equilibrium was reached ≈30 s after switching to the new solution. All experiments were performed at 33–35 °C. The superfusate was equilibrated with 60 mmHg PO2 and 40 mmHg PCO2 in control conditions and CO superfusion was induced by addition of 180 mmHg PCO to the perfusate as described above, by substitution for N2.

Electrophysiology

Experiments were performed on isolated type I cells. All current-clamp experiments were performed using the perforated patch whole-cell recording technique and voltage-clamp experiments were performed using either the perforated patch or the conventional whole-cell recording technique. Electrodes (8–15 MΩ for perforated patch whole-cell recording and 5–7 MΩ for conventional patch whole-cell recording) were made using Clarke GC150F-10 borosilicate glass capillaries (Clarke Electromedical, Reading, UK) and were fire-polished before used. The head stage ground was connected to a Ag-AgCl pellet that was placed in a side bath filled with the pipette solution, connected to the main bath via an agar bridge containing 3 m KCl. The junction potentials between the electrode and the bath were cancelled using the voltage pipette offset control of the amplifier. Electrode capacitances were electronically compensated (≈4 pF). Cell capacitance was calculated by integration of the current obtained during pulses between -60 and -70 mV, divided by the pulse amplitude. Dividing by cell capacitance normalised current amplitudes.

Data acquisition and analysis

Experiments were conducted using an Axopatch-1D (Axon Instruments Inc.) amplifier and currents were filtered with a Bessel low-pass filter. Records were simultaneously displayed on an oscilloscope (Gould 1602), digitised with a Digidata-1200 A/D converter (Axon Instruments Inc.) and stored on disk in a PC computer using the Clampex (for voltage-clamp experiments) and Fetchex (for current-clamp experiments) routines of pCLAMP version 6.02 software (Axon Instruments Inc.).

For perforated patch whole-cell recording the pipette tips were filled by dipping the tip of the pipette into the amphotericin B-free filling solution, before backfilling with amphotericin B-containing solution. After the gigaseal between the pipette and the cell was achieved, the electrical access to the cytoplasm was monitored by applying 10 mV pulses for 10 ms from a holding potential of -70 mV and monitoring the capacitive transient. The current was filtered at 5 kHz and sampled at 50 kHz. Typically, access was gained within 5 min and once access was stable the experiments started. We only recorded perforated patch whole-cell recordings in cells with membrane potentials more negative than -40 mV and with holding currents less than 10 pA at -70 mV. In current-clamp experiments we only recorded cells that responded to acidosis.

Cells were voltage clamped at -70 mV and currents determined from a repetitive voltage ramp protocol between -90 and -30 mV (0.3 mV ms−1 every 6 s). The resultant current records from each cell were averaged (five ramps). Currents were sampled at 1 kHz and filtered at 500 Hz. Membrane conductance was determined by fitting a linear regression to the current-voltage relationship over the range -60 to -50 mV. Using this protocol, quinidine (1 mm) caused a 100 % decrease in the membrane conductance measured between -60 and -50 mV (data not shown), as previously reported (Wyatt & Buckler, 2000; Buckler et al. 2000). Global currents were measured during a 10 mV increment pulse protocol between -90 and +30 mV (average of the last 20 ms of 300 ms duration pulse every 6 s) from a holding potential of -70 mV. Currents were sampled at 5 kHz and filtered at 1 kHz. Sensitive currents were determined by subtracting the current density amplitudes obtained in the presence of CO from those obtained under control conditions.

Analysis of the patch-clamp data was performed using either Clampfit or Fetchan (pCLAMP version 6.02) and Origin 4.1 software (Microcal Inc., Northampton, MA, USA). Data are expressed as means ± s.e.m. (n is the number of observations) and were tested for significant differences (P < 0.05) with regression analysis, paired t tests or one-factor ANOVA with post hoc tests, as appropriate, using Statview II (Abacus Concepts, Berkeley, CA, USA) software.

RESULTS

Ca2+ and light dependence of CO in the in vitro carotid body

Chemoreceptor discharge was recorded from 16 single fibre chemoafferents from adult carotid bodies. In all cases, addition of high CO in the absence of light increased chemodischarge approximately tenfold, from a control value of 0.64 ± 0.15 Hz to a steady level of 6.44 ± 1.75 Hz (P < 0.005). This effect of CO was reversibly photolabile, being reduced immediately and markedly by the presence of cold white light to 34.2 ± 3.5 % (n = 16, P < 0.05) of its maximum value in the dark. The effect of CO was dependent upon the presence of extracellular Ca2+ (Fig. 1). Thus, in the absence of external Ca2+, control discharge was reduced to below control levels and was then subsequently unaffected by CO (0.08 ± 0.04 and 0.09 ± 0.05 Hz, respectively; P > 0.20; n = 5). Discharge responses were fully reversible upon return to normal perfusate Ca2+ levels.

Figure 1. Carbon monoxide sensitivity is light- and extracellular [Ca2+] dependent.

Figure 1

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Single fibre chemoreceptor discharge was recorded and binned into 10 s periods. CO (320 mmHg) was delivered as shown (horizontal bars), initially in the presence of bright white light. After a period of at least 2 min, the light was switched off (light off; horizontal thick bars) for a brief period. On the left can be seen the normal, brisk and intense control response to CO with normal extracellular Ca2+. On the right, in the absence of extracellular Ca2+ (0 Ca; horizontal thin bar), CO was without effect when the light was switched off. Inset shows eight superimposed afferent action potentials from the single fibre recording with scale bars representing 1 mV and 1 ms.

The source of Ca2+ entry during CO-mediated excitation was examined further by use of the non-selective Ca2+ channel antagonist Ni2+ (2 mm, n = 5) and the L-type Ca2+ channel blocker D600 (100 μM, n = 6). Both antagonists reduced spontaneous chemodischarge in hyperoxia and both prevented the CO-mediated elevation in discharge. Thus, control discharge in the presence of Ni2+ was reduced to 0.01 ± 0.005 Hz and was not significantly increased by CO (0.03 ± 0.02 Hz; P > 0.18). Similarly, D600 reduced control discharge to 0.21 ± 0.06 Hz and also prevented a significant effect of CO (0.58 ± 0.21 Hz; P > 0.05).

P2 receptor antagonism prevents CO-mediated chemoexcitation

The potential role of ATP neurotransmission in the mediation of CO-induced chemoexcitation was determined, in a separate series of experiments, by measuring single fibre chemoafferent discharge (n = 9) in the absence and presence of the P2 receptor antagonist PPADS. All experiments were performed in normoxic normocapnia. Control discharge was elevated by 320 mmHg CO, as expected, by more than tenfold (P < 0.05, ANOVA) and in a photolabile manner (Fig. 2). Addition of PPADS (50 μM) to the superfusate solution both reduced baseline discharge and prevented any significant effect of CO (P > 0.05, ANOVA; Fig. 2). This inhibitory effect was completely reversed after washout (Fig. 2).

Figure 2. Effect of ATP receptor antagonism upon carbon monoxide sensitivity.

Figure 2

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Single fibre chemoreceptor afferent discharge was recorded and binned into 10 s periods and steady-state discharge averaged over at least 30 s. All recordings were made in the absence of light. CO (320 mmHg)-stimulated discharge was inhibited by the addition of the ATP receptor antagonist PPADS (50 μM). The inhibitory effect of PPADS was reversed by washout for 30–60 min. Data are given as means ± s.e.m.*P < 0.05, n = 8.

Carbon monoxide depolarises isolated type I cells

As CO excites the adult in vitro carotid body by a Ca2+- and D600-dependent mechanism, this suggested that CO might act presynaptically to depolarise isolated type I cells. We addressed this suggestion by utilising current clamp in perforated patch experiments to measure directly the action of CO upon type I cell resting membrane potentials. We first examined the ability of respiratory acidosis (a well-known chemostimulus in these cells) to depolarise the cells and then tested the effect of CO. We only performed CO experiments on cells that responded to a simulated respiratory acidosis (PCO2 = 76 mmHg, pHo ≈7.16). Figure 3 shows a record of a resting membrane potential of -45 mV that was depolarised to -35 mV after application of acidosis in one cell. In this cell, after full recovery from acidosis, CO also induced a depolarisation of the membrane to -35 mV. This effect was fully and rapidly reversible after washout of CO. In this and five other similar recordings, all made in the absence of light, acidosis and CO both significantly (P < 0.05, ANOVA) depolarised type I cells from -49 ± 3 mV (control) to -37 ± 4 mV for acidosis and to -38 ± 2 mV for CO. The effects of both acidosis and CO were not photolabile and similar results were obtained with or without the presence of cold, white light.

Figure 3. Carbon monoxide depolarises isolated type I cells.

Figure 3

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Example of the effect of acidosis and CO in one cell on the resting membrane potential. The recording was made during current clamp using the amphotericin B, whole-cell configuration. Acidosis (PCO2 = 76 mmHg; PO2 = 60 mmHg) and CO (PCO = 180 mmHg; PO2 = 60 mmHg) both reversibly depolarised the type I cell. Experiments were performed in the absence of light.

Carbon monoxide decreases outward currents in isolated type I cells via an intracellular messenger

In order to determine the effect of CO on net current, we depolarised type I cells between -90 and +30 mV, from a holding potential of -70 mV. Figure 4A (left) shows, from one cell, that this protocol elicited currents that were outward above -50 mV and which increased in amplitude with increasing depolarisation. The application of CO decreased the amplitude of this outward current (Fig. 4A, right). The current density-voltage relationships of this cell averaged with six other cells are shown in Fig. 4B. The current density-voltage relationship showed that CO significantly decreased the amplitude of the current above -50 mV (P < 0.05). Near the resting membrane potential of ≈-50 mV (corresponding to the membrane potential where the current density was 0 pA pF−1), CO induced an inward shift in current (Fig. 4B, inset) corresponding to a depolarisation from -48 to -38 mV (approximate values directly read from the current-voltage relationship). This correlates with our results shown above using current clamp and shows reversal of the CO effect after washout. The current density-voltage relationship of this CO-sensitive current (given as the difference between control and CO currents) is represented in Fig. 4C and shows a reversal potential of -58 mV (approximate value directly read from the current-voltage relationship).

Figure 4. Carbon monoxide decreases the amplitude of outward currents above -50 mV.

Figure 4

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A, an example of outward currents obtained during 300 ms pulses to membrane potentials between -90 and +30 mV from a holding potential of -70 mV, in control conditions (left) and during perfusion with CO (right). Recordings were made using the amphotericin B, whole-cell configuration. B, the mean ± s.e.m. (n = 7) current density-voltage relationships in control conditions (•), in the presence of CO (○) and after recovery from CO (⋆). The inset shows an expanded scale of the same current density-voltage relationships to highlight the effect of CO at membrane potentials where current density was around 0 pA pF−1. C, the mean ± s.e.m. (n = 7) of the CO-sensitive component of the total current density-voltage relationship determined as the difference between the current density amplitudes in control conditions and in the presence of CO. Experiments were performed in the absence of light.

We also performed similar experimental protocols but using the conventional whole-cell configuration of the patch clamp technique. Example currents in control conditions and during CO perfusion from one cell are shown in Fig. 5A. Step depolarisation elicited currents that were outward above -60 mV and which increased in amplitude with increasing depolarisation. Figure 5B represents the mean current density-voltage relationships in seven cells in control conditions and during CO perfusion. CO significantly decreased the amplitude of the current but only above +10 mV (P < 0.05). The degree of inhibition by CO (difference in CO current density/ control current density) was not voltage dependent at potentials greater than +10 mV, with CO decreasing the amplitude of the control current by 25.0 ± 5.7 % at +10 mV and by 25.2 ± 4.7 % at +30 mV and with a mean inhibition between +10 mV and +30 mV of 25.4 ± 3.0 %. Moreover, this inhibition was not photolabile and CO inhibition above +10 mV also occurred in the presence of light. Near the resting membrane potential of ≈-55 mV (corresponding to the membrane potential where the current density was 0 pA pF−1), CO perfusion was without effect on currents (Fig. 5B, inset). The effect of CO was largely reversible, more so at the higher test potentials. The current density-voltage relationship of the CO-sensitive current (given as the difference between control conditions and CO perfusion) is represented in Fig. 5C.

Figure 5. Carbon monoxide decreases the amplitude of outward currents above +10 mV.

Figure 5

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A, example of outward currents obtained during 300 ms pulses to membrane potentials between -90 and +30 mV from a holding potential of -70 mV, in control conditions (left) and during perfusion with CO (right). Recordings were made using the conventional, whole-cell configuration. B, the mean ± s.e.m. (n = 7) current density-voltage relationships in control conditions (•), in the presence of CO (○) and after recovery from CO (⋆). The inset shows an expanded scale of the same current density-voltage relationships to highlight the negligible effect of CO at membrane potentials where current density was around 0 pA pF−1. C, the mean ± s.e.m. (n = 7) of the CO-sensitive component of the total current density-voltage relationship determined as the difference between the current density amplitudes in control conditions and in the presence of CO. Experiments were performed in the absence of light.

Carbon monoxide decreases the resting membrane conductance in isolated type I cells

In order to determine the effect of CO on resting currents in type I cells of the rat carotid body, we measured the membrane conductance between -60 and -50 mV. Type I cells were voltage clamped, using the amphotericin B whole-cell patch-clamp technique, and subjected to repetitive voltage ramps between -90 and -30 mV every 6 s. Figure 6 shows, in one type I cell, representative recordings of resting membrane current under control conditions and during CO perfusion during protocols performed in the dark. CO caused a decrease in the slope of the current-voltage relationship (represented by the ramp currents) that was reversible. The mean membrane conductance determined by fitting a linear regression to the current-voltage relationship over the range -60 to -50 mV was significantly decreased from 293.31 ± 2.13 pS under control conditions to 187.06 ± 2.14 pS after CO perfusion (n = 12, P < 0.05). Experiments performed in the presence of bright white, cold light gave similar results and the membrane conductance in the presence of CO (191.60 ± 1.18 pS) was still significantly less than control (n = 7, P > 0.05) but not significantly different from the response to CO in the absence of light (n = 12, P > 0.05).

Figure 6. Carbon monoxide decreases resting membrane conductance.

Figure 6

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Cells were voltage clamped at -70 mV and subjected to a 2 s voltage ramp from -90 to -30 mV every 6 s. A, voltage protocol and resultant currents during a single experiment. During CO exposure there was a reversible decrease in the amplitude of the current. B, mean current responses to voltage ramps. The lower trace is that during CO exposure. Slope conductances were measured between -50 and -60 mV. Recordings were made using the amphotericin B, whole-cell configuration. Experiments were performed in the absence of light.

When we performed similar experiments, in the dark, but using the conventional whole-cell configuration, CO was without effect on resting membrane conductance between -60 and -50 mV with conductances during control and CO perfusion being 274 ± 39 and 282 ± 40 pS, respectively.

Carbon monoxide decreases a leak K+ resting membrane conductance in isolated type I cells

In order to confirm a role for a background, ‘leak’ current in mediating the outward CO-sensitive current, we performed experiments using a ‘blocking solution’ in which most other currents were blocked (Buckler, 1997). Included in the bath solution was 10 mm TEA, 5 mm 4-AP and 2 mm Ni2+ and external Ca2+ was also removed. The effect of membrane potential was assessed using a voltage-clamp protocol between -90 and +30 mV and a current-voltage relationship for the CO-sensitive current was constructed. This sensitive current showed a reversal potential at -66 mV, close to that of the CO-sensitive current obtained in control conditions (Fig. 7B). This shows that CO can decrease a ‘leak’ resting membrane conductance that is different from that mediated through voltage (Kv)- and Ca2+ (KCa)-activated K+ channels or Ca2+ channels. Nevertheless, the amplitude of this sensitive current was decreased. For example at +30 mV the current density was 24.20 ± 3.90 pA pF−1 in control conditions and just 0.57 ± 0.21 pA pF−1 in the blocking solution. Thus, this suggests that CO can also decrease Kv and/or KCa currents.

Figure 7. Carbon monoxide decreases a K+ resting membrane conductance.

Figure 7

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A, average current-voltage relationships of the CO-sensitive current in the presence of K+ channel blockers, Ni2+ and the absence of Ca2+ with 4.5 mm K+ (•, n = 4) or with 20 mm K+ (○, n = 5). Note the change of the reversal potential between these two different conditions. B, summary mean bar graph of the reversal potential obtained in control conditions (4.5 mm K+, n = 7), in the presence of K+ channel blockers, Ni2+ and the absence of Ca2+ (4.5 mm K+ + blockers, n = 4) and in this last condition but with 20 mm K+ (20 mm K+ + blockers, n = 5). C, plot of the measured shift in reversal potential observed following the increase in K+ (Na+ replacement, •).The open circles indicate calculated shifts in reversal potential expected for a purely K+-selective conductance. Data represent means ± s.e.m. in 4–5 cells. Recordings were made using the amphotericin B, whole-cell configuration and performed in the absence of light.

The current-voltage relationship of this CO-sensitive current (Fig. 4C and Fig. 7) shows a reversal potential at around -61 mV in control conditions, which further suggests that this current was mainly carried by K+. In order to confirm the role of K+ in mediating this CO-sensitive current, current-voltage relationships were constructed at two levels of extracellular K+ and in the presence of K+ channel blockers, Ni2+ and without external Ca2+. Increasing external K+ from 4.5 to 20 mm caused a substantial increase in the reversal potential of the CO-sensitive current (Fig. 7B). The mean reversal potential for the CO-sensitive current was -61 ± 3 mV in control conditions, -66 ± 6 mV in the presence of K+ channel blockers, Ni2+ and in the absence of Ca2+, and -30 ± 3 mV with 20 mm K+. These values followed the predicted values for the equilibrium potential for K+ but with an inward shift of around +30 mV (Fig. 7C).

Discussion

CO was used at high tension to mimic the action of hypoxia upon carotid body discharge. Our data confirmed the fast, excitatory action of high tensions of CO upon chemoreceptor discharge in an in vitro rat carotid body model (Lahiri et al. 1993; Lahiri & Acker, 1999; Lahiri et al. 1999) and we have now demonstrated this action to occur at the level of single fibre chemoafferents, thus removing the potentially complicating effects of fibre recruitment. All chemoafferent fibres recorded from responded to CO and there is no evidence therefore to suggest heterogeneity in responses arising from different fibres.

This excitatory action of CO upon chemodischarge was, to a large extent, reversibly inhibited by light, as previously described (Wilson et al. 1994), and thus, although we did not use monochromatic light to determine action spectra, there is little reason to doubt that the light-sensitive action we observed was not also mediated via CO interaction with cytochromes of the mitochondrial respiratory chain or other mitochondrial or cytosolic haem protein(s) (Prabhakar, 2000). The degree of inhibition by light of the putative CO-haem interaction appears to be critically dependent upon the PCO/PO2 ratio (Lahiri & Acker, 1999) and we deliberately used steady PCO/PO2 ratios of 2 or more throughout this study to avoid the complicating effects of using CO tensions equal to, or lower than, O2 tensions, at which point CO acts, like hyperoxia, to reduce chemodischarge in a non-light-dependent manner (Lahiri et al. 1993; Conway et al. 1997). When given together with hypoxia, CO can also act like hyperoxia to attenuate the inhibitory effect of hypoxia upon K+ channel currents (Lopez-Lopez & Gonzalez, 1992; Riesco-Fagundo et al. 2001), and these findings were taken as evidence for a role of a membrane-bound haem protein in O2 sensing. In our experiments, CO acted like hypoxia rather than hyperoxia and, although we did not examine the effect of CO and hypoxia together specifically, we cannot easily account for the potential difference between our data and those of the previous studies. Nevertheless, we found that the light-sensitive component could not account for all the CO-augmented chemodischarge, leaving a substantial non-light-dependent effect. In addition, we showed both the light-dependent and light-independent actions of CO in the whole organ to be critically dependent upon the presence of external Ca2+. Whilst the use of EGTA to generate a zero [Ca2+] extracellular solution has been criticised in a previous study as leading quickly, in the carotid body, to a run down of intracellular Ca2+ (Lahiri et al. 1997), the effects of Ni2+ and D600 are nevertheless most simply interpreted as defining a significant requirement for Ca2+ entry through voltage, primarily L-type, Ca2+ channels. These data are in agreement with Rozanov et al. (1999), who were able to inhibit afferent chemosensory discharge with 200 μM CdCl2. These findings in the whole organ suggested, at least according to the membrane hypothesis of chemotransduction, an action of CO upon the presynaptically located, neurosecretory type I cell, with CO acting not unlike hypoxia to raise intracellular Ca2+ (Buckler & Vaughan-Jones, 1994) and cause transmitter release. Such an action has been described (Buerk et al. 1997) whereby high CO caused an augmented release of dopamine from cat carotid bodies but this effect was, surprisingly, not light dependent, nor was it correlated in time with chemoafferent discharge. These data were thus interpreted by these authors as strongly suggestive of a dual site of action of CO, with the photolabile component of CO being confined to afferent nerve endings (Buerk et al. 1997; Lahiri & Acker, 1999), and subsequent absorption photometry studies implicated this action to be mediated through a CO-haem interaction that was directly coupled to an ion channel in the afferent nerve terminal (Lahiri et al. 1999). Whilst possible, this schema is not in direct accordance with previous suggestions that an ion channel-coupled haem would act more as a ‘desaturation monitor’ to measure oxygen lack, in which case CO would prevent rather than cause channel closure (Lopez-Lopez & Gonzalez, 1992; Lopez-Barneo et al. 1993). Given the relatively high density of mitochondria located in these nerve terminals, their proximity to the plasma membrane and the presence there of transmitter-like substances (Osborne & Butler, 1975; McDonald, 1981), these data might not rule out an action at this site involving mitochondria and Ca2+-dependent neurotransmission (David et al. 1998).

Whilst our data cannot unequivocally confirm a postsynaptic action of CO at the nerve terminal, it is strongly suggestive of such as we have demonstrated that the presynaptic effect of CO upon the membrane potential of isolated type I cells is not light sensitive. This effect on the membrane potential might, however, account for the CO-mediated neurotransmitter release described by Buerk et al. (1997). The absolute need of CO-mediated chemodischarge for external Ca2+, and to a large extent voltage-gated Ca2+ channel activation, could thus imply a permissive role for transmitter release upon the CO-mediated generation of postsynaptic action potentials, as previously hypothesised (Lahiri & Acker, 1999), but this permissive transmitter is most probably not dopamine (Buerk et al. 1997). However, our data demonstrating a clear prevention of the CO-mediated increase in chemodischarge by application of the P2-receptor antagonist PPADS strongly implicate ATP as the permissive transmitter, perhaps co-localised in synaptic vesicles with another transmitter (Zimmermann, 1994). The dose of PPADS used (50 μM) was selected as the minimum dose that gave maximal block of hypoxia-mediated increases in chemoreceptor discharge (data not shown). Whilst PPADS is perhaps more selective for the P2X ligand-gated ion channel, it appears that it may also block the G-protein-coupled, P2Y receptor (Ralevic & Burnstock, 1998), but in both cases would act to prevent ATP-mediated depolarisation and thus prevent either Ca2+ entry and/or the generation of action potentials. Whilst we have no data to place definitively the stored ATP in either the type I cell and/or the nerve ending, previous studies using immunofluorescence have shown the presence of P2X receptors on the afferent nerve terminals (Zhang et al. 2000). In addition, exogenous ATP has been shown to increase isolated type I cell intracellular [Ca2+], suggesting the presence of purinoceptors also on type I cells (Mokashi et al. 2001).

The depolarisation of membrane type I cells by CO could result from the suppression of an outward current regulating the resting membrane potential, as described above, and/or from an activation of an inward current. The CO-sensitive current reversed at ≈-61 mV and was outward at membrane potentials more positive than this. This reversal potential was relatively close to the predicted (and calculated) equilibrium potential for K+ and increasing the extracellular K+ level to 20 mm caused a positive shift in the reversal potential suggesting that the CO-sensitive current was carried by K+ ions. This shift in reversal potential was observed whilst using a ‘blocking solution’, indicating that the CO acted to decrease the leak conductance described previously in these cells (Buckler, 1997). Nevertheless, since the magnitude of the CO-sensitive current was also decreased when experiments were performed in the presence of both TEA and 4-AP this strongly suggests that CO decreased other K+ conductances mediated, for example, through KCa and Kv channels. In addition to the implied effect at the nerve terminal, our data thus demonstrate a further effect of high tensions of CO upon type I cell leak channel conductances that appears to require intracellular signalling by a diffusible factor(s) and a less indirect effect upon other channel conductances of type I cells. These effects were all light insensitive. Our finding that CO could reduce the leak conductance to ca 60 % of its maximum value and other K+ conductances to ca 75 % of their maximal values is in broad agreement, albeit of lesser magnitude, with the effect of NaCN upon these conductances in previous studies (Peers & O'Donnell, 1990; Wyatt & Buckler, 2000). Only the indirect effect upon the leak channel could explain the observed depolarisation caused by CO, which is similar to the previously reported absence of hypoxia effect upon this channel's conductance in excised patches of type I cells, and there is little reason to believe that it is not also analogous to a two-pore domain potassium channel (Lesage & Lazdunski, 2000), most probably TASK-1, as identified in the carotid body (Buckler et al. 2000). These channels are sensitive to physiological changes in pH with around 90 % of the maximal current inhibited by a drop in extracellular pH to 6.7 (Duprat et al. 1997) and a > 50 % reduction in type I cell leak channel conductance by isocapnic acidosis to pH 6.4 (Buckler et al. 2000). We found no difference in extracellular pH in our superfusates equilibrated with or without high CO and also believe that the effects we observed upon channel conductances were also most probably not acting through a change in intracellular acid, as it has been reported that tensions of CO considerably higher than that used here had no significant effect upon type I cell intracellular pH (Mokashi et al. 1998).

Although relatively close, the reversal potential of the CO-sensitive current was actually not equal to the calculated equilibrium potential for K+ and was more positive by ca 30 mV. This raises the possibility that the CO-sensitive current was also carried by other ions. This is supported by the decrease in the difference between the calculated and measured reversal potential of the CO-sensitive current when using the ‘blocking solution’. Indeed, CO might activate a non-selective cation conductance, as seen in chromaffin cells (Inoue et al. 1998), or an undefined inward current previously reported for mitochondrial uncouplers in type I cells (Buckler & Vaughan-Jones, 1998).

The effect of CO was largely reversible, thus ruling out the possibility of a run down of current. The reversibility was more so perhaps in the perforated patch configuration than in the whole cell, where a slight increase in the magnitude of the inward current after washout of CO might suggest a slow time effect due presumably to internal dialysis. We found a significant current density difference between the two patch configurations, with current densities in the whole-cell configuration being on average some twofold greater than those found in perforated patch experiments. The perforated patch current densities we observed are in close agreement with those reported by others (Stea et al. 1991). We have no explanation for this, but as the only difference between the two configurations should be the presence or absence of intracellular dialysis, we suggest that in the perforated patch configuration there may be the presence of an inhibitor compound acting on IK, when compared with the whole-cell configuration. For example, when using the perforated patch configuration activated protein kinase C may be present, which is known to reduce the outward current in type I cells (Peers & Carpenter, 1998).

Regarding the morphology of the current-voltage relationships, we found no evidence of the ‘hump’ normally attributable to a KCa component of the whole-cell current and we had made no specific attempt to eliminate it. This may be due to differing conditions, as this particular characteristic of the type I cell is most commonly observed when the whole-cell configuration has been used at room temperature and with Hepes as the pH buffer (Hatton & Peers, 1996; Hatton et al. 1997; Peers & Carpenter, 1998). Since we also did not find the hump in either of the whole-cell configurations we used, we suspect that this difference is due to temperature and/or pH buffer. Indeed, a difference between using Hepes or bicarbonate solutions as the pH buffer has been described (Stea & Nurse, 1991). Furthermore, this hump in the current-voltage relationship is often observed, or at least is particularly pronounced, only if experiments are performed using membrane potentials more positive to +30 mV (Stea et al. 1991).

In summary, our data can be taken to show that CO at high tensions relative to O2 acts at the type I cell in a light-insensitive manner and thus, by inference, the light-sensitive effect is most likely to occur at the afferent nerve terminal. At the type I cell the action of CO is to decrease leak channel conductances and other voltage-dependent K+ conductances, leading to cell depolarisation, of significant magnitude to induce voltage-gated Ca2+ entry and neurotransmitter release. We suggest that the critical transmitter released is ATP and this release accounts for some 30 % of the CO-mediated chemoexcitation, presumably acting through purinoceptor-mediated afferent nerve depolarisation. The majority of the CO-mediated chemoexcitation appears, however, to be generated through a direct action of CO at the nerve terminal. Given the absolute requirement for purinoceptor activation for CO-mediated chemoexcitation, it would appear that the action of CO at the afferent nerve terminal is also dependent upon the release of ATP. The origin and site of action of this ATP is not known.

Acknowledgments

We thank the Wellcome Trust for supporting this study through project grant funding and through a European collaborative travel grant. P.K. was a Lister Institute Research Fellow for part of the study, A.F.C. was funded by the University of Birmingham, UK, F.A.-H. is sponsored by the King Khalid University, Saudi Arabia, and E.D. by Agence De l'Environnement et de la MaÎtrise d'Energie and Conseil Regional Centre, France.

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