| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Journal of Physiology (2001), 535.1, pp. 95-106
© Copyright 2001 The Physiological Society
 |
ABSTRACT |
|---|
 TopAbstract IntroductionMethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The carotid body is the principal peripheral chemoreceptor responsible for the cardiorespiratory adaptation to changes in blood PO2, PCO2 and pH. The carotid body contains two specific cell types. Glial-like (type II) cells encapsulate groups of type I (glomus) cells that synapse with carotid sinus nerve endings. It is accepted that the type I cell is the chemosensitive element. Depolarization of the cell membrane, elevation of [Ca2+] in the cytoplasm and neurosecretion followed by an increased discharge rate of the carotid sinus nerve are characteristic events during the response to different stimuli (for a review on carotid body chemoreceptor cells see Gonzalez et al. 1994).
How different stimuli evoke depolarization is still an important question. The intracellular pH (pHi) of chemoreceptor cells strictly follows the extracellular pH (pHo) (Buckler et al. 1991), while the intracellular acidosis during isohydric hypercapnia (an increase in CO2 pressure at constant pHo) is transient only (Buckler et al. 1991). As the pattern of neural response is very similar to that of pHi, it was proposed that a fall in pHi triggers the depolarization of chemoreceptor cells in hypercapnia and/or acidosis (Buckler & Vaughan-Jones, 1994a). The cytoplasmic increase in [H+] is assumed to inhibit the K+ conductance of the plasma membrane, thus evoking membrane depolarization (Buckler & Vaughan-Jones, 1994a; Gonzalez et al. 1994). The only channel that has been shown to be inhibited by cytoplasmic acidification in rat chemoreceptor cell is the large conductance calcium-activated potassium channel (Peers & Green, 1991). However, this channel opens only at elevated intracellular [Ca2+] or at potentials positive to -30 mV. Thus such a change in channel function may augment, but not initiate the acidosis-induced depolarization (Buckler & Vaughan-Jones, 1994b).
Besides pHi, a direct regulatory effect of pHo on the resting membrane potential should be considered as well. One possibility is that an acid-sensing cation channel (Waldmann et al. 1997) contributes to the membrane potential change during acidosis. This channel is most pronouncedly activated at pHo values below 7.0 and its presence in the chemoreceptor cell has not yet been reported. Extracellular pH changes even within the physiological range have been shown to influence ion channel function in a few other cell types (Duprat et al. 1997; Reyes et al. 1998; Clark et al. 1998; Ferroni et al. 2000; Makara et al. 2001). TASK 1 and TASK 2 channels, members of the two-pore domain containing potassium channel family, lack intrinsic voltage sensitivity and are inhibited by extracellular acidosis (Duprat et al. 1997; Reyes et al. 1998). Recently a TASK-like channel, sensitive to acidosis and hypoxia, was described by Buckler et al. (2000). This channel may account for earlier data showing that membrane depolarization provoked by hypercapnic acidosis was primarily the consequence of the inhibition of a potassium conductance in rat chemoreceptor cells (Buckler & Vaughan-Jones, 1994b).
Although K+ channels are undoubtedly important in setting the membrane potential, it should be noted that the resting potential of rat chemoreceptor cells is 30-40 mV more positive than the estimated equilibrium potential of potassium (EK ~-90 mV) (Buckler & Vaughan-Jones, 1994b). This implies that the conductance for other ions besides potassium has to function at resting Em. In this study we provide evidence for the presence of an inwardly rectifying chloride current in these cells, which may contribute to the deviation of Em from EK. This current is directly regulated by pHo in the physiologically relevant pH range. The current, which is augmented by extracellular acidosis and reduced by alkalosis, may participate in the signal transduction during extracellular acidosis.
|
METHODS |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Cell isolation
Experiments were performed on chemoreceptor cells isolated from the carotid bodies of two or three 10- to 16-day-old Wistar rats. The rats were anaesthetized with sodium pentobarbital (5 mg intraperitoneally). The head together with the neck was cut off and placed in ice-cold saline. The carotid bodies were removed and incubated in low-Ca2+ (0.2 mM) phosphate-buffered saline (PBS) containing collagenase (2 mg ml-1, Type 1, Sigma) and trypsin (2 mg ml-1, Difco laboratories, Detroit, MI, USA) at 37 °C for 17 min. The carotid bodies were teased apart with forceps, then digested for a further 5 min. Following trituration the cells were centrifuged at 200 g for 10 min. The pellet was resuspended in culture medium (50 µl per carotid body). Cells in 25 µl suspension were plated on the centre of a poly-D-lysine-coated 35 mm Petri dish or a 22 mm glass coverslip. Two hours were allowed for adhesion in an incubator (5 % CO2, 37 °C), and then 1.5 ml of culture medium was added to the Petri dishes. Cells were cultured for 6-50 h. Unlike background cells, chemoreceptor cells in culture remained phase-bright. Polygonal shape and short processes were signs of good adhesion, good viability and good seal-forming ability.
The experimental protocol was approved by the Animal Care and Ethics Committee of Semmelweis University (No. 17-3/98). All the respective legal and institutional rules for animal protection were followed.
Patch-clamp measurements
Voltage-clamp experiments were performed on single chemoreceptor cells using an RK-400 (Biologic Science Instruments, Claix, France) or Axoptach-1D (Axon Instruments, Foster City, CA, USA) patch-clamp amplifier. Pipettes were pulled from borosilicate glass tubing GC120TF-10 or GC120F-10 (Clark Electromedical, Pangbourne, Reading, UK) using a P-87 puller (Sutter Instrument Co., USA). Pipette resistance was 4-10 M
for whole-cell measurements and 10-20 M for cell-attached measurements when the pipette was filled with the intracellular solution. The bath was earthed using an Ag-AgCl pellet. Cells were locally perfused by a gravity-driven perfusion system from a linear array of five microcapillary plastic tubes located about 0.1 mm from the cell. In tail current and Em measurements, when the chloride concentration in the perfusion solution was changed a Dri-Ref-2SH (World Precision Instruments, Aston, UK) reference electrode was used instead of an Ag-AgCl pellet in order to avoid significant changes in the electrode potential. This electrode utilises 3 M KCl as the liquid junction. The electrode potential shifts resulting from solution changes never exceeded ±3 mV and were not corrected for. Current data were not leak-corrected unless otherwise stated. Currents were low-passed at 1-2 kHz (-3 dB, 5- or 8-pole Bessel filter) and sampled at 2-10 kHz using a Digidata 1200 interface board (Axon Instruments). Experiments, data storage and analysis were performed using software pCLAMP 6 (Axon Instruments) running on a PC/AT computer. For whole-cell experiments, chemoreceptor cells were selected on a morphological (see above) and electrophysiological basis. The mean membrane capacitance of chemoreceptor cells was 5.5 pF as measured with the built-in circuit of the amplifiers. Immediately after reaching the whole-cell configuration in Tyrode solution, activation of a large outward K+ current at potentials positive to -40 mV was observed. Dialysis with K+-free, Cs+-containing pipette solution mostly eliminated this K+ current and unmasked a high-threshold (~-40 mV) Ca2+ current. This Ca2+ current was significantly reduced by the application of a solution containing low [Ca2+], high [Mg2+] and nifedipine (Solution I).
A steady-state activation curve was obtained by analysing the initial amplitude of tail currents at -50 mV following a step back from test potentials ranging from -100 to 10 mV. The normalized conductance values (G/Gmax) were fitted with a single Boltzmann isotherm using the following equation:
where G/Gmax is the relative conductance, V is the test potential, V1/2 is the potential at which the conductance is half-maximally activated, and k is the slope factor.
Fluorimetric measurement of intracellular pH
Cytoplasmic pH was measured with the dual-emission pH-sensitive fluoroprobe, carboxy-SNARF-1 (Molecular Probes, Leiden, The Netherlands). The cells were loaded with 10 µM acetoxymethyl ester of the dye in Hepes-Tyrode solution for 10 min at room temperature. The coverslip with SNARF-loaded cells was placed in an experimetal chamber mounted on the stage of an inverted epifluorescence microscope (Nikon Diaphot). Clusters of 2-10 cells were illuminated at 540 nm and the emitted fluorescence at 580 and 640 nm was measured using two photomultiplier tubes (Model 712, Photon Technology International, Lawrenceville, NJ, USA). The two signals were integrated in 0.5 s intervals and digitally sampled at 10 Hz. The ratio of fluorescence (580/640 nm) was then calculated. For calibration, the standard nigericin technique was applied (Thomas et al. 1979), in which the cells were exposed to nigericin-containing (10 µM) solutions of pH 5.5, 7.4 and 9.5.
Description of the experimental chamber
When bicarbonate-buffered solutions were used the glass coverslip with cells attached was placed on the bottom of a 2 ml Perspex chamber. The chamber contained ~100 µl incubation medium which was exposed to a permanent flow of a prehumidified gas mixture containing 5 % CO2 and 20 % O2 in N2. Solution changes were carried out within 40 s by infusing 1 ml of the new solution pre-equilibrated with the above gas mixture. The excess volume was continuously removed by a suction pump. The steady-state stability of the cell environment was confirmed by repetitive application of the control solution (pH 7.4).
Solutions
The culture medium contained Dulbecco's modified Eagle's medium (GibcoBRL, Csertex kft, Hungary) and Ham's F-12 medium (1:1) supplemented with 10 % heat inactivated fetal calf serum (Protein GMK, Gödöll''o, Hungary), 100 i.u. ml-1 penicillin, 100 µg ml-1 streptomycin and 84 u l-1 insulin (GibcoBRL). Hepes-Tyrode solution contained (mM): NaCl 127, KCl 3.6, CaCl2 2, MgCl2 0.5, glucose 11, Hepes 10, pH adjusted to 7.4 with NaOH. Solutions used for current measurements in the whole-cell mode are shown in Table 1.
For fluorimetric and Em measurements the composition of the bathing solution was (mM): KCl 4.5, NaCl 117, NaHCO3 23, CaCl2 2.5, MgCl2 1, glucose 11, equilibrated with 5 % CO2 and 20 % O2 in N2, pH ~7.4, 300 mosmol l-1. For isocapnic acidosis (pH ~6.8) the HCO3- concentration of the above solution was reduced to 6 mM by Cl- replacement. In Em measurements the pipette solution contained (mM): K2SO4 55, KCl 30, MgCl2 5, EGTA 1, glucose 10, Hepes 20, sucrose 50, pH adjusted to 7.3 with NaOH, 300 mosmol l-1. For pH calibration the solutions contained 140 mM KCl, 1 mM MgCl2 and 20 mM Mes, 10 mM Hepes or 10 mM ethanolamine; the pH was adjusted to 5.5, 7.4 and 9.5, respectively, with NaOH or HCl, as required. The osmolarity of the solutions was checked using a freezing point osmometer (model 3MO, Adveneced Instuments Inc., Norwood, MA, USA).
All experiments were carried out at 28 ± 2 °C. All chemicals were from Sigma, unless otherwise specified.
Statistics
The number of cells (n) indicates the cumulative number of measurements performed at least on two independent cell cultures. Data are presented as means ± S.D. For statistical analysis Student's paired or unpaired t test was used, as appropriate. A value of P < 0.05 was considered statistically significant.
|
RESULTS |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Hyperpolarization-activated inward current
To examine Cl- currents we applied high-chloride containing solutions in which K+ and Na+ were replaced with the impermeant cations NMDG+ and TEA+ (Solution I). The known hyperpolarization-activated and inwardly rectifying cation channels were blocked by extracellular Cs+ (1 mM) (Kubo et al. 1993; Maccaferri et al. 1993). Hyperpolarization-activated inward current could be elicited by step hyperpolarization to -120 mV from the -20 mV holding potential (Fig. 1A). The current consisted of an instantaneous current jump and a time-dependent relaxation (Fig. 1B). The current was usually small (0.5-1 pA pF-1, at -120 mV, at 700 ms) and the activation was slow at the beginning of the experiment (Fig. 1B, trace a). Within 2 min of breaking the patch, the current amplitude began to increase and simultaneously the activation speeded up (current run-up, Fig. 1B and D). The reversal potential (Erev) of the current, as measured after the current run-up during ramp depolarization (Fig. 1C), was near 0 mV. Current characterization was performed after the current run-up was complete. The final current density varied considerably among cells (3-15 pA pF-1, at -120 mV, at 700 ms). No current run-down was observed during the experiments, some of which lasted up to 30 min. The current showed no tendency to inactivate during step hyperpolarization, but deactivated upon depolarization. Using an ATP-free pipette solution, half of the chemoreceptor cells showed marked run-up of the inward current (n > 50), and in preliminary experiments, when 1 mM ATP-containing solution was applied 25 % of the cells also exhibited current run-up (n > 50). The run-up did not influence the sensitivity to pHo, cadmium and 9-AC described later. For current characterization, an ATP-free pipette solution was used so that evoking larger currents required less time and distortion of the current characteristics (e.g. Erev, pHo dependence) by the leak conductance and capacitive transients could be reduced.
 |
View larger version [in this window] [in a new window] |
|
|
Figure 1. Time dependent run-up of hyperpolarization-activated inward current in chemoreceptor cell A, cells were held at -20 mV. Every 15 s, a step hyperpolarization to -120 mV was applied, followed by a ramp depolarization to 60 mV. B, traces were recorded at 60 s (a), 120 s (b), 180 s (c) after breaking the patch. C, current c in panel B recorded during ramp depolarizations as a function of voltage. Note that the current reversal potential at the end of current run-up is close to 0 mV. D, average current contributing to the last 25 ms of step hyperpolarizations is plotted against time. | ||
Ion selectivity
To determine the charge carrier of the inwardly rectifying current we applied tail current measurements. As the applied solutions were designed so that only significant chloride or proton currents were allowed, we had to distinguish between these two charge carriers. Tail currents were recorded during step depolarization from -100 mV to different potentials between -40 and 40 mV (Fig. 2A). For control measurements Solution I was used. Under these conditions ECl was ~0 mV and EH was ~12 mV (pHo = 7.0, pHi = 7.2). The tail current reversal potential (Erev) was -1 ± 2 mV (n = 4, Fig. 2B) suggesting that the current is carried by chloride. Replacing NMDG+ with Na+ (Solution II) was without significant effect on Erev (-2 ± 3 mV, n = 4, Fig. 2C), which excludes the possibility that hyperpolarization-activated cation channels are responsible for this current. Reducing the extracellular pH to 6.6, thus shifting EH to ~36 mV, did not influence the current reversal potential (-2 ± 7 mV, n = 4, Fig. 2D). As the proton currents characterized so far are strictly selective for protons and are outwardly rectifying (DeCoursey, 1998), it is unlikely that this current was flowing through a proton channel. Finally we had to distinguish between a non-selective cation current and an anion current. This was done by isosmotically substituting NMDG-Cl for sucrose (Solution III). Under these conditions Erev shifted by 32 ± 9 mV in a positive direction from the control level of -1 ± 8 mV (n = 5, P < 0.005, Fig. 3). These data confirm that primarily anions (chloride) and not cations are charge carriers of the current.
 |
View larger version [in this window] [in a new window] |
|
|
Figure 2. Effects of changing extracellular pH or cation composition on Erev Cells were held at -20 mV. Tail currents were evoked every 15 s. A, step hyperpolarizations to -100 mV were applied for 1 s followed by step depolarizations to different voltages between -40 and 40 mV. B, for control experiments, tail currents were recorded during step depolarizations in high-Cl-, NMDG+-containing solution at pHo 7.0 (Solution I). Representative tail current traces demonstrate the effect of replacing extracellular NMDG+ with Na+ (Solution II) (C) or changing pHo from 7.0 to 6.6 (D). | ||
 |
View larger version [in this window] [in a new window] |
|
|
Figure 3. Effect of changing extracellular anion composition on Erev The voltage protocol is as described for Fig. 2A except for omission of the -40 mV step. A, representative tail current traces recorded in control solution (Solution I). B, the effect of reducing extracellular chloride from 143 to 16.5 mM by replacing NMDG-Cl with sucrose (Solution III). C, current-voltage relationships of the tail currents recorded in control solution (Solution I, | ||
Voltage dependence
To evaluate whether this hyperpolarization-activated current can be active at the resting potential of chemoreceptor cells (-50 to -60 mV; Gonzalez et al. 1994; Buckler & Vaughan-Jones, 1994b), we determined the steady-state activation curve and current-voltage relationship of the current (Fig. 4). Voltage steps were applied from the -20 mV holding potential to different voltages between -100 and 40 mV (Fig. 4A). A family of hyperpolarization-activated currents in Fig. 4B demonstrates that the time-dependent component was present at voltages negative to -30 mV (Solution I, pHo = 7.0). To gain a steady-state current-voltage relationship, average currents corresponding to the last 25 ms of the voltage steps were plotted against test-voltage in Fig. 4C (n = 5). The I-V curve shows marked inward rectification. The steady-state activation curve (Fig. 4D, n = 3) revealed that current activation was present in the entire negative membrane potential range.
 |
View larger version [in this window] [in a new window] |
|
|
Figure 4. Voltage dependence of the inwardly rectifying anion current A, voltage steps were applied from the -20 mV holding potential every 15 s to different test potentials between -100 and 40 mV, followed by a voltage step to -50 mV. B, family of hyperpolarization-activated current measured at pHo = 7. For demonstration purposes, original current curves are digitally low-passed at 50 Hz (Solution I). C, to gain a steady-state current-voltage relationship average current contributing to the last 25 ms of the test potentials is plotted against voltage (n = 7). D, to obtain the steady-state activation curve, current amplitudes measured at the onset of tail currents at -50 mV were corrected for leak. Leak current amplitude at -50 mV was determined as the amplitude of initial current contributing to the -50 mV test potential. The G/Gmax value is equal to the corrected tail current amplitude (I) following a given test potential divided by the corrected tail current amplitude following the -100 mV test potential (Imax). The mean G/Gmax values (weighted with 1/S.D., n = 3) were fitted with a single Boltzmann isotherm as described in Methods. The current was half-maximally activated at -51.5 mV (V1/2) and the slope factor (k) was -12.6 mV. | ||
pHo sensitivity
To evaluate whether the chloride current can contribute to the membrane potential response to pHo changes, we investigated the influence of pHo on the current amplitude. A change in pHo between 7.0 and 7.8 significantly altered the inward current, the steady-state currents at -120 mV being -46 ± 21 and -34 ± 13 pA, respectively (n = 7, P < 0.01). The graded effect of pHo between pH 6.6 and 7.8 is demonstrated at -100 mV in Fig. 5A-C. Figure 5D demonstrates the effect of pHo on the steady-state current-voltage relationship at pHo 7.0 and 7.8. Extracellular alkalization reduced the inward current at potentials negative to -40 mV. The effect of pHo change on current activation was fast and fully reversible (not shown).
 |
View larger version [in this window] [in a new window] |
|
|
Figure 5. pHo dependence of the inwardly rectifying current A, the holding potential was -20 mV. Every 15 s a 2 s hyperpolarizing voltage step to -100 mV was applied. B, representative traces measured at different pHo values. C, average current contributing to the last 25 ms of the step hyperpolarization at a given pHo was divided by the current amplitude at pHo 6.6. Corrected amplitudes are plotted against pHo (n = 5). D, to examine the pH dependence of the current-voltage relationship, the voltage protocol displayed in Fig. 4A was used. Voltage steps were applied every 15 s from the -20 mV holding potential to different test potentials between -100 and 10 mV. The average current contributing to the last 25 ms of the voltage step is plotted against test potential at pHo = 7.0 ( | ||
Effect of organic and inorganic inhibitors
Divalent cations, such as Cd2+ and Zn2+ were reported to block inwardly rectifying chloride currents when applied at submillimolar concentrations (Ferroni et al. 1997; Clark et al. 1998). The effect of different inhibitors was tested at -100 mV and pH 7.4. Application of 0.1 mM Ba2+, a potent inhibitor of inwardly rectifying K+ channels (Kubo et al. 1993), had no effect on the inward current (not shown). Zn2+ at 0.1 mM reduced the steady-state current amplitude to 85 ± 12 % (from -28 ± 13 to -24 ± 13 pA, n = 5, P < 0.05). Addition of 0.1 mM Cd2+ reduced the current from -21 ± 11 to -6 ± 2 pA (n = 5, P < 0.05, Fig. 6B). Considering that the residual current did not display a time-dependent component and did not differ from the non-specific leak current (see below), it is reasonable to assume that Cd2+ under the conditions applied completely blocked the inwardly rectifying current. The effect of Cd2+ and Zn2+ was fast and fully reversible. We also tested the effect of the chloride channel inhibitor 9-AC, which is relatively specific for hyperpolarization-activated anion channels at low concentrations (Strange et al. 1996). When applied in the extracellular solution at 1 mM, 9-AC reduced the current to 40 ± 15 % (from -31 ± 16 to -11 ± 3 pA, n = 5, P < 0.05, Fig. 6C). The inhibitory effect of 9-AC developed within 2 min and was only partially reversible. The reversal potential of the 9-AC-inhibited current was 5 ± 4 mV in control solution (Solution I, pH = 7.0) and was shifted by 37 ± 7 mV to the positive direction when NMDG-Cl was replaced by sucrose (Solution III, pH= 7.0, n = 4, P < 0.005, Fig. 7). This confirms that 9-AC inhibits an anion (Cl-) current.
 |
View larger version [in this window] [in a new window] |
|
|
Figure 6. Effects of Cd2+ and anthracene-9-carboxylic acid (9-AC) on the chloride current A, step hyperpolarizations were applied every 15 s from the -20 mV holding potential to -100 mV for 1.5 s, followed by a ramp depolarization to 20 mV within 2 s. B, the effect of 0.1 mM Cd2+. C, the effect of 1 mM 9-AC. Representative traces for 5 experiments with similar results (Solution I, pHo = 7.4). | ||
 |
View larger version [in this window] [in a new window] |
|
|
Figure 7. 9-AC inhibits an anion current A, voltage protocol is similar to that described in Fig. 6A. B, 9-AC-sensitive current in control solution (Solution I). C, 9-AC-sensitive current after reducing extracellular chloride from 143 to 16.5 mM by replacing NMDG-Cl with sucrose (Solution III). D, currents recorded during ramp depolarization. Note the ~35 mV positive shift in the Erev of the 9-AC-sensitive current following the replacement of NMDG-Cl by sucrose. Traces are obtained by averaging 4 traces in the presence and after washout of 9-AC. The differences between the averages are displayed. Representative of 4 experiments with similar results. | ||
Single-channel conductance
The low unitary conductance of inwardly rectifying Cl- channels makes it difficult to apply single channel patch-clamp technique for channel characterization (Pusch et al. 1999). To gain a rough estimate of the single channel conductance of the anion channel, fluctuations in the steady-state whole-cell current at -100 mV were analysed before and during Cd2+ block (Hille, 1992) (Fig. 6B). The input resistance during Cd2+ block ranged from 8 to 30 G, as calculated from the cord conductance between -90 and -70 mV during ramp depolarization. These values are in the range of seal resistance (10-70 G), measured before establishing whole-cell configuration. Thus the measured current fluctuation during Cd2+ block was considered as background noise. For stationary fluctuation analysis current data measured during the last 200 ms of the 2 s voltage steps were used. The following equation was applied:
2/[I (1 - p)]}/U,
where g is the single channel conductance, i is the single channel current through an open channel at a given potential U (Em - Erev), 2 is the variance of the steady-state whole-cell current corrected for background noise, I is the leak-corrected mean whole-cell current and p is the probability for a channel being in the open state. Background current variance and mean leak current were determined during Cd2+ block. As Erev is close to 0 mV, U was regarded as -100 mV. Applying a relatively high p value of 0.5 the estimated single channel conductance is 4 ± 2 pS (n = 5) in 140 mM chloride. Even if estimation of p is wrong and current fluctuations are underestimated (because of noise filtering), it is unlikely that single channel conductance is outside the 1-10 pS range.
Effect of 9-AC on intracellular pH
Anion conductance in chemoreceptor cells is probably functionally coupled to the Cl-/HCO3- exchanger and may also have an intrinsic bicarbonate permeability. Thus it can play a role in pHi regulation by influencing bicarbonate extrusion (Buckler & Vaughan-Jones, 1994a). Therefore we examined the effect of 1 mM 9-AC on the resting pHi in CO2/HCO3--buffered media. Intracellular pH changes were measured ratiometrically using the pH-sensitive fluorescent dye carboxy-SNARF-1. The average resting pHi in clusters of chemoreceptor cells was 7.29 ± 0.10 at pHo ~7.4. Following application of 1 mM 9-AC a slow intracellular alkalization could be observed (0.09 ± 0.06 pH units in 5 min, n = 7, P < 0.01, Fig. 8). The effect of 9-AC was fully reversible and the solvent (DMSO) failed to evoke any change in pHi. These data confirm that the 9-AC-sensitive anion channel is involved in pHi regulation in intact cells and this anion conductance (directly or indirectly) contributes to the background acid load.
 |
View larger version [in this window] [in a new window] |
|
|
Figure 8. Effect of 9-AC on pHi in CO2/HCO3--buffered solution Intracellular pH in a cluster of ~10 chemoreceptor cells was measured using carboxy-SNARF-1. The integrity of pH regulation was tested by applying isocapnic acidosis (by reducing HCO3- from 23 to 6 mM at constant 5 % CO2). 9-AC induces a slow and reversible increase in pHi. The effect is not mimicked by application of the solvent (DMSO). | ||
Effect of 9-AC on the membrane potential
Having shown that 9-AC induces alkalization, probably by reducing inwardly rectifying anion conductance, it was tempting to investigate whether 9-AC has any influence on the resting Em. Since we are aware of no data on the exact ionic composition of the cytoplasm in chemoreceptor cells, it was important to estimate the value and the changes of resting Em in fairly intact cells. To cope with this problem we applied measurements in the cell-attached mode. To estimate Em the method used by Verheugen et al. (Verheugen & Vijverberg, 1995; Verheugen et al. 1999) was adapted for chemoreceptor cells. Currents from cell-attached patches were recorded during fast voltage ramps (5 mV ms-1, Fig. 9A) every 500 ms, using a pipette solution containing 140 mM K+. The reversal potential of voltage-gated K+ current was used to determine the mean Em during a 25 s period. With high-K+ solution in the pipette EK across the patch membrane approximated 0 mV. Thus K+ current reversed when the pipette potential equalled Em. Fifty consecutive traces were averaged to gain an average patch current (Figs 9B). Correction was made for the leak component by extrapolation of the linear fit of the current at patch potentials negative to the activation threshold of voltage-dependent K+ channels. Applying the cell-attached method, three of the five tested cells displayed a hyperpolarization of 13.7 ± 4.9 mV from a resting Em of -56.3 ± 5.5 mV (Fig. 9B and C), while in two cells there was no response (the change in Em was less than ±3 mV). The occasional activation of a large-conductance K+ channel confirmed that all the five tested cells were chemoreceptor cells. The above data indicate that a 9-AC-sensitive conductance influences the resting membrane potential in at least a fraction of chemoreceptor cells.
 |
View larger version [in this window] [in a new window] |
|
|
Figure 9. The effect of 9-AC on the membrane potential A, in the cell-attached mode fast voltage ramps were applied from 100 to -120 mV every 500 ms. Note that a negative shift in the pipette potential (Epip) means depolarization of the patch membrane. B, 50 traces were averaged in the absence and presence (dotted line) of 1 mM 9-AC. Leak conductance was considered as the slope conductance (as indicated by the straight lines) before the activation of voltage-dependent K+ channel. C, leak-corrected patch currents are displayed as a function of pipette potential. Application of 1 mM 9-AC (dotted line) evokes membrane hyperpolarization as indicated by the negative shift of the reversal potential of the voltage-dependent K+ current. Representative of 3 experiments with similar results (for further details see text). Arrows indicate the activation threshold of the voltage-gated K+ channel. | ||
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
In the first part of this study, applying the whole-cell voltage-clamp technique, we have shown that chemoreceptor cells of the rat carotid body display a pHo-sensitive anion current. The current is activated by extracellular acidosis and inhibited by alkalosis in the physiologically relevant pH range of 7.0 to 7.8. The steady-state current-voltage relationship shows marked inward rectification and current activation is present in the negative membrane potential range. The current is inhibited by the organic anion channel inhibitor 9-AC. In subsequent studies we tested the participation of this current in the control of intracellular pH and membrane potential of intact cells.
Comparison with other inwardly rectifying chloride channels
It was shown that the cloned hyperpolarization-activated chloride channel (ClC-2) is widely expressed in epithelial and non-epithelial cells (Thiemann et al. 1992). The inwardly rectifying chloride currents are activated by hyperpolarization, acidic pHo, cell swelling (Gründer et al. 1992; Jordt & Jentsch, 1997) and intracellular chloride, and they display a small single channel conductance (Strange et al. 1996; Jentsch & Gunther, 1997; Pusch et al. 1999). Jordt & Jentsch (1997) showed that the pHo sensitivity of ClC-2, expressed in Xenopus oocytes, was most pronounced in the physiological range (between pH 7.0 and 8.0). Two of the hyperpolarization-activated chloride currents were shown to be highly sensitive to extracellular pH, even in the physiologically relevant range in mammalian cells. The inwardly rectifying chloride current in rat superior cervical ganglion neurons (Clark et al. 1998) is also activated by acidosis, and displays marked sensitivity to pHo between pH values of 8 and 6.9. At pHo 6.9 the current activation was present at membrane potentials negative to 0 mV. At -90 mV the current was reduced by 0.1 mM Zn2+ by 90 %, while 0.1 mM Cd2+ and 1 mM 9-AC reduced the current by 78 % and 34 %, respectively. The chloride current described in this study is very similar to this current. Recently Ferroni's group (Ferroni et al. 2000) and our group (Makara et al. 2001) described a current in cultured rat cortical astrocytes with strikingly similar characteristics. Ferroni and co-workers also tested the effect of pHi, which was the opposite of that of pHo (Ferroni et al. 2000). The single-pore conductance of the underlying double-pore channel was 3 pS in 140 mM chloride (Nobile et al. 2000). This value is comparable to that computed in the present study for chemoreceptor cells and to that published recently for the heterologously expressed ClC-2 (2.8 pS in 110 mM Cl-, Weinreich & Jentsch, 2001).
Anion channels in chemoreceptor cells
Carbonic anhydrase is present in chemoreceptor cells (Stea & Nurse, 1989), and the significance of the formation of carbonic acid in signal transduction was indicated by the enhancement of the response to hypoxia by HCO3-, added to the incubation medium (Panisello & Donnelly, 1998). Besides carbonic anhydrase activity, the presence and significance of the Cl-/HCO3- antiporter was demonstrated by the effect of the antiporter blocker 4,4-diidothiocyanatostilbene-2-2'-disulfonic acid (DIDS), which reduced the baseline activity as well as the response of the carotid sinus nerve to hypoxia or hypercapnic acidosis (Iturriaga et al. 1998). In addition to the anion exchanger, anion conducation also plays a significant role in the coupling process, as revealed by the application of 9-AC. This inhibitor of Cl- channels not only inhibited the neural response to acidosis (Iturriaga et al. 1998) but also overcame the aforementioned potentiation of the response to hypoxia by bicarbonate (Panisello & Donnelly, 1998). These data show that the transmembrane transport of bicarbonate and a Cl- conductance are essential in the chemoreceptor function of these cells.
Anion conductance in rat glomus cells has also been examined at the cellular level. Stea & Nurse (1989) characterized a voltage-independent chloride channel in inside-out patches. This channel, which had a large conductance (about 300 pS, in 140 mM Cl-), displayed moderate permeability to bicarbonate. Its opening probability was not influenced by hypercapnic acidosis applied to the internal membrane surface. The current described in this study is activated by hyperpolarization and the single channel conductance is in the range of 1-10 pS (in 140 mM Cl-), so it is unlikely that it flows through the voltage-independent chloride channel described above. Carpenter & Peers (1997) described a swelling- and cAMP-activated, weakly outwardly rectifying chloride current. Large currents (a few hundred picoamps at -80 mV) were measured during hyposmotic cell swelling, but pH dependence was not reported. The chloride current described in this study required neither cAMP in the pipette solution, nor swelling of the cell, and displayed apparent inward rectification, and the swelling activated current must therefore be different from the current described in the present paper.
Possible role of the pH-sensitive chloride channel
According to current hypotheses, stimulation-excitation coupling in chemoreceptor cells takes place via membrane depolarization and calcium influx through voltage-activated calcium channels. It has been proposed that intracellular acidification evoked by acidic and/or hypercapnic stimuli brings about membrane depolarization by inhibiting K+ channels (Peers & Buckler, 1995). Although it was shown that the temporal pattern of changes in pHi and intracellular [Ca2+] display remarkable similarities (Buckler & Vaughan-Jones, 1994a), the ability of intracellular acidosis to evoke depolarization at constant pHo remains to be demonstrated. Besides the role of pHi, other possible mechanisms for contributing to depolarization during acidic challenge should also be considered. The chemoreceptor cells of the rat carotid body display a background K+ conductance which is inhibited by hypoxia and acidosis (Buckler & Vaughan-Jones, 1994b; Buckler, 1997; Buckler et al. 2000). The characteristics of the underlying channel (Buckler et al. 2000) are comparable to that of TASK (Duprat et al. 1997; Reyes et al. 1998). Such a conductance may in fact participate in the acidosis-evoked depolarization. The features of this channel led the authors to the conclusion that both extra- and intracellular pH might be important in triggering depolarization (Buckler et al. 2000). Accepting the significance of inhibition of K+ conductance by intra- and probably extracellular acidosis, important points remain unclear. First, what is the conductance that counteracts the hyperpolarising effect of the resting K+ conductance? Second, how are pHo changes transmitted to the cytoplasm? Third, what is the temporal sequence of membrane depolarization and pHi decrease? The presence of the pH-sensitive, inwardly rectifying Cl- conductance in chemoreceptor cells can provide some further insight into the chemoreceptive process.
1. In most cell types the distribution of Cl- between the extra- and intracellular space is passive and determined by the membrane potential only. However, in cells that possess the Cl--HCO3- antiporter, the cytoplasmic Cl- concentration becomes higher and ECl will be more positive than Em. If the cell also exhibits a Cl- conductance, which is the case in chemoreceptor cells of the carotid body, the resting Em will shift in a positive direction from EK towards ECl, and the extent of the shift will depend on the ratio of the K+ and Cl- conductances. Moreover, once the Cl- conductance is increased and the K+ conductance is decreased, as was achieved by extracellular acidosis in the present study, the shift towards ECl will be more pronounced. Therefore, the presence of a pH-sensitive Cl- channel in the chemoreceptor cell provides a mechanism by which extracellular acidosis depolarizes the plasma membrane.
2. Bicarbonate and also chloride efflux through an anion channel may reduce pHi in a cell expressing the Cl--HCO3- exchanger. The activation of such an anion channel would result in increased extrusion of these anions and increased formation of HCO3- and H+, and thus a fall in pHi. In this way the pH-sensitive anion current can contribute to the adaptation of pHi to pHo.
3. Taking the above considerations together, membrane depolarization (receptor potential) during isocapnic acidosis may not only follow or accompany, but even precede the change in pHi.
In our experiments extracellular acidosis activated an anion current. The inhibition of this current by 9-AC evoked hyperpolarization and intracellular alkalization in intact chemoreceptor cells. In summary, activation of the anion conductance by acidosis, as described here, may participate in membrane depolarization and promote intracellular acidification in rat chemoreceptor cell.
|
REFERENCES |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
| BUCKLER K. J. (1997). A novel oxygen-sensitive potassium current in rat carotid body type I cells. Journal of Physiology 498, 649-662 | [Abstract] |
| BUCKLER K. J. & VAUGHAN-JONES, R. D. (1994a). Role of intracellular pH and [Ca2+]i in acid chemoreception in type-I cells of the carotid body. Advances in Experimental Medicine and Biology 360, 41-55 | [Medline] |
| BUCKLER K. J. & VAUGHAN-JONES, R. D. (1994b). Effects of hypercapnia on membrane potential and intracellular calcium in rat carotid body type I cells. Journal of Physiology 478, 157-171 | [Abstract] |
| BUCKLER K. J., VAUGHAN-JONES, R. D., PEERS, C., LAGADIC, G. D. & NYE, P. C. (1991). Effects of extracellular pH, PCO2 and HCO3- on intracellular pH in isolated type-I cells of the neonatal rat carotid body. Journal of Physiology 444, 703-721 | [Abstract] |
| BUCKLER K. J., WILLIAMS, B. A. & HONORE, E. (2000). An oxygen-, acid- and anaesthetic-sensitive TASK-like background potassium channel in rat arterial chemoreceptor cells. Journal of Physiology 525, 135-142 | [Abstract/Full Text] |
| CARPENTER E. & PEERS, C. (1997). Swelling- and cAMP-activated Cl- currents in isolated rat carotid body type I cells. Journal of Physiology 503, 497-511 | [Abstract] |
| CLARK S., JORDT, S. E., JENTSCH, T. J. & MATHIE, A. (1998). Characterization of the hyperpolarization-activated chloride current in dissociated rat sympathetic neurons. Journal of Physiology 506, 665-678 | [Abstract/Full Text] |
| DECOURSEY T. E. (1998). Four varieties of voltage-gated proton channels. Frontiers in Bioscience 3, 477-482 | |
| DUPRAT F., LESAGE, F., FINK, M., REYES, R., HEURTEAUX, C. & LAZDUNSKI, M. (1997). TASK, a human background K+ channel to sense external pH variations near physiological pH. EMBO Journal 16, 5464-5471 | [Abstract/Full Text] |
| FERRONI S., MARCHINI, C., NOBILE, M. & RAPISARDA, C. (1997). Characterization of an inwardly rectifying chloride conductance expressed by cultured rat cortical astrocytes. Glia 21, 217-227 | [Medline] |
| FERRONI S., NOBILE, M., CAPRINI, M. & RAPISARDA, C. (2000). pH modulation of an inward rectifier chloride current in cultured rat cortical astrocytes. Neuroscience 100, 431-438 | [Medline] |
| GONZALEZ C., ALMARAZ, L., OBESO, A. & RIGUAL, R. (1994). Carotid body chemoreceptors: from natural stimuli to sensory discharges. Physiological Reviews 74, 829-898 | [Medline] |
| GRÜNDER S., THIEMANN, A., PUSCH, M. & JENTSCH, T. J. (1992). Regions involved in the opening of CIC-2 chloride channel by voltage and cell volume. Nature 360, 759-762 | [Medline] |
| HILLE B. (1992). Fluctuations measure the size of elementary events. In Ionic Channels of Excitable Membranes, 2nd edn, pp. 321-326. Sinauer, Sunderland, MA, USA | |
| ITURRIAGA R., MOKASHI, A. & LAHIRI, S. (1998). Anion exchanger and chloride channel in cat carotid body chemotransduction. Journal of the Autonomic Nervous System. 70, 23-31 | [Medline] |
| JENTSCH T. J. & GUNTHER, W. (1997). Chloride channels: an emerging molecular picture. Bioessays 19, 117-126 | [Medline] |
| JORDT S. E. & JENTSCH, T. J. (1997). Molecular dissection of gating in the ClC-2 chloride channel. EMBO Journal 16, 1582-1592 | [Abstract/Full Text] |
| KUBO Y., BALDWIN, T. J., JAN, Y. N. & JAN, L. Y. (1993). Primary structure and functional expression of a mouse inward rectifier. Nature 362, 127-133 | [Medline] |
| MACCAFERRI G., MANGONI, M., LAZZARI, A. & DIFRANCESCO, D. (1993). Properties of the hyperpolarization-activated current in rat hippocampal CA1 pyramidal cells. Journal of Neurophysiology 69, 2129-2136 | [Medline] |
| MAKARA J. K., PETHEÖ, G. L., TÓTH, A. & SPÄT, A. (2001). pH-sensitive inward rectifying chloride current in cultured rat cortical astrocytes. Glia 34, 52-58 | [Medline] |
| NOBILE M., PUSCH, M., RAPISARDA, C. & FERRONI, S. (2000). Single-channel analysis of a ClC-2-like chloride conductance in cultured rat cortical astrocytes. FEBS Letters 479, 10-14 | [Medline] |
| PANISELLO J. M. & DONNELLY, D. F. (1998). Chemotransduction by carotid body chemoreceptors is dependent on bicarbonate currents. Respiratory Physiology 112, 265-281 | |
| PEERS C. & BUCKLER, K. J. (1995). Transduction of chemostimuli by the type I carotid body cell. Journal of Membrane Biology 144, 1-9 | [Medline] |
| PEERS C. & GREEN, F. K. (1991). Inhibition of Ca2+-activated K+ currents by intracellular acidosis in isolated type I cells of the neonatal rat carotid body. Journal of Physiology 437, 589-602 | [Abstract] |
| PUSCH M., JORDT, S. E., STEIN, V. & JENTSCH, T. J. (1999). Chloride dependence of hyperpolarization-activated chloride channel gates. Journal of Physiology 515, 341-353 | [Abstract/Full Text] |
| REYES R., DUPRAT, F., LESAGE, F., FINK, M., SALINAS, M., FARMAN, N. & LAZDUNSKI, M. (1998). Cloning and expression of a novel pH-sensitive two pore domain K+ channel from human kidney. Journal of Biological Chemistry 273, 30863-30869 | [Abstract/Full Text] |
| STEA A. & NURSE, C. A. (1989). Chloride channels in cultured glomus cells of the rat carotid body. American Journal of Physiology 257, C174-181 | [Medline] |
| STRANGE K., EMMA, F. & JACKSON, P. S. (1996). Cellular and molecular physiology of volume-sensitive anion channels. American Journal of Physiology 270, C711-730 | [Medline] |
| THIEMANN A., GRÜNDER, S., PUSCH, M. & JENTSCH, T. J. (1992). A chloride channel widely expressed in epithelial and non-epithelial cells. Nature 356, 57-60 | [Medline] |
| THOMAS J. A., BUCHSBAUM, R. N., ZIMNIAK, A. & RACKER, E. (1979). Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 18, 2210-2218 | [Medline] |
| VERHEUGEN J. A., FRICKER, D. & MILES, R. (1999). Noninvasive measurements of the membrane potential and GABAergic action in hippocampal interneurons. Journal of Neuroscience 19, 2546-2555 | [Abstract/Full Text] |
| VERHEUGEN J. A. & VIJVERBERG, H. P. (1995). Intracellular Ca2+ oscillations and membrane potential fluctuations in intact human T lymphocytes: role of K+ channels in Ca2+ signaling. Cell Calcium 17, 287-300. | [Medline] |
| WALDMANN R., CHAMPIGNY, G., BASSILANA, F., HEURTEAUX, C. & LAZDUNSKI, M. (1997). A proton-gated cation channel involved in acid-sensing. Nature 386, 173-177 | [Medline] |
| WEINREICH F. & JENTSCH, T. J. (2001). Pores formed by single subunits in mixed dimers of different ClC chloride channels. Journal of Biological Chemistry 276, 2347-2353 | [Abstract/Full Text] |
Acknowledgements
The authors thank Ms Anikó Rajki and Mr Csaba Fülöp for their skilful assistance and Dr Felicia Slowik MD PhD and Dr Gábor Nagy MD for their help in the identification of the carotid body and chemoreceptor cells. This work was supported by the Hungarian National Science Foundation (OTKA T 026173), Council for Medical Sciences (ETT 528/96) and the Hungarian Academy of Sciences (AKP 96-236/53).
Corresponding author
A. Spät: Department of Physiology, Semmelweis University, Faculty of Medicine, PO Box 259, H-1444 Budapest, Hungary.
This article has been cited by other articles:
|
|
 |
|
  J. R. Lopez-Lopez and M. T. Perez-Garcia An ASIC Channel for Acid Chemotransduction Circ. Res., November 9, 2007; 101(10): 965 - 967. [Full Text] [PDF]
|
|
|
|
|
|
Z.-Y. Tan, Y. Lu, C. A. Whiteis, C. J. Benson, M. W. Chapleau, and F. M. Abboud Acid-Sensing Ion Channels Contribute to Transduction of Extracellular Acidosis in Rat Carotid Body Glomus Cells Circ. Res., November 9, 2007; 101(10): 1009 - 1019. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. W. Putnam, J. A. Filosa, and N. A. Ritucci Cellular mechanisms involved in CO2 and acid signaling in chemosensitive neurons Am J Physiol Cell Physiol, December 1, 2004; 287(6): C1493 - C1526. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), and a solution in which NMDG-Cl was replaced by sucrose (Solution III, 
). Tail current amplitude was determined by fitting the slow component of the tail (as indicated by the boxes) with a single exponential (white lines). The exponential was extrapolated back to the zero time point of the step. The difference between the current amplitude at zero time and at the end of the fit was considered as the tail current amplitude (n = 5).