Published online before print September 13, 2007, doi: 10.1161/CIRCRESAHA.107.154377
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© 2007 American Heart Association, Inc.
Cellular Biology |
Acid-Sensing Ion Channels Contribute to Transduction of Extracellular Acidosis in Rat Carotid Body Glomus Cells
From the Cardiovascular Center and Departments of Internal Medicine (Z.-Y.T., Y.L., C.A.W., C.J.B., M.W.C., F.M.A.) and Molecular Physiology and Biophysics (M.W.C., F.M.A.), University of Iowa, Iowa City; and Veterans Affairs Medical Center (M.W.C.), Iowa City, Ia.
Correspondence to Francois M. Abboud, MD, Department of Internal Medicine, University of Iowa, 200 Hawkins Dr, 616 MRC, Iowa City, IA 52242. E-mail francois-abboud{at}uiowa.edu
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Abstract |
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Carotid body chemoreceptors sense hypoxemia, hypercapnia, and acidosis and play an important role in cardiorespiratory regulation. The molecular mechanism of pH sensing by chemoreceptors is not clear, although it has been proposed to be mediated by a drop in intracellular pH of carotid body glomus cells, which inhibits a K+ current. Recently, pH-sensitive ion channels have been described in glomus cells that respond directly to extracellular acidosis. In this study, we investigated the possible molecular mechanisms of carotid body pH sensing by recording the responses of glomus cells isolated from rat carotid body to rapid changes in extracellular pH using the whole-cell patch-clamping technique. Extracellular acidosis evoked transient inward current in glomus cells that was inhibited by the acid-sensing ion channel (ASIC) blocker amiloride, absent in Na+-free bathing solution, and enhanced by either Ca2+-free buffer or addition of lactate. In addition, ASIC1 and ASIC3 were shown to be expressed in rat carotid body by quantitative PCR and immunohistochemistry. In the current-clamp mode, extracellular acidosis evoked both a transient and sustained depolarizations. The initial transient component of depolarization was blocked by amiloride, whereas the sustained component was eliminated by removal of K+ from the pipette solution and partially blocked by the TASK (tandem-p-domain, acid-sensitive K+ channel) blockers anandamide and quinidine. The results provide the first evidence that ASICs may contribute to chemotransduction of low pH by carotid body chemoreceptors and that extracellular acidosis directly activates carotid body chemoreceptors through both ASIC and TASK channels.
Key Words: carotid body • ASIC • glomus cell • chemoreceptors • pH sensitivity
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Introduction |
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The carotid bodies function as major peripheral chemoreceptors sensing changes in arterial blood oxygen, carbon dioxide, and pH in mammals.1–3 Glomus type I cells are generally accepted as the chemosensitive elements activated by hypoxemia, hypercapnia, and acidosis.3 The common cellular mechanism for glomus cell transduction is the release of neurotransmitters such as ATP, acetylcholine, and dopamine as a result of an increase in intracellular Ca2+ caused by stimulus-induced membrane depolarization.4–7 This leads to synaptic activation of adjacent carotid nerve endings that elicits both hyperventilation and sympathetic activation.1,8
The molecular identity of the PO2, PCO2, and pH sensors is still an important question. The depolarization induced by hypoxemia has been ascribed to a closure of K+ channels including large-conductance, Ca2+-activated potassium (BK) channels.3,9–12 The transduction of acidosis to membrane depolarization was proposed to be mediated by a drop in intracellular pH which would inhibit 1 or more types of K+ channels, leading to membrane depolarization.3,13,14 The findings of extracellular pH-sensitive ion conductances, including pH-sensitive Cl– currents15 and the non–voltage-gated TASK (tandem-p-domain, acid-sensitive K+ channel)-like currents16 in glomus cells, suggest that extracellular acidosis per se might contribute to membrane depolarization.
In this study, we tested the hypothesis that extracellular acidosis directly activates pH-sensitive ion channels on carotid body glomus cells. We found a transient response that is comparable biophysically and pharmacologically to acid-sensing ion channels (ASICs), as well as the expression of ASIC1 and ASIC3 in carotid bodies. Additionally, we described a sustained extracellular acidosis-evoked response consistent with inhibition of TASK-like channels.
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Materials and Methods |
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Published protocols for isolation of glomus cells from rat carotid bodies were followed.10,17 Conventional whole-cell patch-clamp recordings from glomus cells provided data on membrane currents and voltage potentials during fast exchange of the varying pH solutions extracellularly, whereas intracellular pH was buffered at pH 7.2. Carotid body mRNA and proteins of various ASICs were measured using real-time RT-PCR and immunohistochemistry.
An expanded Materials and Methods section is in the online data supplement at http://circres.ahajournals.org.
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Results |
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Acid-Evoked Currents in Glomus Cells
We used brief applications of acidic solutions while maintaining a high intracellular buffering capacity using Hepes to prevent intracellular pH changes. The cells were voltage clamped at –60 mV. Extracellular solutions at pH 7, 6, 5, and 4 triggered transient, rapidly activating and desensitizing inward currents (Figure 1). The threshold for activation occurred at about pH 7, and the currents were half-activated at pH 6.3, as shown in Figure 1. The Table shows that 34.9% of glomus cells responded to pH 6 with an average inward current of 19.2±4.9 pA and 52.9% responded to pH 5 with 32.5±4.3 pA. The time constant for deactivation at pH 6.0 was 1.8±0.22 seconds. There was no significant difference in the size (cell diameter) and membrane capacitance of responder versus nonresponder glomus cells (Table). During activation of the inward current, a significant increase in the magnitude of current oscillations occurred at peak currents as compared with baseline oscillations at pH 7.4 (Figures 1 and 2
). These waned rapidly as the current inactivated.
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Acid-Evoked Currents in Glomus Cells Display Properties Consistent With ASICs
The pH sensitivity and rapid desensitization suggested that the acid-evoked currents in glomus cells might be generated by ASICs. To further explore this, we tested additional biophysical and pharmacological properties of the currents. Unlike many ligand-gated channels, ASICs preferentially conduct Na+. Removal of Na+ from the extracellular solution significantly reduced inward current amplitudes (Figure 2
A). Note that the Na+-free bath also decreased the baseline current oscillations. Moreover, the degenerin/epithelial Na+ channel blocker amiloride (50 and 200 µmol/L) inhibited in a dose-dependent manner and reversibly the acid-evoked currents (Figure 2B). In addition to this rapidly activating and desensitizing inward current blocked by Na+-free solutions and by amiloride, a small, more sustained inward current remained during the period of exposure to low pH (Figure 2). TRPV1 (transient-receptor potential, vanilloid type 1) channel blocker capsazepine (20 µmol/L) had no effect on both the rapid transient and sustained small inward currents (Figure 2C). Psalmotoxin venom (PcTX1), the blocker of ASIC1a,18 had no effect on the acid-evoked current in glomus cells, but it did reduce it in hippocampus neurons (Figure 2D). Similarly, the BK channel blocker iberiotoxin (IbTX) did not alter the current (Figure 2E).
Previous studies have shown that decreasing extracellular Ca2+ concentration facilitates activation of ASIC currents.19,20 As shown in Figure 3A, currents evoked by pH 6.0 were dramatically enhanced in the absence of extracellular Ca2+. The Table also shows that when Ca2+ was removed from the acid solution of pH 6.0, the percentage of responding cells almost doubled to 66.7% and the peak inward current more than doubled to 56.8±7.9 pA, but the time constant of deactivation was not significantly changed (1.6±0.21 seconds; n=19). A higher concentration of amiloride (400 µmol/L) was necessary to block the enhanced transient current (Figure 3B). Responses to pH 6 with and without Ca2+ appeared to be enhanced when a CO2/HCO3– buffered instead of a Hepes-buffered bath solution was used (see the Table).
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Lactic acid is generated during tissue ischemia and also during exercise. It is known to potentiate ASIC channels probably by reducing extracellular Ca2+ concentration.21 Addition of lactate (30 mmol/L) to the extracellular solution enhanced the pH-sensitive transient current (Figure 3C).
Expression of ASICs in the Carotid Body
ASICs are expressed in various populations of sensory nerves, but their expression in the carotid body has not been previously explored. Using primers specific for individual ASICs, we identified with real time PCR the mRNAs of each ASIC subunit normalized against the expression of GAPDH. Compared with the mRNA level of ASIC3, the expression of ASIC1b was relatively high, whereas that of ASIC2a, ASIC2b, and ASIC1a was low (Figure 4A). We also identified relatively high mRNA levels of the BK
subunit, which is known to be important for oxygen sensing in carotid body.
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To test whether ASIC proteins are expressed in rat glomus cells in situ, we used confocal immunofluorescence analyses. With an antibody directed against both ASIC1a and -1b subunits, we found that the glomus cell clusters showed ASIC1 immunoreactivity (green) (Figure 4B2 and 4B6). Double-staining revealed weak ASIC2 (using antibody directed against the ASIC2a splice variant) (Figure 4B3) and stronger ASIC3 immunoreactivity (red) (Figure 4B7) in most cells that were ASIC1 positive. Intercluster spaces appeared to be devoid of staining, and several lone nuclei suggested that there was some cell selectivity in staining, although the majority appeared to stain positively. The absence of a primary antibody prevented any immunostaining (Figure 4B10). Moreover, the transfection of a COS-7 cell with ASIC2 resulted in significant fluorescence (Figure 4B12), which was absent from the control nontransfected cell (Figure 4B11).
Acid-Induced Transient and Sustained Depolarization of Glomus Cells
The membrane potential of glomus cells was recorded using the conventional whole-cell patch-clamp technique. The resting membrane potential ranged from –20 to –60 mV and averaged –33.2±1.3 mV (n=52). These values were similar to some reports22–24 but were more positive than other reports obtained in neonatal rats and in rabbits using the perforated-patch clamp.25–27 Acidic pH (6.0) triggered a transient depolarization (20.0±1.5 mV) in 28 of 52 glomus cells (53.8%) and a sustained depolarization (20.5±1.2 mV) in all 52 cells (100%) (Figure 5). The transient depolarization was reversibly blocked by amiloride, but the sustained depolarization was not blocked by amiloride (Figure 5A and 5B). The sustained depolarization at pH 6.0 was not significantly different between cells that showed a transient depolarization (n=28) and those that did not (n=24) (20.9±1.8 versus 20.1±1.8 mV, respectively). In addition, the magnitude of the sustained depolarization was pH dependent (Figure 6A) and lasted throughout the period of exposure to low pH.
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mV±SE) recorded during the first 5 seconds of exposure to low pH (transient depolarization, black bars) and the maximal sustained decrease in membrane potential measured during the last 5 to 10 seconds of the period of low pH exposure (shaded bars). Amiloride inhibited the transient but not the sustained depolarization. *P<0.01 for amiloride inhibition of the transient depolarization compared with control and recovery.
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Dependence of Sustained Depolarization on [K+]i
The acid-induced responses in glomus cells and chemosensing neurons have been proposed to be mediated by inhibition of K+ conductance.3,13 To test this hypothesis, intracellular K+ was reduced using a K+-free pipette solution and substituting N-methyl-D-glucamine Cl for KCl. In 5 such cells, pH 6.0 did not cause a sustained depolarization and in fact caused a slight hyperpolarization (Figure 6B). However, as expected, removal of intracellular K+ also produced a predictable depolarization to a positive resting membrane potential (RMP). To test whether the loss of pH-evoked sustained depolarization was attributable to removal of intracellular K+ or the resultant change in RMP from a negative to a positive value, we used normal KCl (140 mmol/L) as the intracellular solution but injected depolarizing current to achieve a positive RMP (13.8±3.5 mV) close to that caused by K+-free pipette solution. As shown in Figure 6C, pH 6 induced the sustained depolarization despite the positive RMP.
To test whether there was a contribution of pH-sensitive Cl– conductance or Na+-K+ ATPase to the sustained depolarization, CdCl2 (blocker of pH-sensitive Cl– current in glomus cell)15 and ouabain (inhibitor of Na+-K+ ATPase) were applied. Neither of them had a significant effect on the pH 6.0–induced sustained depolarization (Figure 6F).
Thus, our data indicate that the low pH-evoked sustained depolarization is attributable to inhibition of K+ conductances. To begin to test the possible contribution of different K+ channels to this sustained depolarization, IbTX (BK channel blocker), tetraethylammonium (voltage-gated K+ channel blocker), anandamide (TASK-1 channel blocker),28 and quinidine (inhibitor of TASK-like currents in glomus cells)16 were used (Figure 6D through 6F). Although IbTX, anandamide, and quinidine depolarized the membrane, only anandamide and quinidine partially inhibited the low pH-induced sustained depolarization.
In Figure I of the online data supplement, we show that both IbTX and low pH suppress the Ca2+-activated component of the K+ currents at positive membrane potentials, yet BK does not appear to contribute to the acid-evoked inward current or depolarizations. These results are discussed below and in the online data supplement.
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Discussion |
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We found that rapid extracellular acidosis generated both transient and sustained currents and depolarizations in isolated glomus cells. The transient responses had biophysical and pharmacological properties consistent with ASICs. Moreover, we found evidence for significant expression of 2 ASIC subunits, ASIC1 and ASIC3, within the carotid body and glomus cells in particular. Additionally, we characterized a sustained acid-evoked depolarization with properties consistent with TASK-like channels as previously described.16,26 Our data provide the first evidence that ASICs function as pH sensors in glomus cells of rat carotid bodies.
ASICs and Their Expression in Glomus/Carotid Body
In recent years, we and others have examined the role of the degenerin/epithelial Na+ channel family of non–voltage-gated cationic channels in sensory signaling.29–31 ASICs are members of this family that are directly gated by acidic extracellular pH.31–33 Three ASIC genes and their alternatively spliced transcripts (ASIC1a, -1b, -2a, -2b, and -3) generate homomultimeric or heteromultimeric acid-activated ion channels, each with different pH sensitivity and current characteristics.34–36 ASIC1a, -1b, and -3 subunits are most sensitive to changes in pH; the half-maximal activation of both channels occurs at approximately pH 6.5,30,34,37 and the threshold of activation of ASIC3 may occur at near neutral pH under certain conditions.37 ASIC2 channels, on the other hand, are far less sensitive to pH change (pH50
4.5).36 ASICs are primarily expressed in neurons of the central nervous system and in peripheral sensory neurons. In the latter, they are purported to transduce sensory stimuli including nociception, mechanoreception, and chemosensation.31–33 In taste cells, ASICs may function as sour-taste receptors.38 In glioma cells, the presence of ASIC2 in the plasma membrane appears to influence cell growth and migration.39
This is the first report of the presence of ASICs in a mammalian arterial chemoreceptor. mRNA encoding of ASIC subunits and ASIC proteins were detected in rat carotid body. In parallel, ASIC currents were recorded from isolated glomus cells. These data lend further support for a generalized role of ASICs in sensory transduction. Interestingly, we found relatively high expression of the more pH-sensitive subunits, ASIC1b and ASIC3, and relatively low expression of the least pH-sensitive ASIC2.30,34–37
ASICs Impart Unique pH Sensory Characteristics to Glomus Cells
Rapid perfusion of extracellular protons triggered a transient current and depolarization up to 66% of glomus cells studied. The currents were carried by extracellular Na+, inhibited by amiloride (a degenerin/epithelial Na+ channel blocker), and potentiated by lowering of extracellular [Ca2+] or addition of lactate. A higher percentage of glomus cells (9/10 cells) responded to low pH at zero Ca2+ when a CO2/HCO3– buffer was used instead of Hepes (Table). This may have been the result of a more stable intracellular pH.40 These properties, as well as the fast-gating kinetics, are all consistent with ASICs. Coincidently, capsazepine, a blocker of another desensitizing pH-activated channel, TRPV1, had no effect on the currents.
Our recorded current properties and expression data provide some insight into the molecular make up of these ASICs. The pH50 of glomus cell currents was 6.3, and pH 7 was sufficient to generate current. This implies a major contribution of ASIC3 to the currents.30,37 Correspondingly, we found high expression of ASIC3 in the carotid body. This also suggests that ASIC2 is not a significant contributor to the currents, as we found very low expression of ASIC2 subunits. This finding fits with the notion that ASIC2 is perhaps more important in mechanosensation, as targeted deletion of ASIC2 alters mechanoreceptive properties in mouse skin,41 gut,42 and aortic baroreceptor neurons.43 Moreover, we found that ASIC2 deletion in mice impairs the carotid occlusion baroreflex, whereas ASIC1 and ASIC3 deletions impair the carotid occlusion chemoreflex.44
We also found significant expression of ASIC1b mRNA in the carotid body, and ASIC1 coimmunostained with ASIC3 in most glomus cells. This implies that ASIC1b could either form separate homomeric channels or it might combine with ASIC3 to form heteromeric channels. In fact, we have previously shown that in mouse sensory neurons from the dorsal root ganglia, most ASIC channels are heteromultimers comprised of multiple subunits.34 However, closer inspection of the ASIC-like current properties in glomus cells suggests the channels are ASIC homomers because their rate of desensitization was relatively slow and it is known that homomeric ASIC channels desensitize slowly compared with heteromeric channels.34,36 We also found that PcTX1, the selective blocker of ASIC1a, did not reduce the acid-evoked current in glomus cells, but it did so in hippocampus neurons, where ASIC1a expression is prevalent. On the whole, our studies suggest that ASIC3 is a principal functional acid sensing subunit and that ASIC1 and ASIC2 may have a lesser contribution to the ASIC current phenotype in glomus cells. Interestingly, we recorded transient acid-evoked responses in 66% of cells, and yet almost all glomus cells appeared immunopositive for ASIC subunits. This discrepancy suggested that ASIC subunits may not form functional acid sensitive channels in all glomus cells. On the other hand, when a HCO3– instead of Hepes buffer was used, 9 of 10 glomus cells revealed an inward current, possibly reflecting a more effective buffering of intracellular pH with HCO3–.40,45
We also recognize that the reliability of quantitative comparisons between the relative expression of different ASICs may be questioned. However, we find support for our conclusion by contrasting the expression of the same ASIC subunit in nodose ganglion neurons to that in the carotid body. The very low expression of ASIC2a and -2b mRNA in the carotid body contrasts with their high expression in the nodose ganglia, where they may function primarily as mechanosensors.43,46 Using the anti-ASIC2a antibody, we also observed greater immunofluorescence activity in nodose ganglia than in carotid body.46 The relatively high expression of ASIC3 message and protein in glomus cells may reflect their function as primary ASIC sensors of low pH.
The capacity of lactate to facilitate ASIC currents in glomus cells has potentially important physiological implications. A decrease in extracellular Ca2+ concentration has been found to facilitate proton gating of ASICs.19,20 In addition, lactate enhances ASIC currents by chelating extracellular Ca2+, potentially sensitizing cardiac sensory neurons to sense ischemia.21 In the present study, we found a similar phenomenon: ASIC activation in glomus cells was dramatically enhanced in Ca2+-free solution and significantly increased by lactate. The peripheral chemoreceptor reflexes have been found to contribute to ventilatory stimulation during exercise-mediated metabolic acidosis as well.3,7,47–49 Moreover, lactate concentration in the blood reaches levels during maximal exercise (8 to 20 mmol/L)50 that are known to potentiate ASIC channels.21 Thus, the unique capacity of ASIC channels to integrate both H+ and lactate suggests that they might be particularly important pH sensors during metabolic acidosis associated with tissue ischemia or maximal exercise.
Most ASIC channels desensitize despite the continued presence of low pH. This property may limit the transduction of low pH by ASICs compared with other purported pH sensors in glomus cells. However, many substances are known to modulate desensitization rates in ASICs,37,51 and this might afford a means for more sustained pH sensing properties of glomus cells. Although we did not record sustained amiloride-sensitive responses in our studies, ASIC channels can generate sustained acid-evoked responses under certain conditions such as myocardial ischemia,37 which may be important in mediating persistent responses to prolonged stimuli.
Role of Non–Voltage-Gated TASK-Like Channels in Acid Sensing
We also found that extracellular acidosis triggered a sustained, non-ASIC mediated depolarization of glomus cells throughout the period of exposure to low pH. Ion-substitution experiments suggested that inhibition of a K+ current is responsible for this sustained depolarization. Moreover, responses to a variety of channel blockers suggested to us that the underlying channel may be a TASK-like family member. This finding is consistent with results described earlier by Buckler and colleagues.16,26,52
Role of Ca2+-Activated K+ Channels in Acid Sensing
Transduction of acidosis in glomus cells may also be induced by inhibition of Ca2+-activated K+ channels (BK). Intracellular14 and extracellular53 acidosis have been shown to inhibit BK in type 1 glomus cells from rat and rabbit. This BK inhibition may occur without any effect on Ca2+ channels,53 implying a direct inhibitory effect of acidosis on BK. In other studies on glomus cells from adult rabbits, direct effects on L-type Ca2+ currents were described.54 Low pH of the extra- or the intracellular solutions inhibit Ca2+ currents by 43±4% and 13±1%, respectively. A similar inhibitory effect of acidosis on Ca2+ currents has been described also in other tissues55 and would be compatible with an inhibitory effect on BK.
Our results in supplemental Figure IA through IC show that IbTX (n=8) and extracellular acidosis (n=4) both suppress significantly the Ca2+-activated component of the K+ current seen predominantly at the positive membrane potentials on the current–voltage relationship curve. However, neither the acid-evoked inward-current nor the acid-evoked depolarizations, which are induced from negative RMPs, were affected by IbTX (Figures 2E, 6E, and 6F). Hence our results favor a more dominant role of the non–voltage-gated depolarizing currents ASICs and TASK as mediators of depolarizations of glomus cells during extracellular acidosis.
O2 and pH/CO2 Sensing in Glomus Cells Involve Multiple Molecular Sensors
Our data, along with other reports on chemosensory by voltage-gated BK and Ca2+ channels as well as non–voltage-gated Na+ (ASIC) and K+ (TASK) currents, underline the complexity of the effects of hypoxia, hypercapnia, and acidosis at this juncture. The online data supplement includes supplemental results and discussion of the effect of acidosis and hypoxia on BK, ASICs, and TASK.
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Acknowledgments |
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We thank Shawn M. Averkamp for typing the manuscript and Shawn Roach for preparation of the figures. We also thank Dr John Wemmie for providing us with an early supply of PCTX venom and Dr Michael Welsh for his critical review of the manuscript.
Sources of Funding
This work was supported by NIH grant HL14388-36.
Disclosures
None.
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Footnotes |
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Original received April 20, 2007; revision received August 29, 2007; accepted September 5, 2007.
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