acid-sensing ion channel 3
high conductance Ca2+-activated K+ channel
two-pore-domain acid-sensitive potassium channels
Carotid body glomus cells are activated by hypoxia and acidosis, but their capacity to differentiate between the two has been undefined.
This is the first work to quantify a differential sensory transduction of hypoxia and acidosis with reciprocal responses in individual glomus cells.
Cytoplasmic [Ca2+] in clusters of glomus cells indicates 68% of glomus cells respond to both hypoxia and acidosis but are selectively more sensitive to one or the other; the rest respond to either hypoxia (19%) or acidosis (13%).
This uncoupling/reciprocal response was recapitulated in a mouse model by genetically altering the expression of ASIC3, an acid-sensing ion channel that we had identified in earlier studies as a mediator of pH sensitivity in carotid body.
We speculate that selective sensory transduction of glomus cells to either hypoxia or acidosis may result in activation of afferents preferentially more sensitive to hypoxia or acidosis, perhaps evoking more specific autonomic adjustments to each stimulus.
Abstract Carotid body glomus cells are the primary sites of chemotransduction of hypoxaemia and acidosis in peripheral arterial chemoreceptors. They exhibit pronounced morphological heterogeneity. A quantitative assessment of their functional capacity to differentiate between these two major chemical signals has remained undefined. We tested the hypothesis that there is a differential sensory transduction of hypoxia and acidosis at the level of glomus cells. We measured cytoplasmic Ca2+ concentration in individual glomus cells, isolated in clusters from rat carotid bodies, in response to hypoxia ( mmHg) and to acidosis at pH 6.8. More than two-thirds (68%) were sensitive to both hypoxia and acidosis, 19% were exclusively sensitive to hypoxia and 13% exclusively sensitive to acidosis. Those sensitive to both revealed significant preferential sensitivity to either hypoxia or to acidosis. This uncoupling and reciprocity was recapitulated in a mouse model by altering the expression of the acid-sensing ion channel 3 (ASIC3) which we had identified earlier in glomus cells. Increased expression of ASIC3 in transgenic mice increased pH sensitivity while reducing cyanide sensitivity. Conversely, deletion of ASIC3 in the knockout mouse reduced pH sensitivity while the relative sensitivity to cyanide or to hypoxia was increased. In this work, we quantify functional differences among glomus cells and show reciprocal sensitivity to acidosis and hypoxia in most glomus cells. We speculate that this selective chemotransduction of glomus cells by either stimulus may result in the activation of different afferents that are preferentially more sensitive to either hypoxia or acidosis, and thus may evoke different and more specific autonomic adjustments to either stimulus.
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The activation of peripheral chemoreceptors, as a result of a drop in or pH, initiates a powerful neurogenic reflex which causes hyperventilation to restore and pH, and regional autonomic circulatory adjustments to preserve oxygenation of vital organs. In the carotid body the glossopharyngeal nerve endings are the chemosensory afferents of neurons in the petrosal ganglia. Action potentials are triggered in those terminals and relayed to the nucleus of the tractus solitarius in the medulla to evoke the chemoreceptor reflex (Pallot, 1987; Lopez-Barneo et al. 1988; Prabhakar & Peng, 2004; Lahiri et al. 2006; Kumar, 2009; Peers et al. 2010; Kumar & Prabhakar, 2012). A most comprehensive review on peripheral chemoreceptors and the function and plasticity of the carotid body was published earlier this year (Kumar & Prabhakar, 2012).
A unique feature of chemoreceptor signalling is the primary site of signal transduction which consists of clusters of small round cells approximately 10μm in diameter known as glomus type I cells.
A drop in or pH causes their depolarization (Buckler & Vaughan-Jones, 1994a, b; Weiss & Donnelly, 1996; Buckler et al. 2000; Tan et al. 2007). Several ion channels are involved in this depolarization which is associated with a rise in intracellular Ca2+ concentration (Lopez-Lopez et al. 1997; Summers et al. 2002; Tan et al. 2007, 2010; Buckler, 2007; Liu et al. 2011). The rise in [Ca2+]i evokes the vesicular release of a variety of transmitters including acetylcholine, adenosine triphosphate, dopamine and noradrenaline (norepinephrine) that act on the sensory nerve terminals (Vicario et al. 2000; Nurse, 2005; Prabhakar, 2006).
The glomus cells exhibit a profound degree of morphological heterogeneity in several species (Morita et al. 1969; Hellström, 1975; Schamel & Verna, 1992). Their functional heterogeneity has also been reported. Biscoe et al. (1970) were first to show that the same chemosensory fibre may be activated by both hypoxia and acidosis. Dasso et al. (2000) reported that hypoxia, CO2 and acidosis can activate a majority of neonatal rat glomus cells.
An important question therefore is whether all type I glomus cells are equally sensitive to hypoxia and acidosis or is there a differential sensitivity that allows the distinctive sensory recognition of each signal? The inability to distinguish these two major chemical signals seems functionally inefficient. Therefore we tested the hypothesis that there is a differential sensory transduction of these two stimuli at the level of glomus cells. The fact that the physiological reflex responses to activation of the carotid body by these two stimuli appear to be similar suggests that the glomus cells are equally sensitive to them (Prabhakar & Peng, 2004; Kumar, 2009; Peers et al. 2010). Otherwise, one might have expected a different efferent response to these two markedly different sensory signals. Moreover, the numerous gap junctions connecting glomus cells (Pallot, 1987; Abudara et al. 2002; Kumar & Prabhakar, 2012) suggest that there might be a rapid spread of depolarization between glomus cells in the same cluster provoking a generalized pattern of activation with both hypoxia and acidosis.
On the other hand several observations favour a non-uniform activation of glomus cells. First their structural heterogeneity is pronounced and some have suggested several different types based on the density of vesicles and their size (Morita et al. 1969). Second is the frequently reported additive or synergistic effect of hypoxia and acidosis. Hypoxia enhances significantly the effect of acidosis on chemoreceptor nerve activity (Lahiri & DeLaney, 1975). This synergism suggests that different ionic or molecular pathways may be selectively activated by hypoxia or acidosis (Lahiri & Forster, 2003; Kumar & Prabhakar, 2012) and their expression may vary in different glomus cells or even in different clusters.
In the work presented here, we tested the responsiveness of glomus cells to hypoxia and acidosis, and identified a pattern of reciprocal sensitivity which we replicated by altering the gene expression of the acid-sensing ion channels (Tan et al. 2007, 2010). We speculate that such a selectivity at the level of sensory transduction would be expected to evoke differential and more optimal reflex autonomic responses to hypoxia and acidosis that have yet to be confirmed.
University of Iowa policy and federal regulations require a review of projects for the humane treatment and safe use of vertebrate animals in research and teaching by all University of Iowa staff on or off campus. At the University of Iowa, the review is conducted by the Institutional Animal Care and Use Committee. All requirements have been followed and approved in regards to the work in this paper.
Experiments were carried out on isolated clusters of carotid body glomus cells from rats and from genetically modified mice.
Isolation and culture of glomus cells
The experiments were performed according to previously published protocols (Lopez-Lopez et al. 1997; Xu et al. 2003; Tan et al. 2007). All procedures carried out were approved by the University of Iowa Animal Care and Use Committee and followed the guidelines of the American Physiological Society.
Briefly, rats or mice were deeply anaesthetized with isoflurane inhalation and then decapitated. The region of the common carotid artery bifurcation including the carotid bodies was excised via a midline surgical approach. Both carotid bodies were removed and incubated with Ca2+- and Mg2+-free Tyrode solution containing collagenase (2 mg ml−1, Type IV; Sigma), deoxyribonuclease (DNase II, 0.5 mg ml−1; Sigma) and trypsin (0.4 mg ml−1) for 25 min at 37°C. The tissue was then gently triturated and cultured. (See extended methods in the online Supplemental material for more details regarding the isolation of clusters of glomus cells in mice versus rats.)
The cells were loaded with the acetoxymethyl ester form of Fluo-4, Fluo-4/AM (5 μm; Invitrogen- Molecular Probes), a single wavelength fluorescent probe, for 30 min at room temperature (23°C), in the bath solution containing 0.05% Pluronic-127 (see extended methods in online supplement).
The advantages of the use of Fluo-4 as a non-ratiometric probe compared to indo-1 or fura-2 and its stability over the experimental range from pH 6.0 to 7.4 are reviewed in the online Supplemental material (see supplemental fig 2).
Subsequently, cells were washed and incubated in bath solution at room temperature for 45 min in order to de-esterify the intracellular Fluo-4/AM prior to the experiments. The calcium ionophore 20 μm ionomycin and 2 mm EGTA (Sigma) were used to determine maximum and minimum fluorescence intensity (Fmax and Fmin), respectively (Grynkiewicz et al. 1985; Payne & Demas, 2000).
Sensitivity of imaging system
The resolution of our imaging system allowed us to discriminate quantitatively the responsiveness of individual cells and we were able to detect significant differences between neighbouring cells in the same cluster that were reliable and reproducible (see extended methods in online supplement material).
We could therefore conclude that despite the presence of symmetrical junctions (desmosomes) between glomus cells (Hess, 1975; Abudara et al. 2002; Hayashida & Hirakawa, 2002) the electrogenic coupling that might have been expected to unify the response possibly in all the cells of the same cluster did not take place.
The normal bath solution (, ∼140 mmHg; pH 7.4) was prepared from Hank's balanced salt solution (HBSS, purchased from GIBCO Invitrogen) consisting of (millimolar concentrations): 136.9 NaCl, 5.4 KCl, 4.2 NaHCO3, 1.3 CaCl2, 0.8 MgSO4, 0.4 NaH2PO4, 5.6 d-glucose, 10 Hepes. The hypoxic solution was the normal solution bubbled with 95% N2+ 5% CO2 (, 15 mmHg; pH 7.4). The low pH solution was obtained by adding acetic acid to reach a level of pH 6.8 or 6.0 (see online supplemental material on the composition of solutions and the stability of pH).
Selection of pH 6.8/6.0 as the acidotic stimulus
It could be suggested that if we had lowered the pH to levels below 6.8 the responses of rat glomus cells would have been greater. We felt, however, that pH levels closer to 6.8 would be more meaningful from a functional perspective in contrasting them to hypoxic levels of 15 mmHg. Moreover, several cells had a very pronounced increase in [Ca2+]i in response to pH 6.8, exceeding the response to hypoxia (Fig. 3C). In other cells that were less sensitive to pH than to hypoxia, a pH of 6.0 did not induce a higher Ca2+ transient than hypoxia (Fig. 3A).
In glomus cells from the genetically modified mice, responses to acidosis were studied at pH 6.0. Responses to pH 6.8 were generally smaller in mice than in rats, and we used a pH level of 6.0 to ascertain the maximal activation of ASICs in the three genotypes and establish a clear association of the acid-sensitive phenotype with the genotype. The association was confirmed (see Fig. 5A). Responses to pH 6.0 were significantly reduced in ASIC3 knockout (KO) and enhanced in ASIC3 transgenic (Tg) compared to wild-type (WT) mice. The reduction in ASIC3 KO could not be ascribed to a submaximal pH-induced activation but rather to the absence of ASIC3.
The response to 1 mm of the metabolic inhibitor sodium cyanide (NaCN; Sigma) was contrasted with that to pH 6.0 in each genotype (Wyatt & Buckler, 2004). (See details in online Supplemental material.)
A coverslip with attached clusters of glomus cells was transferred to a RC-20 recording and perfusion chamber (Warner Instruments) and mounted on an inverted microscope (Nikon TE2000-u, Melville, NY, USA). The chamber was perfused by gravity from a 50 ml syringe containing the normal solution (, ∼140 mmHg; pH 7.4). Other solutions were perfused through the chamber by gravity from different syringes connected to a manually controlled multiple channel switch stop. (See details of preparation in online Supplemental material.)
Generation of ASIC3 KO mice
The ASIC3 KO mutants used in this study were generated as previously described by Drew et al. (2004). (See online Supplemental fig 1 that shows the gel identifying bands of WT and mutant alleles in ASIC3 KO mice.)
Generation of ASIC3 Tg mice
The ASIC3 Tg mice were generated in the Welsh lab of the University of Iowa. The procedures were similar to those published by Vralsted et al. (2011). (See online Supplemental material for details.)
We used the appropriate analysis tools provided by Excel. The probability associated with Student's t test determined whether two samples were likely to have come from the same population. The ANOVA provided the probability that the distribution of the data for single factors in three sample groups was the same. The linear regression produced the slope of a line that best fitted a single set of correlations between two variables. A slope of 1 is the ‘line of identity’ when the two variables are identical. The R2 value of the correlation coefficient shows how well the data points fit the linear regression line.
Clusters of carotid body glomus cells were cultured and loaded with Fluo-4/AM and exposed to solutions with varying and pH (see extended methods in online Supplemental material).
The switching from a normal perfusate ( mmHg, pH 7.4) to the hypoxic one ( mmHg, pH 7.4) caused a rapid drop of in the chamber solution to 15 mmHg (Fig. 1A), and a rapid [Ca2+]i transient (Fig. 1B) which returned quickly to baseline. After a 3 min wash period with a normal solution, the low pH solution ( mmHg, pH 6.8) was started (Fig. 1C), causing another [Ca2+]i transient. Peak responses were generally seen within 30 s of the change in solutions and returned to baseline. The responses to low and low pH differed while the response to 5 μm ionomycin was reproducible when given after low and low pH.
The repeated consecutive exposures of the same cell to pH 6.8 caused nearly identical [Ca2+]i responses (Fig. 1D). These findings add credence to the validity of quantitative comparisons of responses.
Responses of glomus cells within clusters
Bright field and fluorescence images were obtained on several individual cells in each cluster simultaneously. Under normoxic conditions at pH 7.4, baseline cytosolic [Ca2+]i ranged from 50 to 80 nm (Fig. 2A and B). After exposure to pH 6.8, variable increases in [Ca2+]i occurred in most cells. The increases did not correlate with the baseline values. They peaked within 30–50 s and gradually returned to control levels within 2–3 min.
A schematic diagram (Fig. 2C) portrays the peripheral chemoreceptor complex with the carotid body, a cluster of glomus cells with sensory nerve endings, and a single glomus cell with representative ion channels as the primary site of chemotransduction.
Reciprocal sensitivities to hypoxia and acidosis among clusters and glomus cells within each cluster
Figure 3A shows a cluster of glomus cells in a bright field exposure and the heterogeneous fluorescence among individual cells of the same cluster at the time of the peak responses to acidosis (pH 6.0), hypoxia (, 15 mmHg), and ionomycin (5 μm). In this cluster, maximal responses to low pH were smaller than responses to hypoxia.
Contrasting responses of two different clusters are also seen in Fig. 3B and C. Two important results are portrayed. The first is that individual glomus cells in the same cluster respond very differently to the same stimulus be it hypoxia or acidosis. The magnitude of responses varied significantly. The cells ranged from unresponsive to very sensitive and could reach over 250 nm[Ca2+]i in response to either hypoxia or acidosis. The second result is that individual clusters differ markedly in their relative sensitivity to hypoxia and acidosis. Cluster I, for example, was relatively insensitive to low pH but very sensitive to hypoxia, while Cluster II was preferentially sensitive to low pH except for one glomus cell which was unresponsive to low pH but most sensitive to hypoxia. The pattern of responses also differed. Many cells had rapid elevations of [Ca2+]i to a peak followed by a rapid decline to baseline. Others had a more sustained [Ca2+]i level after the peak, and still others had a slow rise and fall (Fig. 3B and C).
A total of 137 glomus cells in 15 clusters obtained from 20 carotid bodies of 10 rats were tested. The resting cytosolic [Ca2+]i at normoxic conditions was 71 ± 6 nm (n= 137). Of the 137 cells 28 (∼20%) were unresponsive. More than two-thirds of the 109 responsive cells were sensitive to both hypoxia and acidosis (n= 74, 68%), while 21 cells (19%) responded only to hypoxia and 14 cells (13%) responded only to acidosis (Fig. 3D). The hypoxic response was greater (Δ 40 ± 2 nm, n= 95) than the response to acidosis (Δ 28 ± 3 nm, n= 88) (P= 0.015).
Figure 3E shows the number of cells that responded to both low pH and hypoxia at each level of decadal increment of [Ca2+]i ranging from Δ 10 nm to Δ 160 nm. Of 74 cells, the largest number had a small increase of 10–20 nm[Ca2+]i in response to low pH (n= 50). In contrast, only 13 cells had a similarly low response to hypoxia. Conversely, a large number of cells had a large increase of 30–40 nm[Ca2+]i in response to hypoxia (n= 28), and only 8 cells had a similarly large response to acidosis. Larger increases in [Ca2+]i from 41 to 160 nm were seen in 29 cells in response to hypoxia but in only 6 cells in response to low pH. The results indicate a reciprocity in the responsiveness of most glomus cells to hypoxia and acidosis. The cells that were more sensitive to hypoxia were less so in response to low pH and vice versa.
ASIC3 expression alters responses to low pH and to cyanide in a reciprocal manner
In earlier studies, we had identified ASIC3 as an ion channel in carotid body glomus cells important in the mediation of acid sensitivity (Tan et al. 2007). We also associated increased sensitivity to pH in glomus cells of spontaneously hypertensive rats with increased expression of ASIC3 (Tan et al. 2010). We wondered, therefore, whether we could demonstrate the phenomenon of reciprocity between responses to hypoxia and acidosis by altering expression of ASIC3 in glomus cells.
Thus, to explore the mechanism of this reciprocity, we measured the increases in cytoplasmic [Ca2+]i in clusters of glomus cells isolated from carotid bodies of three groups of mice: ASIC3 null (KO), wild-type (WT), and ASIC3 transgenic mice (Tg). Responses to pH 6.0 were compared to the responses to the metabolic inhibitor NaCN (1 mm) (Krylov & Anichkov, 1968; Sato et al. 1991; Wyatt & Buckler, 2004).
Baseline values of [Ca2+]i were (mean ± SEM; nm) 57 ± 2 (n= 205), 56 ± 3 (n= 207) and 55 ± 5 (n= 32) in KO, WT and Tg mice, respectively. The tracings in Fig. 4 represent increases in [Ca2+]i in response to pH 6.0 and 1 mm NaCN in individual cells of the same cluster. The variability between cells and the reciprocal sensitivities to pH and NaCN are evident. Note, for example, that Cell 1 had the largest response to low pH but did not respond to CN. Conversely, Cell 4 had the largest response to CN but a small response to pH. Similarly, reciprocal responses were seen in most cells (Fig. 4).
An analysis of variance of the [Ca2+]i transients in response to pH 6.0 revealed a significant difference between the three genotypes (P < 0.01) with incremental increases in pH sensitivity from the lowest values in ASIC3 KO to the highest values in ASIC3 Tg (Fig. 5). Contrasting responses to low pH with those to CN confirmed the specificity of the positive correlation of pH sensitivity with the ASIC3 genotype since responses to CN showed no such correlation. In fact, responses to CN were drastically reduced in ASIC3 Tg mice (P < 0.01) while responses to acid pH were significantly enhanced (Fig. 5).
The results also indicate that the majority of cells of WT mice responded to pH 6.0 (75%) and to CN (81%). ASIC3-KO mice, however, had a significantly (P < 0.05) reduced percentage of cells responsive to low pH (41%) while the percentage responding to NaCN was not altered significantly (71%). An opposite trend was seen in ASIC3 Tg mice with a lower percentage of cells responding to CN (59%) compared to 69% responsive to low pH. Thus, the ratios of CN over low pH-responsive cells were 1.73, 1.08 and 0.85 in ASIC3 KO, WT and ASIC3 Tg, respectively.
Correlations of responses of individual glomus cells to CN and to low pH within each genotype
Figure 6A, B and C shows the scatter plots of responses of each glomus cell from each of the three genotypes to CN (Y-axis) and low pH (X-axis) in ASIC3 KO, C57 (WT) and ASIC3 Tg mice, respectively. Two linear regressions were obtained in each group. The slope of the linear regressions of cells that were more sensitive to CN (CN/pH >1.0) were significantly different and greater than those of cells more sensitive to low pH (CN/pH <1.0), suggesting two distinct cell populations in each of the three genotypes. Thus, the reciprocal nature of the responses was evident within each genotype.
The reciprocal responses to CN and pH were also evident when comparisons were made between the genotypes (Fig. 6D). As pH responses were enhanced in the Tg mice, the slope of the linear regressions of the more CN-sensitive cells decreased from Y= 2.8X (R2= 0.34) in ASIC3 KO to Y= 1.25X (R2= 0.92) in the Tg, indicating a decline in CN sensitivity that was reciprocal to the increased pH sensitivity. Similarly, in the more pH-sensitive cells, the slope of the linear regressions declined from Y= 0.45X (R2= 0.32) in ASIC3 KO to Y= 0.16 X (R2=−1.27) in the Tg (Fig. 6D), also reflecting the reciprocal decline in CN sensitivity across genotypes (see also Table 1 in supplemental material).
When the ratios of responses to CN over pH 6.0 in all glomus cells of each genotype were averaged and compared by analysis of variance, those ratios declined significantly and progressively from a high level in the ASIC3 KO group to a low level in the ASIC3 Tg group (Fig. 6E).
Responses of glomus cells of WT and ASIC3-KO mice to hypoxia and pH 6.0
Additional experiments were done to see if the increase in the ratio of responses to CN/pH 6.0 in ASIC3-KO vs. WT would be replicated with hypoxia instead of CN.
We measured the responses of individual mouse glomus cells to hypoxia (15 mmHg) and low pH (6.0). Glomus cells were obtained from WT and ASIC3-KO mice. The mean group responses to hypoxia in WT and ASIC3 KO mice were comparable (Fig. 7A and C) but the responses to low pH were significantly reduced in the KO compared to the WT (Fig. 7B and C). The ratios of responses to hypoxia over low pH were more than doubled in the ASIC3 KO group (Fig. 7D), reflecting responses obtained with CN and confirming induction of reciprocal sensitivity with ASIC3 deletion.
The pronounced morphological diversity among glomus type I cells (Morita et al. 1969; Hellström, 1975; McDonald & Mitchell, 1975; Pallot, 1987; Schamel & Verna, 1992; Kumar & Prabhakar, 2012) and their heterogeneous chemosensitivity have been amply supported in the literature (Pang & Eyzaguirre, 1992, 1993; Bright et al. 1996). Varying responses of glomus cells to hypoxia using imaging of whole mouse carotid bodies have also been recently reported by Wotzlaw et al. (2011).
The reported ionic and metabolic pathways that are activated by hypoxia and acidosis suggest that glomus cells are capable of responding to both stimuli (Lopez-Lopez et al. 1997; Abudara et al. 2002; Summers et al. 2002; Lahiri & Forster, 2003; Xu et al. 2003; Prabhakar & Peng, 2004; Nurse, 2005; Prabhakar, 2006; Lahiri et al. 2006; Tan et al. 2007, 2010; Buckler, 2007; Peers et al. 2010).
The majority of the work indicates that the same glomus cell, or neuron, or nerve fibre may respond to more than one stimulus, be it hypoxia, low pH or high CO2. The results have also focused on interactions between stimuli that may be additive or multiplicative (Lahiri & DeLaney, 1975; Dasso et al. 2000; Prasad et al. 2001) or even absent, depending on age and development (Bamford et al. 1999). Thus, the current understanding is that the carotid chemoreceptor units respond to both hypoxia and acidosis without any indication of selective differential activation that allows the distinctive sensory recognition of each of these two major chemical signals. The absence of selective signalling seems functionally inefficient and precludes selective reflex responses.
Differential sensitivity of glomus cells to hypoxia and acidosis
Our studies define quantitatively the relative sensitivity of glomus cells to hypoxia vs. acidosis. We find, as others did, that a majority (68%) of glomus cells respond to both stimuli but in addition we report that the remaining cells are exclusively sensitive to either hypoxia (19%) or to acidosis (13%). We also report a significant difference in the relative sensitivities to either hypoxia or to acidosis that is evident in the individual cells as well as in the clusters of cells that respond to both stimuli. Thus responses of glomus cells to hypoxia or acidosis are uncoupled and reciprocal with most cells being either predominantly or exclusively sensitive to hypoxia or predominantly or exclusively sensitive to acidosis. To our knowledge this concept of reciprocal sensitivity of glomus cells is novel and we propose that it has significant functional implications.
In exploring the mechanism of this reciprocal sensitivity we were able to reproduce it in glomus cells of mice by enhancing or suppressing their genetic expression of ASIC3 and thereby inducing a corresponding change in their acid sensitivity.
Selective sensory transduction in peripheral chemoreceptors
Although both hypoxia and acidosis may activate the same glomus cells, they are often initiated independently in different pathological states. For example, metabolic acidosis may occur without hypoxaemia and vice versa. In fact activation of chemoreceptors with acidosis may, by inducing hyperventilation, result in hyperoxia and hypocapnia which suppress the chemoreceptors (Biscoe et al. 1970). Conversely, hypoxic hyperventilation may result in respiratory alkalosis which inhibits the chemoreceptor activity (Somers et al. 1989). If glomus cells were homogeneously and equally sensitive to hypoxaemia and acidosis a negative feedback would quickly suppress the response to either stimulus. One can argue therefore that uncoupling the sensory transduction of the two stimuli is essential.
Quantitative assessment of selectivity and reciprocal sensitivity of glomus cells to hypoxia vs. acidosis
Uncoupling of responses to hypoxia and acidosis was evident in rat carotid bodies at several levels and strongly suggested the presence of two distinct populations of glomus cells.
First, the responses of clusters of glomus cells to hypoxia and acidosis appeared to differ. In some clusters the glomus cells were predominantly sensitive to hypoxia and insensitive to pH 6.8 or even pH 6.0 while in others the cells were very sensitive to low pH but not to hypoxia.
Second, at the level of individual glomus cells within the clusters there was a wide variability with some responses reaching increases in [Ca2+]i of over 250 nm and others with increases of 10 nm or less. The responders could thus be stratified into two groups: those that are primarily or exclusively sensitive to hypoxia and those that are primarily or exclusively sensitive to low pH.
Third, the increases in cytosolic calcium with hypoxia were significantly greater in a larger number of cells than the responses to acidosis. When the rat glomus cells are stratified as low versus high responders in terms of the magnitude of the [Ca2+]i transients a significantly larger number of low responders (Δ <20 nm) were activated by a low pH than by hypoxia in a ratio close to 3:1. Conversely a significantly larger number of high responders were activated by hypoxia than low pH in a ratio of 6:1 with increases of 40–160 nm[Ca2+]i.
Functional significance of reciprocal sensitivity of glomus cells to hypoxia and acidosis
We believe that the functional significance of this selective sensitivity to either hypoxia or acidosis is to allow the glomus cell to sustain its activity despite the early rapid ventilatory responses which would tend to suppress it. We and others have reported, for example, that in humans the ventilatory response, as well as the increase in sympathetic nerve activity during acute hypoxia, is augmented with the addition of CO2 to maintain isocapnia compared to hypocapnia (Somers et al. 1989). This indicates that hypocapnia and alkalosis may partially buffer the hypoxic response by suppressing pH-sensitive glomus cells.
It is also of interest that the early work of Biscoe et al. (1970) indicates that selectivity may extend to single chemoreceptor fibres from the same strand. They report responses of two single fibres that reveal one to be much more sensitive to hypoxia than the other. We would speculate that one fibre may be receiving input from glomus cells that are predominantly hypoxia sensitive.
Another potentially important functional significance is that the selectively higher sensitivity of certain afferent fibres to either hypoxia or acidosis may result in different chemoreceptor reflex regulation of autonomic drive to provide the optimal adjustment (for example, in blood flow distribution) that would be different in response to hypoxia and to acidosis. With acidosis for example blood flow to the kidney might be maintained whereas in hypoxia blood flow would more optimally be distributed to brain and coronary circulations.
Thus our results on the carotid body in rats have allowed us to quantify the relative sensitivities of individual glomus cells to hypoxia vs. acidosis and establish the notion that the responses are uncoupled and reciprocal in the majority of glomus cells. The concept that emerges is one of differential chemosensory transduction of hypoxia and acidosis with selective sensing and possibly selective chemoreflex adjustments to hypoxia vs. acidosis which have significant functional implications.
Use of mice with genetically modified ASIC3 expression
The goal here was to see whether the reciprocal sensitivity of glomus cells to hypoxia vs. acidosis which we identified in the first part of the study could be reproduced in glomus cells of mice by enhancing or suppressing the expression of genes that would modulate either O2 sensitivity or pH sensitivity. We elected to alter pH sensitivity rather than O2 sensitivity for two reasons. One is the complexity and diversity of the mechanisms that determine O2 sensitivity. Second is the fact that we had identified ASIC3 as an important mediator of pH sensitivity in glomus cells (Tan et al. 2007). This channel was overexpressed in the carotid body of spontaneously hypertensive rats and was associated with the increased chemoreceptor sensitivity (Tan et al. 2010).
We studied the responses of isolated glomus cells from the three genotypes and contrasted responses to low pH and to NaCN.
Phenotypic expression of reciprocal sensitivity
By contrasting the maximal responses to low pH and to CN we were able to define the three phenotypes of acid sensitivity that correlated well with the three genotypes (Fig. 5A), and to establish that the most pH-sensitive group (Tg mice) was the least sensitive to CN. Moreover, in each genotype we found a clear separation between cells that were more sensitive to CN and those more sensitive to pH, confirming the reciprocal nature of the response to those two stimuli in two separate populations of glomus cells (Fig. 6A, B and C).
Within the more CN-sensitive group, the CN/pH sensitivity was least in the Tg (1.25) and highest in the KO (2.8) mice. Moreover, within the more pH-sensitive group, CN/pH sensitivity was least in the Tg (0.16) (Fig. 6D and online Supplemental Table 1).
As anticipated, the ratio of responses of all glomus cells in each genotype to CN/pH was highest in ASIC3 KO (3.7), less high in WT (2.0), and lowest in ASIC3 Tg (0.8) (Fig. 6E). Because of concerns regarding side effects of the high dose of CN (1 mm) we carried out additional experiments in which we tested the effect of hypoxia instead of CN in the ASIC3 KO and found that the ratio of responses to hypoxia/pH more than doubled that in WT as was the case with the ratios of CN/pH (Fig. 7).
Thus, in summary, we believe that the phenomenon of reciprocal sensitivity of glomus cells to pH and hypoxia is supported by the results in normal rats and confirmed in genetically modified mice. We speculate first on its functional significance in allowing the selective sensory transduction and differentiation between the two major chemical stimuli. We also speculate on its significance in sustaining the activation of glomus cells that are selectively activated by either hypoxia or acidosis as the buffering ventilatory responses are triggered, and on the possibility of activating differentially sensitive afferents that might evoke more optimal and specific adjustments to either hypoxia or acidosis.
In the remaining discussion we will briefly review the mechanisms of activation of chemoreceptors, the role of AMP kinase and hypoxia in inhibiting mitochondrial electron transport as well as BKCa and leak K+ currents, the inhibiting interaction between ASIC3 and K+ channels, and the potential release of different transmitters from glomus cells by hypoxia and acidosis.
Mechanisms of activation of chemoreceptors
We believe that ion channels and metabolic pathways responsive to either hypoxia or acidosis or both must be expressed selectively in glomus cells and their expression may vary quantitatively to account for differential sensitivities.
Proton-sensitive non-voltage-gated channels Our previous studies and extensive work by others showed that two non-voltage-gated acid-sensitive Na+ and K+ channels, namely ASICs and the two-pore-domain acid-sensitive potassium channels (TASKs), are expressed in glomus cells and involved in the depolarization evoked by extracellular acidosis. At low pH, the opening of ASICs results in an inward Na+ current, and closure of TASK blocks an outward K+ current, both of which cause depolarization. In our current experiments a persistent smaller Ca2+ transient in response to low pH in ASIC3 KO probably represents the inhibition of TASK channels.
O2-regulated voltage-gated channels Although there is ample evidence that hypoxia, acidosis and cyanide inhibit TASK-like K+ current, other voltage-gated ion channels are also involved (Krylov & Anichkov, 1968; Eyzaguirre & Fidone, 1980; Lopez-Barneo et al. 1988; Sato et al. 1991; Lopez-Lopez et al. 1997; Summers et al. 2002; J. Xu et al. 2003; Hoshi & Lahiri, 2004; Wyatt & Buckler, 2004; Nurse, 2005; Prabhakar, 2006; Buckler, 2007; Tan et al. 2007; Duprat et al. 2007; F. Xu et al. 2007; Tan et al. 2010; Kumar & Prabhakar, 2012). Specifically, several studies have indicated an inhibitory effect of hypoxia on the Ca2+-activated K+ channel (BK) (Lopez-Barneo et al. 1988; Peers & O’Donnel, 1990; Lopez-Lopez et al. 1997; Patel & Honore, 2001; Hoshi & Lahiri, 2004; Lahiri et al. 2006; Peers et al. 2010; Kumar & Prabhakar, 2012).
Mitochondrial electron transport and oxidative phosphorylation: role of AMPK Inhibition of O2-regulated K+ channels may result from inhibition of both mitochondrial electron transport and oxidative phosphorylation (Ortega-Sáenz, 2003; Peers et al. 2010) which occurs with both hypoxia and cyanide. Peers et al. (2010) conclude that ‘AMPK activation provides the basis for unifying the ‘membrane’ and ‘mitochondrial hypothesis’ (previously regarded as disparate) to account for hypoxic chemotransduction. Both processes converge on depolarization and increase Ca2+ transients.’ These authors report that AMPK activation, like hypoxia, inhibited both BKCa and leak K+ currents in type I cells, causing voltage-gated Ca2+ entry, and phosphorylated recombinant BKCa channels directly (Wyatt & Buckler, 2004).
Effect of altered ASIC3 gene expression on responses to low pH and NaCN
We tested the possibility that alteration of pH sensitivity by altering ASIC3 expression would modify the sensitivity to hypoxia or mitochondrial inhibition. We found a strong reciprocal relation between the pH response and that to NaCN after altering ASIC3 expression. Specifically, the overexpression of ASIC3 resulted in a significant decline in the NaCN response.
We wondered about the possibility of an inhibitory interaction between ASIC3, a mediator of proton sensitivity, and the high conductance Ca2+-activated K+ channel (BK) which mediates in part the response to hypoxia.
We had reported a surprising regulation of ion channels where two very different types of channels, ASIC1a Na+ channels and BK K+ channels closely associated in the cell membrane and regulated each other's activity (Petroff et al. 2008). ASIC1a inhibited BK currents. Other ASIC channels, including ASIC3, also inhibited BK as well as other K+ channels, thus providing a possible mechanism for the reciprocal regulation we identified in glomus cells. That interaction was explained by the fact that within the large extracellular domain of ASICs a sequence was identified that resembled one in scorpion toxin that inhibits K+ channels (Petroff et al. 2008). More recently we confirmed this inhibitory interaction between ASICs and BK in neurons (Petroff et al. 2012).
At this point we can only speculate that the physiological consequences of overexpression of ASIC3 may be linked to the inhibition of BK or other K+ channels (which may also be inhibited by hypoxia) thereby explaining at least in part the enhanced response to pH and the reciprocal reduction in response to hypoxia in glomus cells from ASIC3 Tg.
Preferential release of transmitters by hypoxia and acidosis
Of interest are the results obtained by Pokorski & Lahiri (1983) on the effects of metabolic acidosis and hypoxia on aortic chemoreceptor afferents in anaesthetized ventilated cats. Reduction of arterial pH to 7.15 with lactic acid caused a modest increase in nerve activity from 0.3 ± 0.1 to 1.8 ± 0.3 impulses s−1 during normoxia. But with the addition of hypoxia (32 ± 1 mmHg) the increase in nerve activity with lactic acid was enhanced severalfold and reached 9.8 ± 1.9 impulses s−1. Our findings in the isolated glomus cells, which are the site of chemoelectrical transduction, reflect the nerve activity data and show that hypoxia may cause more than twice the increase in [Ca2+]i than the response to low pH, and engage more glomus cells given that many are essentially unresponsive to low pH, yet very sensitive to hypoxia. In addition to the selective activation of different glomus cells with hypoxia and acidosis, the release of different neurotransmitters with hypoxia and acidosis may activate selectively different sensory nerve terminals. Kim et al. (2004) have reported preferential release of ACh from the carotid body in response to acidosis as acidic pH inhibits acetylcholine esterase. Conversely, a low may preferentially release ATP or H2S (Peng et al. 2010), and contribute to the selectivity of sensory transduction.
We conclude that glomus cells may be differentiated into two groups, one is selectively or predominantly sensitive to hypoxia and the other selectively or predominantly sensitive to acidosis. This functional distinction coupled with putative release of different transmitters would further define the selectivity of sensory transduction.
This selective transduction of afferent signals should be expected to evoke different reflex autonomic responses to hypoxia and acidosis as the signals are processed centrally. For example, the reflex response to hypoxia might result in greater sympathetic constriction of renal, splanchnic, cutaneous or muscular vascular beds to maintain perfusion of organs more vulnerable during hypoxia such as coronary and cerebral vascular beds. Conversely during acidosis perfusion of the kidney to favour acid excretion, and of skeletal muscle to reduce a major source of lactic acid, would be teleologically reasonable. That would only be possible if the sensory neural signals were different and the differentiation at the level of sensory transduction would be critical.
Although such a distinction of autonomic responses to hypoxia and acidosis has not been established, our results provide a conceptually valid argument for its possibility.
We arrive at these conclusions with the full appreciation of the complexity of O2-sensing mechanisms and their interaction with ion channels (Kumar & Prabhakar, 2012). We did not address the effects of CO2 and intracellular acidosis or the role of other acid-sensitive ion channels, which may also be sensitive to hypoxia. These and other factors may further define the differential identity of glomus cells and their selective sensitivity to hypoxia or acidosis which we are reporting.
Although our measurements targeted selectively the large and circular type I cells, we recognize the possibility that some of the responsive cells may be type II cells which, though not responsive to hypoxia or acidosis, are responsive to ATP released from type I cells (Xu et al. 2003; Piskuric & Nurse 2012). It is also possible that some of the non-responding cells shown in Fig. 3D are type II cells.
All work was done in the Cardiovascular Research Centre of the University of Iowa. C.W. contributed to the manuscript by preparing glomus cell cultures and collecting the data. K.A.S. participated in the conception of the work related to ASIC3 and provision of the ASIC3 KO mice. Y.L., performed the experiments, and with M.W.C. and F.M.A. contributed to the conception, design, analysis and drafting of the article for content. All authors approved the final version of the article.
We thank the Welsh laboratory at the University of Iowa for providing the ASIC3 transgenic mice and their congenic strain, and Dr Sluka's lab for making available the ASIC3 KO mice. We thank Sally Knipfer and Arlinda LaRose for assistance in preparing the manuscript and Shawn Roach for assistance in formatting the figures (Dept of Internal Medicine, University of Iowa). The work was supported by NIH program project grant HL 14388, and a Merit Review Award from the Department of Veterans Affairs.