Responses of glomus cells to hypoxia and acidosis are uncoupled, reciprocal and linked to ASIC3 expression: selectivity of chemosensory transduction

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 Ca(2+) 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.

Figure 3. Contrasting responses of individual rat glomus cells in different clusters to hypoxia and acidosis: heterogeneity and reciprocity characterize these responses

A, bright field and maximal fluorescence of rat glomus cells of the same cluster in response to low pH (6.0), hypoxia (, 15 mmHg) and ionomycin (5 μm). The response of each cell to pH 6.0 was smaller than its corresponding response to hypoxia. B and C, responses to hypoxia (, 15 mmHg) and acidosis (pH 6.8) in two different clusters (I and II) from a rat carotid body reveal heterogeneity and reciprocal sensitivities to the two stimuli between the two clusters and in individual cells within each cluster. B, all cells in Cluster I are more sensitive to hypoxia than to low pH. C, four of five cells in Cluster II are very sensitive to low pH 6.8 and much less sensitive to hypoxia. One cell in this cluster, however, responds well to hypoxia and not low pH. D, diagram identifies the number of non-responsive and responsive cells and their distribution in terms of responders to hypoxia, low pH, or to both out of a total of 137 glomus cells. E, number of glomus cells separated according to the magnitude of their increase in [Ca²⁺]_i with hypoxia and acidosis. The horizontal bars indicate the number of responding cells at each decadal level of increases in [Ca²⁺]_i from Δ 10 nm to Δ 160 nm. Blue bars represent the cells responding to low pH and the red bars are those responding to hypoxia. Larger Δ[Ca²⁺]_i were seen in more cells with hypoxia, while smaller Δ[Ca²⁺]_i were seen in the majority of cells in response to acidosis.

Figure 5. Responses of glomus cells obtained from three genotypes of mice: ASIC3 knockout (ASIC3 KO), wild-type C57 (WT) and ASIC3 transgenic (ASIC3 Tg)

A, box plot of [Ca²⁺]_i transient responses to pH 6.0. The upper hinge of the box indicates the 75th percentile of the data set; the lower hinge indicates the 25th percentile. The vertical lines encompass the maximum and minimum responses. Δ[Ca²⁺]_i responses of glomus cells to pH 6.0 correlate positively with the expression of ASIC3. The linear regression was significant between the three genotypes. Larger responses are seen in Tg mice compared to WT and smaller responses are seen in KO (ANOVA, *P < 0.01). B, box plot of [Ca²⁺]_i responses to NaCN. The least response is found in ASIC3 Tg compared to WT and ASIC3-KO (*P < 0.01).

Figure 1. Records of [Ca²⁺]_i transients in isolated rat glomus cells in response to hypoxia, acidosis, and ionomycin

(A), The in the bathing medium drops rapidly to hypoxic levels as low as 15 mmHg within 30 sec upon switching from the normal perfusate ( mmHg, pH 7.4) to the hypoxic one ( mmHg, pH 7.4). (B) Hypoxia initiates a rapid increase in [Ca²⁺]_i. (C) Similarly a drop in pH from 7.4 to 6.8 increases [Ca²⁺]_i. Responses to ionomycin (5 μM) were reproducible following hypoxia in (B) and following low pH in (C). (D) Responses to pH 6.8 were reproducible with repeated exposures.

Figure 2. Temporal changes in [Ca²⁺]_i of individual rat glomus cells in a cluster following exposure to pH 6.8

A, bright field and sequential fluorescence images of individual glomus cells within a cluster from rat carotid body showing maximal fluorescence at 0.6 min after exposure to pH 6.8 and subsequent gradual decline over 2.2 min to control levels. B, the corresponding individual tracings of [Ca²⁺]_i. The system allows the tracking of individual cells over time with precision before, during and after the response as the fluorescence returns to baseline. The baseline [Ca²⁺]_i varied between cells and the magnitude of the responses was not a function of the baseline levels. C, schematic representation of the carotid body at the bifurcation of the carotid artery and a cluster of glomus cells with sensory nerve terminals. A single type I glomus cell with selected representative ion channels (TASK, ASIC3, BK, Ca²⁺) responds to low pH and low with the release of ACh and ATP. NTS, nucleus of the tractus solitarius.

Figure 4. Responses of individual mouse glomus cells in the same cluster to low pH (6.0) and to 1 mm NaCN

This cluster is from a carotid body of a WT mouse and had 7 glomus cells. The variability between cells and the reciprocal sensitivities to pH and NaCN are evident. Cell 1 had the largest response to pH but did not respond to CN. Cell 4 had the largest response to CN but a small response to pH. Cell 6 also had a large CN response but no pH response. Cells 2, 3 and 5 had good pH responses and very small CN responses. Cell 7 did not respond to either pH or CN.

Figure 6. Correlations of responses of individual mouse glomus cells to 1 mm NaCN and to pH 6.0 are shown for the three genotypes (ASIC3 KO, C57 WT and ASIC3 Tg): two distinctive populations of glomus cells are apparent in each genotype

A, B and C, the scatter plots of individual cells represent responses to CN (Y-axis) and to low pH (X-axis). The red dots represent cells that responded more to CN (above the dashed line of identity) and the blue dots represent cells that responded more to low pH (below the dashed line of identify). The regression lines of these two groups were adjusted to go through zero and were found to be significantly different in each genotype (in ASIC3 KO P= 0.0084; in WT P= 0.33 × 10⁻⁹; in ASIC3 Tg P= 0.0004), thus identifying two distinctive populations of glomus cells with reciprocal responses within each genotype. D, this graph contrasts the responses across the genotypes. Three regressions above the line of identity (red lines) represent responses of the more CN-sensitive cells in the KO, WT and Tg mice, and the three others in blue represent the more pH-sensitive cells in the three genotypes. In both groups of cells as pH sensitivity increased from the ASIC3 KO to the ASIC3 Tg mice (seen along the X-axis), it was associated with a decline in sensitivity to CN (seen along the Y-axis). In the CN-sensitive group, the responses to CN decreased progressively from ASIC3 KO to WT, and from WT to ASIC3 Tg as pH sensitivity increased (ANOVA P < 0.01). In the pH-sensitive group, the CN sensitivities of the KO and WT cells were not significantly different, but the CN sensitivity of the Tg cells was markedly reduced compared to both WT and KO (P < 0.001). Thus, reciprocity was evident in glomus cells within the same genotypes as well as across genotypes. The unadjusted regression equations that correspond to these linear regressions are shown in Supplemental Table 1 and support the same conclusions. E, the bars represent the means ± SEM of the ratios of Δ[Ca²⁺]_i in response to CN/pH for all glomus cells combined in each group. Those ratios were largest in ASIC3 KO cells and declined progressively from the KO to the Tg group (ANOVA, P < 0.01) and reflected the reciprocal changes in sensitivities to hypoxia and acidosis.

Figure 7. Responses of glomus cells of WT and ASIC3-KO mice to hypoxia and pH 6.0

Tracings indicate intracellular calcium responses to hypoxia (, 15 mmHg) and to low pH (6.0) of single isolated glomus cells from WT (A) and ASIC3 KO (B). Bar graphs indicate means ± SEM of intracellular calcium responses of 13 glomus cells from WT and ASIC3 KO mice to hypoxia and to pH 6.0 (C), and the means ± SEM of the ratios of those responses (hypoxia/pH 6.0) (D). The decreased response to low pH is significant in glomus cells from ASIC3 KO mice (*P < 0.05). This and the preservation or enhancement of responses to hypoxia resulted in more than a doubling of the ratios of responses to hypoxia/pH 6.0 in ASIC3-KO compared to the ratios in WT.