We appreciate the opportunity to clarify the queries regarding the analysis of our data raised in the Letter to the Editor in this issue from Buckler (2013). The reciprocal sensitivity to hypoxia (cyanide – CN) vs. low pH (acidic solution) that we have reported is represented in the regression analyses (Y=aX+b) of responses of individual glomus cells to CN (Y) vs. acid (X). Changes in the slopes (a) were inversely related to increased or decreased sensitivity to acid. These regression analyses are portrayed in Fig. 6 and in the Supplemental Table 1. The data in Fig. 6 are adjusted to go through zero, and the data in the Supplemental Table 1 include both the adjusted as well as the unadjusted regressions and their Y-intercepts. Both sets of analyses support our interpretation. The data on rat cells do define three distinct populations of cells. Of a total of 109 cells, one group responded only to hypoxia (19% of cells), another responded only to acid (13%), and a third responded to both (68%; Figs 3B–D). Figure 3E portrays the range of responses to acid vs. hypoxia by decadal increments in Δ[Ca2+]i. The description and analyses of those results as reported reveal two points: One is that the responses to hypoxia were significantly larger (Δ40 ± 2 nm, n= 95) than those to acid (Δ28 ± 3 nm, n= 88) with a P value of 0.015. The second point is evident in Fig. 3E. In the group of low responding cells (Δ10 to 20 nm), there were 4 times as many cells responding to acid as to hypoxia (n= 50 vs. 13); and conversely, in the high responding cells (Δ30–160 nm) there were 4 times as many cells responding to hypoxia as to acidosis (n= 57 vs. 14). The choice of Δ30 nm[Ca2+]i was not arbitrary. It seemed to uncouple most of the more intensively cells responsive to hypoxia from most of the responsive cells to acid. A χ2 test analysis of the distribution of responsive cells to hypoxia or to acid, based on the intensity of the Ca2+ signal being higher or lower than Δ30 nm, gave a very significant P value of 5.21 × 10−9. We had not reported this statistical analysis in the legend of Fig. 3 and should have done so. With respect to the data in mice, in Fig. 4 we first show individual cells in the same cluster responding with greater intensity to either CN or pH 6.0. In Fig. 5, we show progressive increases in responses to pH 6.0, which are small in ASIC3-KO (n= 205), higher in WT (n= 207), and still higher in ASIC3-Tg (n= 32). The responses to CN were reciprocally reduced significantly in the ASIC-Tg. In Fig. 6, responses of each cell to CN vs. acid were plotted and regression analyses were carried out in each of the three genotypes. By genetically increasing or decreasing the sensitivity to acid, we saw inverse (reciprocal) responses to CN. These changes are reflected in the plotted regression lines and the changes in the slopes of these regressions. Our main finding is that in both populations of responsive cells, the CN > acid (gradient >1 – red cells) and the CN acid groups (gradient >1), the slopes of the regressions were at a high of 2.8 in ASIC3−/− and decreased to 2.1 in WT and to 1.2 in ASIC3-Tg, notwithstanding the admittedly underpowered Tg group. The corresponding ‘unadjusted’ regressions also showed the inverse relation. These results, published in the Supplemental Table 1, were 2.07, 1.56, and 1.1, respectively. In the CN < acid groups (gradient <1), the slopes were 0.45 in ASIC3−/−, 0.60 in WT, and decreased significantly to 0.16 in ASIC3-Tg. The corresponding ‘unadjusted’ regressions in Table 1 were 0.37, 0.48, and −0.04. In this group of cells with low CN sensitivity, the reciprocity was not evident between ASIC3−/− and WT, but was pronounced in the ASIC3-Tg cells. When as seen in Fig. 6E, the responses of all cells in each genotype are lumped together without any separation of high or low CN responders and the ratios of Ca2+ responses to CN/acid in each genotype are averaged, the results are more compelling. The pronounced directional decrease in the ratio across the three genotypes is a function of both an increased response to acid and a decreased response to CN. The undeniable variability of individual responses has been minimized by studying mutant mice with the same genetic background, age, environment, gender, and state of health. Thus, we reaffirm our interpretation of the results. As intriguing as these results may be, the functional meaning of a preferential sensitivity of glomus cells to either hypoxia or acidosis will need to be tested in vivo by demonstrating distinctive autonomic responses to carotid body activation by hypoxia and acidosis.
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Jun 27, 2024
Nancy Q Zhao + 7
Open Access
