Postnatal maturation of carotid body and type I cell chemoreception in the rat

Owen S. Bamford, Laura M. Sterni, Michael J. Wasicko, Marshall H. Montrose, and John L. Carroll

Department of Pediatrics, The Johns Hopkins School of Medicine, Baltimore, Maryland 21287-2533


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The site of postnatal maturation of carotid body chemoreception is unclear. To test the hypothesis that maturation occurs synchronously in type I cells and the whole carotid body, the development of changes in the intracellular Ca2+ concentration responses to hypoxia, CO2, and combined challenges was studied with fluorescence microscopy in type I cells and compared with the development of carotid sinus nerve (CSN) responses recorded in vitro from term fetal to 3-wk animals. Type I cell responses to all challenges increased between 1 and 8 days and then remained constant, with no multiplicative O2-CO2 interaction at any age. The CSN response to hypoxia also matured by 8 days, but CSN responses to CO2 did not change significantly with age. Multiplicative O2-CO2 interaction occurred in the CSN response at 2-3 wk but not in younger groups. We conclude that type I cell maturation underlies maturation of the CSN response to hypoxia. However, because development of responses to CO2 and combined hypoxia-CO2 challenges differed between type I cells and the CSN, responses to these stimuli must mature at other, unidentified sites within the developing carotid body.

development; carbon dioxide; stimulus interaction; intracellular calcium


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE CAROTID CHEMORECEPTORS are largely responsible for sensory input to ventilatory and arousal responses to hypoxia and supply a component of CO2-mediated ventilatory drive (11). The chemosensor is generally considered to be the carotid body type I cell that responds to hypoxia and CO2 with a rise in intracellular Ca2+ (2). The Ca2+ increase is thought to initiate neurotransmitter release and excitation of the carotid sinus nerve (CSN), although the details are still controversial.

In every species studied to date, including human, ventilatory sensitivity to hypoxia is low at birth and matures with age. The neural response of the carotid body to hypoxia also shows postnatal maturation both in vivo and in vitro (6, 16, 19). Although the carotid body is generally considered to be an O2 sensor, it is also sensitive to CO2. Moreover, CO2 increases sensitivity to hypoxia and vice versa (18), so that the response to hypoxia depends partly on the level of CO2. This phenomenon of O2-CO2 interaction also matures postnatally in the carotid bodies of the cat in vivo (6) and the rat in vitro (16).

Little is known about the site or mechanism of carotid body maturation. There have been no studies on the maturation of the Ca2+ response to CO2 or on O2-CO2 interaction at the cell level. However, we have evidence for maturation of the O2 response in type I cells. Previous work from our laboratory (7, 27, 28) showed in rabbits and rats that type I cell intracellular Ca2+ concentration ([Ca2+]i) responses to anoxia are weak in cells from newborns and increase with age. It is possible therefore that maturation of type I cells underlies maturation of carotid body function. We tested the hypothesis that maturation of type I cell function occurs on a schedule consistent with the maturation of hypoxic responses and O2-CO2 interaction in the whole carotid body. We therefore measured [Ca2+]i responses to O2, CO2, and O2-CO2 interaction in type I cells and the neural responses of whole in vitro carotid bodies over the age range from term fetal to 2-3 wk.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Isolation

For cell studies, carotid bodies were harvested from groups of rats in 4 age ranges: 20 day fetal (-1 day), birth-1 day (1 day), 7-8 days (8 days), and 2-3 wk postnatal (14 days). All procedures were approved by the Animal Care and Use Committee of John Hopkins Medical School (Baltimore, MD). Rat pups were anesthetized by inhalation of methoxyflurane (Metofane) and decapitated. The carotid bifurcations were rapidly removed and placed in ice-cold phosphate-buffered saline (PBS). On a cold stage, the carotid bodies were dissected out in PBS and then dissociated with the method described by Buckler and Vaughn-Jones (4). Briefly, the carotid bodies were incubated at 37°C with trypsin-collagenase in PBS for 10-15 min, then teased apart with forceps and digested for a further 5 min before trituration with a glass pipette. After a 5-min centrifugation, the supernatant was removed, and the pellet was resuspended in growth medium before plating at 30 µl onto poly-D-lysine-coated glass coverslips. The cells were left for at least 1.5-2 h to recover from dissociation before study; most cells were studied after an overnight incubation. Type I cells have a high catecholamine content, and in preliminary studies, characteristic type I cell morphology (~10- to 15-µm diameter, rounded shape, and a tendency to occur in clusters) was found to be correlated with the presence of catecholamines by using glyoxylic acid-induced amine fluorescence (21). In subsequent studies, the cells were identified by morphology.

Superfusion Solutions

The standard buffered salt solution (BSS) superfusate contained (mM) 118 NaCl, 24 NaHCO3, 3 KCl, 2 KH2PO4, 1.2 CaCl2, 1 MgCl2, and 10 glucose. The baseline solution was equilibrated with 5% CO2-20% O2 and contained propidium iodide (PI; 5 µg/ml). All solutions were filter sterilized at 0.22 µm (Millipore).

Intracellular Ca2+ and pH Measurements

Before study, the cells were examined for PI fluorescence. Cells with damaged membranes take up PI, which binds to nucleic acids to form a fluorescent complex with an absorbance peak at 535 nm and an emission peak at 617 nm. Cells with nuclear PI fluorescence were rejected, and coverslips with a high proportion of PI-positive cells were not studied.

[Ca2+]i was measured with the Ca2+-sensitive fluorescent indicator fura 2 (12). The cells were studied in a closed chamber through which BSS flowed by gravity at ~1 ml/min. All tubing was stainless steel to minimize gas exchange. A valve allowed switching of the superfusate between baseline and reservoirs equilibrated with different gas mixtures. The reservoirs, microscope stage, and superfusate entering the chamber were temperature controlled at 35-36°C by circulating water.

The methods used for the Ca2+ studies were based on those described by Sterni et al. (27). Dissociated cells attached to a coverslip were loaded by incubation for 8 min at 37°C in BSS containing 4 µM fura 2-AM. Fura 2-AM is membrane permeant and is deesterified to the [Ca2+]i-sensitive fura 2 within the cell. Fura 2-AM was stored at -20°C in DMSO at 0.8 µg/µl before dilution to the working concentration in BSS. Incubation was in a 5% CO2 atmosphere to maintain the correct BSS pH. After the cells were loaded, the coverslip with the attached cells formed the floor of a closed superfusion chamber placed on a Zeiss Axiovert TV135 microscope with a ×40 Fluar (Zeiss) objective. Excitation light was provided by a 75-W xenon light source attenuated to <20% full power by neutral density filters. Under these conditions, photobleaching was minimal over the ~30-min experiment duration. Fura 2 fluorescence emission was measured at 505-540 nm while the cells were illuminated alternately by a computer-controlled filter wheel at 340 and 380 nm. Image pairs were collected <1 s apart at intervals of 8-12 s.

Intracellular pH (pHi) was measured with the fluorescent indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) (25). Coverslips with attached cells were incubated in 6.5 µM BCECF-AM (Molecular Probes, Eugene, OR) in BSS for 8 min at 37°C. The coverslip was then mounted in a closed superfusion chamber as for the Ca2+ measurements. Fluorescence emission was measured at 505-540 nm. Excitation illumination was at 440 and 495 nm. Image pairs were collected <1 s apart at intervals of 12-20 s.

Pairs of fluorescence images were recorded with a Videoscope intensifier (model VS31845) and charge-coupled device camera (model 200E) at a constant intensifier and camera gain. Images were collected as eight-frame averages, digitized, and stored for later analysis with Metafluor (Universal Imaging). Background images were acquired at the end of each experiment from an area with no cells and subtracted pixel by pixel from the experimental images. In preliminary experiments, autofluorescence was found to be negligible (<1% of intensity) with the filter sets and image intensifier gain settings used. Fluorescence intensity data from each of the selected cells in the field were collected and stored separately and treated as independent observations. [Ca2+]i was calculated with in vitro calibration according to the method of Grynkeiwicz et al. (12). Briefly, this method finds the fluorescence intensity ratios in free fura 2 saturated with Ca2+ (maximum ratio) and in Ca2+-free solution (minimum ratio). These values are then used by substitution to find the dissociation constant. By knowing the maximum and minimum ratios and the dissociation constant, [Ca2+]i can be calculated for any observed value of the fluorescence intensity ratio. pHi was calculated from a calibration function obtained with the high-K+-nigericin method (25). Metafluor software (Universal Imaging) was used for measurements of fluorescence intensities in selected cells within fields, ratio calculations, and image and graphic displays.

Measurement of [Ca2+]i depends on deesterification of fura 2-AM inside the cell. Preliminary studies were done to confirm that the cells were able to deesterify fura 2-AM when prepared as described here. Cells from newborn and 2-wk rats were given a nonspecific depolarizing challenge of BSS, with 40 mM K+ replacing an equimolar amount of Na+. Cells from both age groups responded with an increase in [Ca2+]i of ~500-600 nM, confirming both that fura 2 was present in the Ca2+-sensitive form and that Ca2+ entry could occur on depolarization. Some cell fields in both age groups were also treated with the Ca2+ ionophore ionomycin. Intracellular Ca2+ rose to micromolar levels in both groups, demonstrating effective deesterification of fura 2-AM.

Superfusate PO2 was measured immediately upstream from the cell chamber with an in-line electrode (Microelectrodes). Preliminary tests showed no significant leakage of O2 through the short length of flexible tubing between the electrode and chamber at the superfusate flow rate used in these studies.

Experimental Protocol

Ca2+ and pH studies. Fields of cells were selected for study by visual criteria. Isolated and clustered cells were selected for study. Challenges were given by switching from baseline BSS superfusate equilibrated with 20% O2-5% CO2 to BSS equilibrated with one of the following gas mixtures: hypoxia (5% CO2-1% O2), hypercapnia (15% CO2-20% O2), combined hypercarbia plus hypoxia (1% O2-15% CO2), and anoxia (5% CO2-balance N2 and 0.5 mM sodium dithionite). Fifteen percent CO2 was used to allow comparison with published studies that used 10-20% CO2 (e.g., Ref. 4). Challenges were for 2 min and were followed by a 5-min recovery period. Preliminary tests showed that a 5-min recovery was enough for full recovery from all challenges except anoxia, which required up to 10 min. Consequently, the order of presentation was not randomized, but anoxia was always presented last. Cells showing unstable Ca2+ concentration ([Ca2+]) values under baseline conditions and cells with no significant response to anoxia were excluded from the study.

Electrophysiological recording. Recordings were made from preparations taken from 28 rats in four age groups: 20-day fetal (n = 4), 1-3 days postnatal (n = 4), 8-11 days (8 days; n = 8), and 16-21 days (18 days; n = 10). However, there was no significant difference between the fetal and 1-day groups in any of their responses, and the two groups were therefore combined to give a new 1-day group (n = 8). CSN activity was recorded with the technique described by Kholwadwala and Donnelly (16). The carotid bifurcation was removed, and the carotid body with its attached nerve was dissected out on a cold stage under a microscope. The carotid body was transferred to an open chamber mounted on an inverted microscope and superfused with BSS at 35°C equilibrated with 5% CO2-air. A fine needle was used to hold the carotid body in position on the floor of the bath. A suction electrode was positioned at the cut end of the CSN, and a small bundle of fibers was sucked into the tip. Amplification and display were conventional. A window discriminator was used to select impulses of at least two times the noise level. Impulses were counted in 1-s bins. All pulse counts were digitized and stored as a computer file. In some experiments, the number of active fibers present was small enough for a single-fiber impulse to be selected with the window discriminator. It was found, however, that such single-fiber recordings showed response profiles very similar to those seen in few fiber recordings, and they were not used routinely. A multiway valve block allowed switching of the superfusate between baseline and challenges.

Experimental Protocol: Electrophysiological Recording

Baseline superfusate was BSS equilibrated with 5% CO2-20% O2. Challenge superfusates were BSS equilibrated with 5% CO2-1% O2 (hypoxia), 15% CO2-20% O2 (hypercarbia), or 15% CO2-1% O2 (combined hypoxia plus hypercarbia). The hypoxia level of 1% was chosen to produce a severe challenge without anoxic depression. Challenges were given for 2 min, followed by a recovery period of baseline perfusion for at least 5 min. Preliminary studies showed that a 5-min recovery period allowed complete recovery of the response and that the order of presentation did not affect the response. To simplify the analysis procedure, challenges were usually given in a standardized order: first hypoxia, then CO2, and finally the combined challenge. The sequence was then repeated at least once, and data were rejected if peak responses to the same challenge differed by >20%.

Data Analysis

Ca2+ studies. The number of independent preparations was 3, 4, 5, and 7 in the four age groups (-1, 1, 8, and 14 days, respectively). Each cell preparation was from carotid bodies harvested from 4-6 rats, and data were obtained from 1-3 cell fields made from each preparation. Responses were calculated for each cell as the change in (Delta ) [Ca2+]i in nanomoles per liter between the mean baseline value during the preceding minute and the peak value recorded during the challenge. Each data point represents the mean of the maximum values of Delta [Ca2+]i for all the responding cells in a single field. All the fields studied in an age group were combined to derive a mean ± SE for each age group and challenge. Statistical comparisons were made between age groups. Values are given as means ± SE. The value of n is the number of cell fields from which data were obtained.

Electrophysiological recording. Peak neural responses to the challenges are expressed as absolute changes (Delta impulses/s) from baseline, which was defined as the mean impulse frequency during the 1-min period preceding each response. A 5-s moving average was applied as a smoothing function to the impulse rate to avoid results being skewed by a single high count.

O2-CO2 interaction. For the study of O2-CO2 interaction, normoxic and hypoxic responses to CO2 were measured. The normoxic response was the difference between [Ca2+]i or CSN activity at baseline (20% O2-5% CO2) and in hypercapnia (20% O2-15% CO2). The hypoxic CO2 response was the difference between [Ca2+]i or CSN activity in hypoxia (1% O2-5% CO2) and the combined challenge (1% O2-15% CO2). A significant difference between normoxic and hypoxic CO2 responses indicated that hypoxia increased the sensitivity to CO2, i.e., that multiplicative interaction was occurring.

Statistical Analysis

Similar methods were used for statistical analysis of both cell Ca2+ and nerve recording data. Significant effects of age on the responses were detected by ANOVA followed by post hoc testing (Bonferroni) to determine homogeneous subsets of data. Where appropriate, a paired t-test was used to compare paired sets of data. A P value of <0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Chemoreceptor Cell Ca2+ Responses

Responses of the cells to all challenges are summarized in Table 1. Figure 1 shows the averaged [Ca2+]i response profiles for typical single fields of type I cells from rats at 1 (A) and 21 (B) days challenged in turn with hypoxia, CO2, both stimuli together, and anoxia. The average Delta [Ca2+]i values for each age group and stimulus are shown in Table 1. In general, the [Ca2+]i responses to all stimuli were weak or absent in cells from fetal and 1-day-old rats and increased with age. Both clustered and single cells showed responses to challenges.

                              
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Table 1.   Baseline, peak, and change in [Ca2+]i responses in carotid body type I cells to hypoxia and CO2 challenges in 4 age groups



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Fig. 1.   A: mean intracellular Ca2+ concentration ([Ca2+]i) responses to hypoxia, hypercapnia, a combined challenge, and anoxia recorded from a field of 19 type I cells from newborn rats (<24 h). Ca2+ values were calculated from 340- to 380-nm fluorescence intensity ratios after correction for background. Challenges were presented by superfusion with buffered salt solution equilibrated with appropriate gas mixtures. Values are means ± SE. B: same as A but showing data from a field of 21 cells prepared from 21-day rats. Horizontal bars, times for each challenge. All responses were significantly greater than those in newborn cells.

Anoxia

Anoxia evoked significant responses at all ages (Fig. 2A). The Delta [Ca2+]i response increased significantly with age (P < 0.001 by ANOVA) from 115 nM in the -1-day group to 542 nM at 14 days. Post hoc testing showed significant increases occurred between 1 (180 nM) and 8 (434 nM) days, with no significant change between the 8- and 14-day groups.


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Fig. 2.   Change in (Delta ) Ca2+ concentration ([Ca2+]) in response to anoxia (0% O2 and dithionite; A), hypoxia (1% O2; B), hypercapnia (15% CO2; C), and combined hypoxia plus hypercapnia (D). 1d, 8d, and 14d, 1, 8, and 14 days, respectively. Values are means ± SE. Note that vertical scale in B-D is larger than scale in A. Horizontal bars below x-axis indicate homogeneous subgroups of data.

Delta [Ca2+]i Response to Hypoxia

The Delta [Ca2+]i response to 1% O2 (PO2 ~7 mmHg) increased significantly with age (P < 0.001 by ANOVA; Fig. 2B). In cells from the -1- and 1-day groups, the mean Delta [Ca2+]i responses to hypoxia were 16 and 22 nM, respectively. In the 8- and 14-day groups, the mean Delta [Ca2+]i responses were 106 and 144 nM, respectively. Thus there was an approximately fivefold difference between 1 and 8 days, with little development before or after this period.

High CO2-Low pH

Equilibration of BSS with 15% CO2 resulted in a combined high CO2-low pH challenge. Mean extracellular pH (pHo) at baseline (5% CO2) was 7.45 ± 0.02, falling to 6.95 ± 0.02 with 15% CO2. The mean Delta pHo when CO2 was changed from 5 to 15% was 0.50 ± 0.013. Fluorescence measurements with BCECF showed that pHi also fell as CO2 concentration increased. In cells from the 2- to 3-wk group, the mean Delta pHi was 0.26 ± 0.02 when CO2 increased from 5 to 15%. The mean value of the slope Delta pHi/Delta pHo was 0.59 ± 0.03 at 2-3 wk and 0.71 ± 0.05 at 0-1 day (not significant); thus the effect of CO2 on pHi did not change significantly with age and could not account for the observed maturation in the [Ca2+]i response to CO2.

The Delta [Ca2+]i response to 15% CO2 increased significantly with age (P < 0.001 by ANOVA; Fig. 2C). The response increased fourfold (from 32 to 127 nM) between 1 and 8 days. Unexpectedly, there was a decrease in the 14-day CO2 response to 81 nM.

Combined 15% CO2-1% O2

Responses to the combined stimulus were significantly greater than to either stimulus alone at 8 and 14 days (P < 0.01 by paired t-test) but not at earlier ages (Fig. 2D). The mean Delta [Ca2+]i responses were 44 nM at -1 day and 47 nM at 1 day. By 8 days, the response increased to 244 nM, with no further significant increase at 14 days (273 nM). Post hoc testing confirmed that development occurred mainly between 1 and 8 days, with no significant changes occurring before or after this period.

Interaction of CO2 and O2 Effects on the Type I Cell

Stimulus interaction was examined by comparing the Delta [Ca2+] response between 5 and 15% CO2 in 20% O2 (normoxia) with the Delta [Ca2+] response between 5 and 15% CO2 in 1% O2 (hypoxia). The difference between the normoxic and hypoxic Delta [Ca2+] responses to CO2 was used as a measure of O2-CO2 interaction.

The results are shown in Fig. 3. In the two youngest age groups, hypoxic and normoxic CO2 responses were identical. In the 8- and 14-day groups, the group mean for the hypoxic CO2 response was larger than that for the normoxic group but in neither case was the difference significant by t-test. We found no significant multiplicative interaction between CO2 and hypoxia in the type I cell Delta [Ca2+] response. The Delta [Ca2+] responses to hypoxia and CO2 appear to be additive at ages between term fetal and 2-3 wks.


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Fig. 3.   Effect of hypoxia on response to CO2. Values are means ± SE of Delta [Ca2+] responses of type I cells to 15% CO2 under normoxic and hypoxic conditions. See text for details. Hypoxia did not significantly affect response to CO2 at any age.

Electrophysiological Recordings From the CSN

Figure 4 shows the impulse frequency against time in a typical experiment, in this case a 2-wk rat in vitro carotid body preparation. Responses are shown to hypoxia (1% O2), hypercarbia (15% CO2), and a combined challenge. Responses usually showed an initial rise to a plateau that was sustained briefly before a decline to a lower level. The response was taken as the maximum frequency observed during the 2-min challenge after a 5-s moving average function was applied to reduce the effect of anomalous single counts. No maturation of response was evident for any challenge between term fetal and 3-day postnatal rats, and for statistical analysis, all preparations within this range were combined into a single group.


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Fig. 4.   Change in carotid sinus nerve (CSN) activity recorded in an in vitro preparation from a 2-wk rat in response to 2-min challenges as indicated. Each challenge was presented twice. Response to combined challenge (1% O2 + 15% CO2) was larger than sum of responses to individual challenges. imp, Impulse.

Hypoxia

Figure 5A shows the means ± SE for the plateau response to hypoxia in the three age groups. The response to hypoxia increased significantly with age (P = 0.012 by ANOVA), but only the difference between the 1- and 8-day groups was significant by post hoc testing.


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Fig. 5.   CSN responses to hypoxia (A), hypercapnia (B), and combined hypoxia plus hypercapnia (C). Values are means ± SE of peak - baseline frequency. There was significant maturation of response to hypoxia and to combined challenge but not to hypercapnia. Horizontal bars, homogeneous subgroups of data. f-3, Fetal to 3 days. See text for details.

CO2

The response to hypercarbia (15% CO2, pH 7.0) was consistently smaller than that to 1% O2. There was typically a slow rise to a new level of CSN activity that was sustained for the duration of the challenge. Figure 5B shows the mean responses to CO2 for each age group. There was no significant difference between age groups by ANOVA (P = 0.46). Thus we did not find evidence for maturation of the CSN response to 15% CO2 over the range from the late-gestation fetus to 3 wk postnatal.

Combined CO2 and Hypoxia

The combined challenge evoked a greater increase in impulse frequency than either challenge given alone (Fig. 5C). The response profile was usually a transient peak followed by a decline toward baseline. The size of the neural response to the combined challenge increased significantly with age (P = 0.002 by ANOVA). The response in the 1-day group was significantly less than that in the 8- or 16-day groups, which were not significantly different from each other.

Relative Amplitude of Responses to Hypoxia and CO2

The CSN response to CO2 showed no significant increase with postnatal age, whereas the response to hypoxia increased significantly with age. Because some of the factors that contribute to interpreparation variability, such as the number of active fibers in the preparation, affect both responses, their effect can be reduced by taking the ratio of responses to the two challenges. Figure 6 shows the ratio of hypoxia to CO2 responses against age. The hypoxia-to-CO2 response ratio was significantly correlated with age over the range studied (r = 0.90; P < 0.001). The reduced variability allowed significant differences to be detected between all groups by post hoc testing, although the largest difference between groups occurred between 3 and 8 days. The ratio of responses to standardized hypoxic and hypercapnic stimuli appears to be a useful comparative measure for maturation of O2 sensing in the carotid body.


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Fig. 6.   Ratio of CSN responses to hypoxia (1% O2) and hypercarbia (15% CO2). Values are group means ± SE. Post hoc testing showed significant increases with each age group.

Development of O2-CO2 Interaction

The development of stimulus interaction was studied by measuring the effect of hypoxia on the response to CO2 in each age group. For each preparation, the hypoxic CO2 response was calculated as the difference between the responses (in Delta impulses/s) to 5% CO2-1% O2 and to 15% CO2-1% O2. The CO2 response in normoxia was determined previously (see CO2). Normoxic and hypoxic CSN responses to CO2 were then compared for each age group (Fig. 7). Group mean values for hypoxic and normoxic CO2 responses are shown in Fig. 7. Hypoxic and normoxic CO2 responses were significantly different in the 16-day group (P = 0.007 by paired t-test) but not at any earlier ages. This is in contrast to our findings on cells in which no significant stimulus interaction occurred at any age.


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Fig. 7.   CSN response to 15% CO2 in normoxic (20% O2) and hypoxic (1% O2) conditions. Values are means ± SE of peak - baseline frequency. Hypoxia caused a significant increase in response to CO2 in the oldest age group. * Significant difference between hypoxic and normoxic responses.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study provides new data on the site of chemoreceptor maturation in the carotid body. If maturation occurs mainly at the type I cell, then the time course of maturation in cell and whole carotid body responses should be consistent. This was true for the hypoxic responses. Neural and Delta [Ca2+]i responses to hypoxia developed at about the same time, increasing primarily between 1 and 8 days in both. However, we found two marked discrepancies in maturation of CO2-related responses. First, type I cell [Ca2+]i responses to CO2 increased approximately fivefold within the first week of life, whereas the carotid body neural response to CO2 did not change during postnatal development. Second, multiplicative O2-CO2 interaction occurred in carotid body neural responses of the 16-day group but not in the type I cell Delta [Ca2+]i response at any age. Thus O2-CO2 interaction and its maturation must occur at a site other than the type I cell [Ca2+] response. Because the response to hypoxia is determined partly by CO2, it follows that the maturation of chemoreceptor function cannot be completely characterized by studying [Ca2+] responses of isolated type I cells. The site and mechanism of multiplicative stimulus interaction remain unknown.

Technical Considerations

This study was on the development of function. The findings therefore depend on comparisons between age groups and are not greatly affected by any systematic errors in absolute values. However, some potential sources of systematic error should be discussed.

Because cells of different ages were compared, it is important to rule out artifacts of preparation that could have affected groups differently. Differences in dye loading or intracellular deesterification of fura 2 seems unlikely. The first would cause a difference in fluorescence intensity, and the second would lead to variation of the Delta [Ca2+] response to a maximal stimulus such as 40 mM K+. Absolute intensity showed no systematic difference between groups, and the preliminary experiments described in METHODS showed no variation between groups in the Delta [Ca2+] response to high K+ or ionomycin.

The CSN recordings were made with a superfused in vitro preparation. It has been reported that a PO2 gradient exists from the surface to the core of a superfused carotid body so that the challenge varies unpredictably with depth (9), and CSN data are therefore difficult to interpret. However, it is unlikely that internal gradients invalidated our developmental data. The data of Delpiano and Acker (9) show that such a gradient would be greatest under baseline conditions. Thus an increase might be expected in baseline activity with age, yet we found no correlation between baseline activity and age over the range studied (r = 0.26; P = 0.2). Perez-Garcia et al. (24) have reported that the dose-response relationship for the release of dopamine in hypoxia is almost identical for isolated cells and intact superfused carotid bodies, suggesting that any effect of O2 gradients within the superfused carotid body is minor. Moreover, our developmental profile for hypoxic responses is consistent with previous findings by Carroll et al. (6) in cat carotid bodies in vivo and by Kholwadwala and Donnelly (16), who used anoxia as a challenge. In both cases, the carotid body PO2 can be assumed to be uniform. Overall, it appears that even if an internal PO2 gradient affected the absolute level of CSN responses, our developmental findings are probably valid.

Theoretically, the core of a nonperfused preparation might become anoxic in a challenge with 1% O2, and anoxia causes depression of neural activity. However, anoxic depression does not appear to have limited our peak responses because the response to a hypoxia challenge (1% O2) was always increased by CO2. If anoxic depression occurred, it would reduce the apparent response so that the developmental increase in response would appear smaller than it really was. Anoxic depression could not produce an artifact that gave the impression of development.

To summarize, although there are uncertainties about the distribution of PO2 within the in vitro carotid body, data obtained with this preparation are consistent in important respects with findings in vivo. Moreover, the in vitro superfused carotid body preparation offers the only practical, reliable method of recording quantitative carotid sinus activity in the rat. Although the challenges seen by type I cells in the in vitro carotid body and in plated isolated cells were probably not identical, there is no reason to assume that the developmental profiles recorded cannot be usefully compared.

The cell preparation methods were based extensively on published work, particularly that of Buckler and Vaughn-Jones (4). It is known that the responses of type I cells to challenges are affected by the preparative methods used so that quantitative comparisons between different studies may show differing levels of response. However, the same methods for preparation and cell recording were used throughout this study, and data on developmental changes are therefore valid.

Site and Mechanism of Chemoreceptor Maturation

The site and mechanism of chemoreceptor maturation are unknown. Previous studies of possible mechanisms of carotid body development suggest that maturation is not due to perinatal structural alterations affecting blood flow and PO2 gradients (1), vascular changes (8, 20), efferent modulation (6), or changes in circulating modulators (16, 23). Perinatal changes in carotid body innervation have been reported (15), but their importance in response maturation is unknown. Other candidates include changes in type I cell innervation, trophic influences of carotid body innervation (13), excitatory neurotransmitter production, and postsynaptic changes in nerve terminals. Maturational changes could also occur as cell-cell interaction develops in the carotid glomi (22). Sterni et al. (27) have reported that isolated type I cells of the adult rabbit show [Ca2+]i responses to hypoxia that are three- to fivefold larger compared with those in cells isolated from newborns and studied under identical conditions. More recently, we (28) have reported on the developmental time course of the response to graded hypoxia in the rat type I cell. These findings were consistent with the proposal that maturation of the response to hypoxia occurred, at least in part, at the type I cell. Neither study addressed the responses to CO2 nor was there any attempt to relate development in the type I cell and the whole carotid body.

Recently, Pepper et al. (23) proposed that postnatal maturation of hypoxic chemosensitivity could be due to maturation of O2-CO2 interaction rather than development of the hypoxic response per se. Using an in vitro superfused preparation, they found that hypoxia increased the CO2 sensitivity of carotid bodies of adult but not of 5-day rats. The developmental profile was not studied, and no study to date has determined whether O2-CO2 interaction or development of CO2 sensitivity occurs at the level of the type I cell.

We found no evidence of a major change in sensitivity occurring at the time of birth in either cell Delta [Ca2+]i or CSN responses. However, there is strong evidence that the rise in PO2 at birth modulates carotid chemoreceptor resetting (3). The mechanism is unknown but may involve O2-modulated gene expression. PO2 modulates the expression of many cellular components, including enzymes such as tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis (10, 17). PO2 regulates gene expression, leading to peri- and postnatal changes in protein synthesis, synthesis of new receptors, or enzymes that may require a few days, consistent with our observations. However, it is not clear how O2-mediated changes in gene expression at birth can account for the appearance of O2-CO2 interaction in the CSN response after 16 days.

Developmental Profile of Responses in Type I Cells and the CSN

In the present study, the carotid body CSN response to hypoxia, like the type I cell [Ca2+]i response, increased between the term fetal newborn and 8-day groups and did not significantly increase further thereafter (Fig. 6). These findings are consistent with those of Kholwadwala and Donnelly (16). The observation that maturation in type I cell [Ca2+]i responses and CSN activity responses to hypoxia follow a similar time course is consistent with the hypothesis that maturation of type I cell sensitivity underlies, at least in part, overall development of the carotid chemoreceptor response to hypoxia.

Although [Ca2+]i responses to CO2 challenges have been reported by previous investigators (2, 5, 26) and their mechanism was discussed extensively by Buckler and Vaughn-Jones (5), the developmental profile has not previously been studied. We found that type I cell [Ca2+]i responses to 15% CO2 (pH ~7.0) developed at the same time as responses to hypoxia. They were weak in the fetus and at 1 day postnatal, increased between 1 and 8 days, and did not change further in the 2- to 3-wk group. In the absence of measurements from adults, we cannot be certain that no further maturation occurs between 3 and 4 wk and adult, but it seems likely that, in the rat, the type I cell [Ca2+]i response to both CO2 and hypoxia is nearly mature by ~8 days. Thus the 8- to 10-day cells used by many investigators and termed "neonatal" are in fact mature or almost so.

In the rat, the CSN response to CO2 in normoxia does not appear to change with age. This is consistent with Pepper et al. (23), who found no difference between newborn and adult responses. This finding for the neural response contrasts sharply with our results at the type I cell level where the Delta [Ca2+]i response to 15% CO2 increased fivefold between 1 and 8 days and did not change thereafter. This dissociation between maturation of the CO2 response in cell [Ca2+]i and CSN suggests that the link between type I cell [Ca2+]i and nerve activity differs between hypoxic and CO2-acid challenges. Because the mechanisms generating CSN activity in response to hypoxia and CO2 are not understood, we can only speculate concerning potential explanations. If the type I cell is the site of O2 and CO2 sensing in the carotid body, one possibility is that CO2 raises [Ca2+]i in the type I cell but does not lead to neurosecretion as hypoxia does. In support of this possibility, a recent study by Iturriaga and Alcayaga (14) reported that in the in vitro perfused and superfused cat carotid body, both hypoxia and CO2 increased CSN activity; however, although hypoxia also increased catecholamine secretion, CO2 did not. This finding implies that the type I cells respond to hypoxia and CO2 in different ways, even though both responses include an increase in [Ca2+]i. It is possible that the type I cell is not the major site of CO2 sensitivity in the carotid body.

O2-CO2 Interaction in Type I Cells and the CSN Response

Interaction between O2 and CO2 was determined by measuring the effect of hypoxia on the sensitivity to CO2. In [Ca2+] responses, multiplicative O2-CO2 interaction was insignificant at any age, whereas in CSN recordings, significant multiplicative interaction occurred in the oldest group. Clearly, there is an important difference between response properties at the level of the type I cell and the whole carotid body. The question therefore arises of where O2-CO2 interaction occurs if not at the type I cell. At present, only speculation is possible. Candidates include pH-dependent changes in the release or turnover of an excitatory neurotransmitter or effects of pH on postsynaptic responses to neurotransmitters.

Pepper et al. (23), using a similar in vitro nerve preparation but a different method of analysis, have previously reported significant multiplicative O2-CO2 interaction in adult rats but not in rat pups 5-7 days of age. In a previous study from our laboratory (6), O2-CO2 interaction in the in vivo cat carotid body was absent at 1 wk but clearly demonstrable in older kittens and adults. In contrast, in 2- to 11-day piglets, ventilatory measurements show that muliplicative O2-CO2 interaction was present shortly after birth and either did not increase (29) or decreased with age (30). Therefore, in rats and cats, but not in piglets, multiplicative O2-CO2 interaction in carotid body responses shows postnatal maturation. The mechanism underlying this species difference is unknown but may be related to maturity at birth. No similar studies have been reported in human infants.

The development of multiplicative interaction in the neural response showed a time course different from that for individual stimuli. Although the neural response to hypoxia clearly increased between 1 and 8 days, no multiplicative O2-CO2 interaction was detectable before 16-21 days. These data are not consistent with the hypothesis that postnatal resetting of carotid body hypoxic sensitivity is due to development of O2-CO2 interaction. Instead, the present data suggest that maturation of hypoxic sensitivity precedes maturation of O2-CO2 interaction. These findings are consistent with a previous study by our laboratory (6) on cat carotid bodies in vivo. Sensitivity to hypoxia was weakest in 1-wk-old kittens but increased to nearly adult levels by 4 wk of age. In contrast, O2-CO2 interaction in the cat carotid body did not become significant until after 4 wk. Thus, in both the rat and cat, maturation of hypoxic sensitivity precedes maturation of O2-CO2 interaction in carotid chemoreceptors. Indeed, in the cat carotid chemoreceptor, the effect of hypoxia on CO2 sensitivity continued to develop between 8 wk and adult, whereas the response to hypoxia appeared mature much earlier (6).

In conclusion, the responses of type I cells to O2 and CO2 show the greatest maturation between 3 and 8 days postnatal. The development of responses to hypoxia in the type I cell and CSN occurred on similar time courses, but the responses to CO2 and O2-CO2 interaction showed developental differences between type I cells and the CSN. Multiplicative interaction between O2 and CO2 was significant in the whole carotid body but could not be demonstrated in type I cells at any age. The maturation of carotid body function appears to be a complex and stimulus-specific process that occurs, in part, at the type I cell but also involves other so far unidentified mechanisms.


    ACKNOWLEDGEMENTS

This study was supported by National Heart, Lung, and Blood Institute Grant RO1-HL-54621-01 (to J. Carroll).


    FOOTNOTES

O. Bamford was partly supported by Research Grant SPOO17 from the Sudden Infant Death Syndrome Alliance.

Present address of M. H. Montrose: Dept. of Physiology and Biophysics, Indiana Univ., Indianapolis, IN 46202.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: O. S. Bamford, Pediatric Pulmonary Div., Park 316, The Johns Hopkins Children's Center, 600 North Wolfe St., Baltimore, MD 21287-2533 (E-mail: obamford{at}welchlink.welch.jhu.edu).

Received 5 October 1998; accepted in final form 28 January 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 276(5):L875-L884
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