1University of Arkansas for Medical Sciences, College of Medicine, Department of Pediatrics, and 2Arkansas Children's Hospital, Little Rock, Arkansas; and 3Johns Hopkins University School of Medicine, Johns Hopkins Children's Center, Baltimore, Maryland
Submitted 26 November 2003 ; accepted in final form 27 December 2004
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ABSTRACT |
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dopamine receptors; hypoxia; development; chemoreceptor
In neonates, but not adults, carotid denervation leads to high mortality rates and abnormalities of respiratory control (13, 15, 18, 30, 31), suggesting a vulnerable period during mammalian postnatal maturation during which the CB is important for survival and normal maturation of breathing control. Despite their importance in the developing infant, the carotid chemoreceptors have low sensitivity to hypoxia at birth and become more sensitive over the first few days or weeks of life (9, 10, 14, 17, 33, 36, 38), a process termed "resetting." The mechanism(s) underlying resetting are unknown, but some have postulated involvement of inhibitory neuromodulators such as dopamine (DA) (28, 29).
In rats and rabbits, an age-related increase in O2 sensitivity occurs at the type 1 cell level (41, 45). Both the [Ca2+]i rise and the neurotransmitter (catecholamine) release in response to hypoxia increase with maturation, exhibiting the same time course as CSN response maturation, strongly suggesting that a significant proportion of CB development occurs at the presynaptic level (16). However, the mechanisms that modulate this postnatal increase in presynaptic O2 sensitivity are poorly understood. Although DA may be excitatory in the CB under some conditions (27), most studies indicate that DA functions as an inhibitory modulator of CB activity, mainly via inhibitory D2 autoreceptors on the type 1 cell (25, 34, 37).
D2 DA receptor and tyrosine hydroxylase messenger RNAs are colocalized (24) in type 1 cells, D2 DA receptor messenger RNA levels significantly increase with postnatal age in several species, and DA release in vitro is modulated by D2 autoreceptors in an age-dependent fashion (2, 4, 5, 24). Because the inhibitory effects of DA likely involve signaling mechanisms that may change functionally during development, the known postnatal changes in D2 receptor mRNA expression do not necessarily predict the functional effects of D2 stimulation on the type 1 cell response to hypoxia. Although DA has been shown to inhibit CB type 1 cell [Ca2+]i responses to hypoxia in type 1 cell clusters from mature rats (32), developmental changes in the presynaptic effects of D2 receptor stimulation on CB type 1 cells have not been reported. Here we explore the effects of D2 receptor stimulation on type 1 cell [Ca2+]i responses to hypoxia in type 1 cell clusters from newborn and developing rats.
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METHODS |
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Rat pups were anesthetized with methoxyflurane and decapitated, and the head was placed in ice-cold saline. The carotid bifurcations were dissected and placed in ice-cold PBS (Sigma). The CB were dissected from the bifurcations and placed in 1 ml of enzymatic solution composed of 0.6 ml of PBS with 50 µM Ca2+, 0.2 ml of trypsin (1 mg/ml, Sigma), and 0.2 ml of type I collagenase (5 mg/ml, Sigma). The CB were incubated in the enzymatic solution at 37°C in 21% O2/5% CO2 for 20 min. The CB, along with the enzyme solution, were transferred to an Eppendorf tube with a fire-polished glass pipette and incubated for an additional 5 min. After the second incubation, the cells were dispersed by gentle rocking of the Eppendorf tube. The tissue was pelleted at 200 g for 2 min and resuspended in 1 ml of growth media composed of Ham's F-12 (Mediatech) with 1% fetal calf serum, 33 mM glucose, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 1% Fungizone, and 0.08 U/ml insulin. The cells were centrifuged a second time at 200 g for 2 min, the supernatant was removed, and growth media were added at 35 µl/coverslip (typically, 34 coverslips were used per experiment). The cells were plated on poly-D-lysine-coated glass coverslips at 30 µl/coverslip and incubated at 37°C in 21% O2/5% CO2 until use. Clusters of type 1 cells were studied between 4 and 8 h after plating.
Measurement of intracellular calcium. [Ca2+]i was measured by quantitative fluorescence imaging using the Ca2+-sensitive dye fura-2 (26). Cells attached to the coverslip were loaded with fura-2 by incubation for 8 min at 37°C in 21% O2/5% CO2 with 4 mM of fura-2 AM (Molecular Probes). Fura-2 fluorescent emission was measured at 510 nm in response to alternating excitation at 340 and 380 nm (Metafluor, Universal Imaging).
For each coverslip, the background light levels were determined and subtracted, pixel by pixel, from each image before measurement of the fluorescence intensity ratio at 340 nm/380 nm. [Ca2+]i was determined using the 340/380 fluorescence ratio and the following equation
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Experimental protocol.
After the cells were loaded with fura-2, the coverslip was placed in a closed microscope chamber and perfused with a bicarbonate-buffered balanced salt solution (BSS) containing (in mM): 118 NaCl, 23 NaHCO3, 3 KCl, 2 KH2PO4, 1.2 CaCl2, 1 MgCl2, and 10 glucose and equilibrated with 21% O2, 5% CO2, balance N2. Type 1 cells for study were identified by their characteristic appearance (rounded, occurrence in clusters) and size (10 µm) as previously reported (45). Only cells in typical clusters were studied. To further confirm type 1 cell identification, at the end of experiments, cells were challenged with 40 mM KCl to determine that they had a brisk [Ca2+]i rise in response to elevated extracellular potassium, as previously reported for CB type 1 cells for rats from birth to 21 days of age (45).
The 0% O2 hypoxia challenge superfusate consisted of the above BSS equilibrated with 0% O2 in 5% CO2/balance N2. The superfusate for milder hypoxia challenge was identical except for having been equilibrated with 1% O2 in 5% CO2/balance N2. All superfusates were maintained at 35°C. The perfusion rate was 1 ml/min. The oxygen tension of the superfusate entering the chamber was measured with a flow-through oxygen electrode (Microelectrodes, Londonderry, NH). Superfusate PO2 measured at the chamber inlet was
23 mmHg with the 0% O2 solution and
79 mmHg with the 1% O2 solution. The low superfusate PO2 levels were achieved by using stainless steel tubing throughout, oxygen-impermeable valves, and a closed imaging chamber.
The protocol used for cells from rats in the 1-, 3-, and 11- to 16-day-old age groups consisted of three challenges, as follows. While fura-2 fluorescence was measured at 340 and 380 nm every 8 s, cells were superfused with normoxic BSS for 5 min, challenged by suddenly switching the superfusate to the 0% O2 BSS described above for 90 s, and then returned to superfusion with normoxic BSS. After 5 min, the cells were challenged again with 0% O2 superfusate containing 10 µM quinpirole (RBI or Tocris) for 90 s and then returned to superfusion with normoxic BSS. After 5 min, the cells were challenged a third time with 0% O2 superfusate containing 10 µM quinpirole and 10 µM sulpiride (RBI) for 90 s and then returned to superfusion with normoxic BSS. In the 11- to 16-day group only, the protocol outlined above was performed in an identical manner except using 1% O2.
The rationale for the above protocol was to measure the type 1 cell [Ca2+]i response to hypoxia, to determine the degree of hypoxia response inhibition caused by 10 µM quinpirole, a specific DA D2 receptor agonist, and to block the effects of quinpirole with the specific DA D2 receptor antagonist sulpiride. The rationale for using a milder stimulus (1% O2) was to determine whether the effect of quinpirole was related to stimulus strength (i.e., greater effect during submaximal stimulation).
To assess variability of responses to sequential hypoxia challenge, a separate group of type 1 cells from 1-day-old and 11- to 14-day-old rats was exposed, using the same superfusion system, to repeated hypoxia challenges in the absence of quinpirole or sulpiride. While fura-2 fluorescence was measured at 340 and 380 nm every 8 s, cells were superfused with normoxic BSS for 5 min and then challenged with three consecutive 90-s exposures to the 0% O2 BSS, separated by 5 min of normoxic superfusion between challenges.
Freshly isolated CB type 1 cells tend to form two to six cell "clusters" on poly-L-lysine-coated glass. These are not three-dimensional clusters; rather, they are flat, one-cell-thick groupings of cells that contact each other only at their margins. In addition, our chamber superfusion fluid turnover was >10 times/min. Therefore, a significant effect of endogenous DA release from type 1 cells was considered highly unlikely. To determine whether endogenous DA could potentially influence our studies, experiments were conducted by comparing type 1 cell [Ca2+]i responses to 0% O2 (control) and 0% O2 plus 10 µM sulpiride in a subset of 3-day-old and 11- to 13-day-old rats. A small subset of cells was also tested using 10 µM sulpiride in normoxia.
Data analysis.
We studied only clusters of type 1 cells, which ranged in size from two to ten cells. For a given cluster of type 1 cells, fluorescence ratio data were averaged and counted as n = 1. The experimental protocol was performed only once per coverslip. Baseline values were calculated as the average [Ca2+]i during the 1-min period before a challenge, and peak values of [Ca2+]i were the maximum obtained during a 90-s challenge. [Ca2+]i was calculated as peak [Ca2+]i baseline [Ca2+]i. Values are presented as means ± SE for analysis of hypoxia response repeatability. The time to response onset was defined as the time (seconds) elapsed between turning the valve (to superfuse with 0% O2 challenge solution) and the first [Ca2+]i value >25% above prechallenge baseline. Comparisons at a given age between conditions were performed using ANOVA for repeated measures with the Tukey-Kramer multiple comparisons test for post hoc significance testing. Comparisons between ages were made using one-way ANOVA with the Tukey-Kramer multiple comparisons test for post hoc significance testing. Age-related changes in proportion of responses inhibited by quinpirole were compared using the Chi-square test for trend. Testing for differences in [Ca2+]i responses to 0% O2 with vs. without sulpiride was performed using Wilcoxon's signed rank test. All statistics were performed using GraphPad InStat or Prism (GraphPad Software, San Diego, CA). A P value <0.05 was considered statistically significant.
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RESULTS |
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Effect of sulpiride alone.
In 3-day-old rats, the [Ca2+]i response to 0% O2 was 104 ± 37 nM without sulpiride vs. 108 ± 34 nM in the presence of 10 µM sulpiride [n = 6, not significant (ns)]. In type 1 cells from 11- to 13-day-old rats, the
[Ca2+]i response to 0% O2 was 414 ± 64 nM without sulpiride vs. 442 ± 69 nM in the presence of 10 µM sulpiride (n = 3, ns). In addition, in type 1 cells from 11- to 16-day-old rats, neither 10 µM (n = 2) nor 100 µM sulpiride (n = 2) had any effect on resting [Ca2+]i level in normoxia. In the representative tracing shown (Fig. 3A), 10 µM sulpiride alone had little effect on the[Ca2+]i response to 0% O2, whereas in the same cluster, the response was markedly reduced by 10 µM quinpirole. These findings suggest little effect of endogenous DA in type 1 cell clusters under these experimental conditions.
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The effect of quinpirole on the average peak [Ca2+]i response to hypoxia in type 1 cells from the 11- to 16-day-old group is shown in Fig. 4. In cells from mature rats, quinpirole (10 µM) significantly decreased the mean peak [Ca2+]i response to 0% O2 from 561 ± 52 to 372 ± 38 nm (Fig. 4). Sulpiride (10 µM) significantly restored the peak [Ca2+]i response to 493 ± 47 nM, which was not statistically significantly different from the control [Ca2+]i response to 0% O2 (Fig. 4). Analysis as [Ca2+]i response to 0% O2 showed that quinpirole (10 µM) significantly decreased the mean
[Ca2+]i response to 0% O2 from 439 ± 51 to 254 ± 39 nm (P < 0.001), and sulpiride (10 µM) significantly restored the
[Ca2+]i response to 362 ± 46 nM (P < 0.05 different from quinpirole alone), which was not statistically significantly different from the control
[Ca2+]i response.
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Age-related effects of quinpirole. As previously reported (45), [Ca2+]i responses to 0% O2 were significantly smaller in type 1 cells from rats 13 days old (Figs. 1 and 4). The inhibitory effect of quinpirole on the type 1 cell [Ca2+]i response to 0% O2 increased with age. In the 1-day-old group, the 12% decrease in peak [Ca2+]i response in the presence of quinpirole (second hypoxia challenge) was significant (P < 0.05) compared with the first hypoxia challenge but was not significantly different from the third response, with sulpiride (Fig. 4). In type 1 cells from the 3-day-old group, quinpirole decreased the peak [Ca2+]i response to 0% O2 challenge by 18%, and 10 µM sulpiride restored the response to near control levels (Fig. 4). In contrast, in the 14-day-old group, quinpirole decreased the peak [Ca2+]i response to 0% O2 challenge by 34%, and, in the presence of sulpiride, peak [Ca2+]i was restored to a level similar to the first control challenge. Figure 7 plots the effect of age on inhibition of the peak [Ca2+]i response to 0% O2 and was expressed in two ways as follows: 1) % inhibition relative to first hypoxia challenge, and 2) % inhibition relative to the [Ca2+]i response magnitude in the third hypoxia challenge (in the presence of sulpiride). The % inhibition of the hypoxia response caused by quinpirole in the mature group was significantly greater than at 1 and 3 days (Fig. 7).
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DISCUSSION |
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Methodological issues. The purpose of this study was to determine whether DA inhibits the carotid chemoreceptor type 1 cell [Ca2+]i response to hypoxia in an age-dependent manner. Quinpirole, a selective DA D2 receptor agonist, was used instead of DA because of its greater stability in solution and selectivity for the D2 receptor. Although DA D1 receptors are present in the CB, their location and potential function are unclear (1, 3). Because D1 receptor expression, and therefore the D1:D2 ratio, may change with development, questions concerning developmental changes in agonist-mediated effects are better addressed using a selective agonist. Quinpirole is also an agonist for D3 and D4 receptors. However, D3/D4 receptors have not been described in the CB.
CB type 1 cells have a characteristic morphology when enzymatically dissociated and studied within 12 h. In previous studies, we correlated morphology with [Ca2+]i responses to elevated extracellular potassium and with catecholamine staining using the formaldehyde-glutaraldehyde method (41, 45). In addition, cells in this study exhibited a brisk [Ca2+]i rise in response to hypoxia challenge, which is characteristic of dissociated type 1 cells; other cell types in the CB are not O2 sensitive. Therefore, because we studied O2-sensitive cells from the CB, it is highly likely that they were type 1 cells.
For this study, we chose to use only CB type 1 cell clusters and to exclude single isolated cells. Single isolated cells vs. cells in clusters may exhibit different response characteristics, theoretically, due to cell-cell coupling (20). In addition, single isolated type 1 cells are more difficult to distinguish from other cell types than cells in clusters with characteristic cluster morphology. Bright et al. (11) reported that adult rat type 1 cells in clusters never responded to hypoxia. However, that study used superfusate equilibrated to 35 mmHg PO2 as the hypoxia stimulus. In our experience, a lower PO2 consistently elicits a [Ca2+]i rise in clustered type 1 cells (6, 45).
Another consideration is whether clusters of type 1 cells secrete endogenous DA in sufficient concentrations to locally inhibit the [Ca2+]i response to hypoxia. This is unlikely because freshly isolated type 1 cell clusters are only one cell deep (monolayer) and the superfusion fluid turnover rate was >10 times/min in our imaging chamber. Although we did not address this question systematically, pilot studies indicated that the [Ca2+]i response to 0% O2 was not significantly increased by 10 µM sulpiride alone in the type 1 cells from 11- to 13-day-old rats. In addition, in type 1 cells from 11- to 16-day-old rats, neither 10 µM nor 100 µM sulpiride had any effect on resting [Ca2+]i level in normoxia. Finally, the [Ca2+]i response to hypoxia in the presence of sulpiride was not larger than the control [Ca2+]i response to hypoxia, which may be expected if a large inhibitory effect of endogenous DA was present. However, we cannot rule out a small effect of endogenous DA.
Interpretation of results. The rationale behind the study design was to measure the effects, on the type 1 cell [Ca2+]i response to hypoxia, of DA D2 receptor stimulation at different ages. If the resulting inhibition was a D2 receptor-mediated phenomenon, then a selective D2 receptor antagonist should reverse the inhibitory effect, at least partially. Although this is the most plausible explanation for our findings, the possibility must be considered that sulpiride restored the [Ca2+]i response magnitude by some other mechanism. To our knowledge, an effect of sulpiride on ether-à-go-go-related gene (K+ channel) (erg)-like or human erg (HERG)-like K+ currents has not been described, although D2 receptor antagonists such as domperidone and droperidol have been reported to be inhibitors of erg-like currents in other cell types (19, 42) and an inhibitor of HERG-like currents has been shown to increase [Ca2+]i in rabbit CB type 1 cells (39). However, in our study, sulpiride only enhanced the [Ca2+]i response magnitude in the presence of quinpirole. In addition, in the 1-day-old group where the inhibitory effect of quinpirole was minimal, sulpiride also had a minimal effect (Figs. 1 and 4). Together, these findings strongly suggest a D2 receptor-specific mechanism of action for sulpiride in this setting.
The mechanism by which D2 receptor stimulation decreases the [Ca2+]i response to hypoxia is unknown and has received little attention. In the only other report of DA receptor agonist effects on [Ca2+]i in CB type 1 cells, the DA agonist tyramine inhibited the [Ca2+]i increase caused by mild hypoxia in some glomus cells dissociated from adult rats (46). Benot and Lopez-Barneo (8) reported that voltage-dependent Ca2+ current in CB type 1 cells was inhibited by DA, without affecting K+ or Na+ currents. In PC-12 cells, 10 µM quinpirole markedly inhibited the [Ca2+]i rise in response to hypoxia without affecting hypoxia-induced membrane potential depolarization, suggesting that quinpirole inhibits Ca2+ influx in that cell type (47).
The type 1 cell [Ca2+]i response to hypoxia increased with age, consistent with our previous reports in developing rabbits and rats (6, 41, 45), and age-related changes in the effects of quinpirole were also evident. Quinpirole had a minimal effect on the [Ca2+]i response to 0% O2 in the 1-day-old group. By 3 days of age, the [Ca2+]i response amplitude was still small, relative to 1116 days, but quinpirole did have a small inhibitory effect that was abolished by equimolar sulpiride (Fig. 4). By 1116 days of age, the inhibitory effects of quinpirole are clear for the 0% O2 challenge (Fig. 1) and more apparent for the 1% O2 challenge (Fig. 6). Thus there was a clear age-related increase in the inhibitory effect of DA D2 receptor stimulation by a selective agonist.
It is difficult to speculate on the mechanism of the age-related changes we observed in the inhibitory effects of DA on the type 1 cell [Ca2+]i response to hypoxia. The inhibitory effects of DA could be mediated by age-related changes in D2 receptor expression, G protein modulation of Ca2+ currents, ratios of DA-sensitive Ca2+ channel subtypes, or age-dependent effects on K+ currents.
As shown in Figs. 1, 5, and 6A, quinpirole delayed the onset of the [Ca2+]i response to hypoxia by 1520 s compared with control. Equimolar sulpiride partially restored the onset and speed of the [Ca2+]i response, especially for the 1% O2 challenge. The response onset slowing was evident in individual cell cluster measurements as well as in composite tracings (Fig. 6A). The mechanism underlying this effect is unknown, although DA has been shown to inhibit voltage-dependent Ca2+ currents of CB type 1 cells (8), and, in other cell types, quinpirole not only reduces Ca2+ current amplitude but slows activation kinetics (40, 44). Slowing of the [Ca2+]i response onset is consistent with G protein-mediated inhibition of voltage-dependent Ca2+ currents (7).
D2 receptor stimulation likely alters Ca2+ influx in neurons, at least in part, by altering the current-voltage relationship of voltage-dependent Ca2+ channels (7). Therefore, given that the PO2 in the chamber required time (seconds) to reach steady state and that the cells take time (seconds) to depolarize, the slower response onset in the presence of quinpirole may result from a rightward shift in the current-voltage relationship of Ca2+ currents. That is, in the presence of quinpirole, more time would be required to reach the level of depolarization necessary to initiate voltage-dependent Ca2+ entry. With the 1% O2 challenge, the response-onset slowing effect was completely abolished by equimolar sulpiride (Fig. 6A), strongly suggesting that it was a D2 receptor-mediated effect.
Functional implications. Our findings indicate that, in type 1 cells from mature rats, stimulation of D2 receptors reversibly slows and inhibits the [Ca2+]i response to hypoxia. If the present consensus view of the O2 chemotransduction cascade was correct, DA would be expected to decrease exocytosis of excitatory neurotransmitters, and, therefore, CSN activity in response to a given level of hypoxia. Our results provide additional support for the view that DA serves an inhibitory autoregulatory function in the CB.
From a developmental perspective, the main question posed by this study was whether DA is a plausible modulator of postnatal O2 sensitivity resetting. In rat CB, DA turnover rate is high at birth and then declines (28, 29). However, for changes in DA neurosecretion to explain postnatal resetting of carotid chemoreceptor O2 sensitivity, DA levels should be high in newborns and decline with age on a time course consistent with the known developmental time course of the hypoxia response (2 wk in rats). To the contrary, in the CB of developing rats, baseline and anoxia-stimulated free tissue catecholamine levels have been found to be low from 16 days, approximately doubled by 10 days, and approximately doubled again by 2030 days (17). Not only are CB DA levels and release low in newborns, but our findings indicate that the inhibitory effects of DA on the [Ca2+]i response to hypoxia are also minimal at this age. In addition, as baseline and hypoxia-stimulated DA levels in the rat CB increase with age (17), the inhibitory effects of DA on [Ca2+]i also increase along a similar time course. Together, these data make it unlikely that DA is a mediator of postnatal CB O2 sensitivity resetting. More likely, dopaminergic inhibition matures along with CB O2 sensitivity and serves other functions, such as modulating CB O2 sensitivity under conditions of chronic hypoxia (35, 43).
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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REFERENCES |
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