Department of Physiology, University of Adelaide, Adelaide 5005, Australia
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ABSTRACT |
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The hypoxia-evoked secretion of catecholamines from the noninnervated fetal adrenal gland is essential for surviving intrauterine hypoxemia. The ion channels responsible for the initial depolarization that leads to catecholamine secretion have not been identified. Patch-clamp studies of adrenal chromaffin cells isolated from fetal and adult sheep revealed the presence of a Ca2+-dependent K+ current that was reduced by hypoxia. Apamin, a blocker of small-conductance K+ (SK) channels, reduced the Ca2+-dependent K+ current, and the sensitivity of the channels to apamin indicated that the channels involved were of the SK2 subtype. In the presence of apamin, the hypoxia-evoked change in K+ currents was largely eliminated. Both hypoxia and apamin blocked a K+ current responsible for maintaining the resting potential of the cell, and the depolarization resulting from both led to an influx of Ca2+. Simultaneous application of hypoxia and apamin did not potentiate the increase in cytosolic Ca2+ concentration beyond that seen with either agent alone. Similar results were seen with curare, another blocker of SK channels. These results indicate that closure of SK2 channels would be the initiating event in the hypoxia-evoked catecholamine secretion in the adrenal medulla.
hypoxia; potassium channels; calcium
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INTRODUCTION |
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IN THE ADRENAL MEDULLA of the sheep fetus and rat newborn, before the development of a functional innervation, hypoxia acts directly on the chromaffin cells to stimulate catecholamine secretion (4, 5, 21). This direct response of the adrenal medullary cells to hypoxia results in a redirection of blood to the heart, brain, and adrenal glands and is crucial for the survival of the fetus (18). Once innervation has developed (by 130 days of gestation in the sheep), the direct response of the adrenal medullary cells to low PO2 disappears (4); however, it is restored upon denervation, as demonstrated by the hypoxia-evoked secretion of catecholamines from the perfused whole adrenal gland isolated from the adult sheep (1).
It is believed that the mechanism of oxygen sensing in the adrenal chromaffin cells is similar to that in the glomus cells of the carotid body, the major chemoreceptor cells in the adult mammals, where it involves oxygen-sensitive K+ (KO2) channels. These channels close in hypoxic conditions, leading to depolarization, opening of voltage-dependent Ca2+ channels, and exocytosis of neurotransmitters subsequent to the elevation of the cytosolic Ca2+ concentration ([Ca2+]i) (reviewed in Ref. 19). In both the carotid body and the adrenal medulla, there is debate over what types of KO2 channels are responsible for the initial hypoxia-evoked depolarization of the cell membrane.
In adrenal chromaffin cells of the sheep, a current through Ca2+-dependent K+ (KCa) channels was shown to be reduced during hypoxia (20), but the types of KCa channels involved were not fully identified. Large-conductance K+ (BK) channels are present in the adrenal chromaffin cells of many species (13, 14), and at least part of the oxygen-sensitive current in the sheep adrenal medulla is through BK channels (20). Closure of these channels is unlikely to cause the hypoxia-evoked depolarization, however, because BK channels are strongly voltage dependent and normally are closed at resting potential (11). KCa channels that are open at resting potential and might be responsible for the sensitivity of the chromaffin cells to hypoxia are intermediate-conductance K+ (IK) channels and the small-conductance K+ (SK) channels. IK channels are not present in the human adrenal medulla (8) and do not appear to have been described in the adrenal of other species. Voltage-independent SK channels have been found in bovine and rat chromaffin cells (13, 14), and mRNA for SK2 channels, a subtype of the SK channel family, has been isolated from the rat adrenal medulla (9). Recently, SK channels were suggested to play the role of the oxygen sensor in rat adrenal chromaffin cells (10).
In this report, the results of patch-clamp studies and intracellular Ca2+ measurements show that more than one channel type is involved in the response of the sheep adrenal medulla to hypoxic stimuli, with the SK channel being responsible for the initial depolarization.
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MATERIALS AND METHODS |
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Isolation of adrenal chromaffin cells. Pregnant Border Leicester × Merino cross ewes between 137 and 142 days of gestation (term: 147 ± 3 days) were used for these experiments, which were approved by the Adelaide University Animal Ethics Committee. Ewes were killed with intravenous pentobarbitone (8.1 g), and the fetus was removed through a laparotomy incision and decapitated. The fetal adrenal gland was removed, and the medulla, dissected free of the cortex, was minced and incubated at 37°C for 45 min in a Ca2+-free Locke's solution consisting of (in mM) 154 NaCl, 5.6 KCl, 3.6 NaHCO3, 5.6 glucose, and 5.0 HEPES, pH 7.4, supplemented with collagenase (Worthington type II, 0.1%) and deoxyribonuclease (type IV, 100 U/ml; Sigma). Repeated pipetting mechanically dispersed the tissue, and the cells were washed twice in Ca2+-free Locke's solution. The cells were then resuspended in DMEM containing 10% charcoal-absorbed fetal bovine serum (Trace Biosciences, Sydney, Australia), 100 U/ml penicillin, and 0.5 mg/ml streptomycin, plated on glass coverslips, and maintained in culture. Adult chromaffin cells were isolated by the same technique, with the only modification being the doubling of the collagenase concentration to 0.2%.
Electrophysiology.
Whole cell recordings were conducted on cells 1-3 days after
plating, always at room temperature. Pipettes with a resistance between
2 and 4 M were used, and series resistance was 80-95% compensated. Currents were recorded with an EPC-9 amplifier (HEKA), and
data acquisition and analysis were performed on an IBM-compatible computer using Pulse and Pulsefit software (v8.11; HEKA). Corrections for liquid junction potential between the bath and electrode solutions were estimated by using JPCalc (2). Adrenal chromaffin
cells, in a bath of ~500 µl, were superfused continuously at
2-3 ml/min with a solution containing (in mM) 140 NaCl, 4 KCl, 2 CaCl2, 2 MgCl2, and 10 HEPES, adjusted to pH
7.4 with NaOH. The bath solution was equilibrated with either room air
or 100% N2. PO2 was measured with
an O2 microelectrode (World Precision Instruments) placed near the recording site. With hypoxic superfusion,
PO2 reached a level of 7-10 mmHg. For
electrophysiological recordings, cells were exposed to these low
PO2 levels for between 1 and 5 min. When
Ca2+ currents were recorded, cells were bathed in a
solution containing (in mM) 10 CaCl2, 2 MgCl2,
70 NaCl, 70 tetraethylammonium (TEA)-Cl, 10 HEPES, 3 × 10
4 TTX, and 0.1 5-nitro-2-(3-phenylpropylamino)benzoic
acid (NPPB). Whole cell K+ currents were recorded with a
pipette-filling solution containing (in mM) 60 KCl, 75 K-glutamate, 2 MgCl2, 2 Na2ATP, 2 EGTA, and 10 HEPES, adjusted
to pH 7.2 with KOH. For perforated-patch experiments, Na2ATP and EGTA were omitted from this pipette solution and
amphotericin (500 µg/ml) was added. When Ca2+ currents
were recorded, the pipette-filling solution consisted of (in mM) 60 TEA-Cl, 65 Cs-glutamate, 4 MgATP, 10 EGTA, and 10 HEPES, adjusted to pH
7.2 with TEA-OH.
Ca2+ imaging.
Adrenal chromaffin cells were loaded with the Ca2+
indicator by incubating coverslips in a mixture of 20 µM fluo 3-AM
and 0.0025% Pluronic acid in DMEM at 37°C for 30 min. Confocal
microscopy with a water-immersion lens was applied to single cells with
the use of an argon ion laser (Optiscan, Notting Hill, Australia) scanning at a peak of 488 nm. Images were captured with confocal software (F900e, v1.6; Optiscan) and analyzed with Scion Image (Scion,
Frederick, MD). Laser intensity was reduced to 1% of maximum by using
neutral density filters, and the number of scans per cell was kept to a
minimum to avoid photobleaching. Changes in intracellular
Ca2+ levels were taken as the ratio of the increase in the
mean pixel value of the whole cell compared with that in the control
period, where the pixel values are a gray scale ranging from 0 to 255. All values of increased fluorescence were taken 60 s after the start of application of either hypoxic solution or specific channel antagonists through the perfusion system. This ratio is calculated according to the equation R = (F Fmin)/Fmin, where R is the ratio of
Ca2+ fluorescence change, Fmin is the mean
fluorescence intensity level during the control period, and F is the
mean fluorescence intensity level after 1 min of stimulation. This
ratio was then multiplied by 100 to express any fluorescence changes as
a percentage of the resting fluorescence level.
Drugs. Apamin extracted from bee venom, d-tubocurarine, TEA-Cl, NPPB, amphotericin, and DMEM were obtained from Sigma. Fluo 3-AM and Pluronic F-127 were supplied by Molecular Probes (Eugene, OR).
Statistics. Results are expressed as means ± SE. The effects of individual treatments, such as hypoxia, were tested by the Student's paired t-test, and the significance of differences between treatments was tested by an ANOVA and Tukey's post hoc test. P < 0.05 was taken as the minimum level of significance.
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RESULTS |
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Oxygen-sensitive K+ currents in fetal
adrenal chromaffin cells.
Of the 40 adrenal chromaffin cells tested from 137- to 142-day fetal
sheep, in 32 cells the outward current was suppressed significantly by
acute hypoxia. The current-voltage (I-V) relationship in
these cells showed a local maximum at about +40 mV, a characteristic of
Ca2+-dependent K+ currents, and hypoxic
suppression was significant in the region of 0-60 mV
(P < 0.05), the region where the
Ca2+-dependent current is obvious (Fig.
1A). The peak current in these cells was reduced by 36 ± 7% by hypoxia at 40 mV
(n = 11). This hypoxic suppression of current was
reversible on return to normoxic conditions, and these cells were
classed as oxygen sensitive. In perforated-patch recording mode,
similar currents were present, and hypoxia produced a similar reduction
in the outward current that was reversed on return to normoxic
conditions (results not shown).
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Effect of hypoxia on voltage-dependent
Ca2+ current.
The reduction in the Ca2+-dependent K+ current
by hypoxia could be through a direct action on the K+
channels or secondary to a reduced Ca2+ influx. The
I-V relationship of Ca2+ current in fetal
adrenal chromaffin cells was not shifted by hypoxia, and at none of the
voltage steps was a significant difference seen between the
Ca2+ currents in control and hypoxic conditions. The peak
current amplitude of Ca2+ current observed when stepping
from a holding potential of 80 to 10 mV in control conditions was
395 ± 36 pA (Fig. 1B, n = 7), which
was not significantly different from the current amplitude at 10 mV of
391 ± 36 pA seen in hypoxia (n = 7).
Contribution of different types of K+
channels to the oxygen-sensitive current.
Apamin, an inhibitor of SK channels, reduced the outward current in
fetal adrenal chromaffin cells (Fig.
2A). This reduction was
significant at voltages from 0 to 60 mV, and at 40 mV, 200 nM apamin
reduced the current by 36 ± 8% (n = 9).
Comparison of the effects of different concentrations of apamin on the
K+ current showed that 1, 10, and 200 nM all caused
significant decreases in whole cell current from 20 to 60 mV
(P < 0.05), but at no potential were the effects of
these three concentrations different from each other (Fig.
2D).
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Measurement of reversal potential.
For KO2 channels to participate in initiation of the
hypoxic response, closure of those channels must first depolarize the cell membrane to stimulate Ca2+ influx and catecholamine
secretion. A ramp protocol from 120 to +60 mV over 100 ms from a
holding potential of
60 mV was used to investigate the effect of
hypoxia, apamin, and curare on the reversal potential
(Erev) of the membrane current of the adult adrenal chromaffin cells; Erev can be used as a
measure of the resting membrane potential. While the most obvious
effect of all three treatments was seen at positive membrane
potentials, each of them also altered Erev (Fig.
4, A-C).
Erev in control conditions was
55.1 ± 3.0 mV (n = 18), and this shifted to more positive potentials during exposure to hypoxia (
46.4 ± 4.4 mV,
n = 5, P < 0.05), apamin (
43.4 ± 5.1 mV, n = 5, P < 0.05), or curare (
44.7 ± 5.2 mV, n = 8, P < 0.05). These shifts in Erev indicate that SK
channels contribute to the resting potential of chromaffin cells and
that closure of these channels by hypoxia or either of the blocking
agents can cause depolarization of the cell membrane. In contrast to
apamin and curare, 1 mM TEA failed to shift the Erev of the I-V plot of the adrenal
chromaffin cells (n = 4), despite the significant block
of the K+ currents at more positive potentials (Fig.
4D).
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Ca2+ imaging.
The ability of membrane depolarization subsequent to closure of SK
channels to stimulate Ca2+ influx in sheep fetal adrenal
chromaffin cells was investigated with the use of fluo 3 (Fig.
5A). Fluorescence intensity
was increased by treatment with hypoxia (27 ± 4%,
n = 10; P < 0.05) or 200 nM apamin
(24 ± 2%, n = 7; P < 0.05). The
increase in [Ca2+]i, as indicated by the
change in fluorescence, was not significantly different between cells
treated with hypoxia or apamin. Simultaneous treatment with apamin and
hypoxia produced an increase in fluorescence of 21 ± 5%
(n = 5), which was not significantly different from that produced by either treatment alone.
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DISCUSSION |
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The ability of the fetal adrenal medulla to secrete catecholamines in response to hypoxemia implies that the adrenal chromaffin cells possess an oxygen sensor. In the glomus cells of the carotid body, a major chemoreceptor in adult mammals, the role of the oxygen sensor is played by KO2 channels that close in hypoxic conditions. It is well established that closure of KO2 channels in glomus cells causes depolarization of the cell membrane, resulting in Ca2+ influx into the cytosol and exocytosis of neurotransmitters (reviewed in Ref. 19). There are a number of different types of K+ channels in glomus cells of different species that are inhibited by hypoxia. Moreover, a certain degree of variation exists even between different preparations within the same species. For example, in isolated glomus cells of the rat, closure of TASK-1-like K+ channels by hypoxia was found to be responsible for the cell depolarization, and 10 mM TEA was ineffective in mimicking the hypoxic response (3). In thin slices of rat carotid body, however, a Ca2+-dependent BK channel was implicated as an oxygen sensor, and, in contrast to the previous study, application of 5 mM TEA depolarized the cell membrane and caused release of catecholamines (16). In the rabbit carotid body, it has been suggested that closure of human ether-à-go-go-related gene (HERG)-like channels that regulate resting potential may be responsible for the initial depolarization (19) and that an oxygen-sensitive transient K+ current modulates the response (7, 15). In PC-12 cells, derived from a tumor of the rat adrenal medulla but often used as a model of the glomus cell, 1 mM TEA stimulated the secretion of catecholamines apparently through membrane depolarization due to block of K+ channels (23). The KO2 channel in this cell type has been identified as Kv1.2 from the Shaker subfamily of voltage-gated K+ channels (6).
The carotid body and the adrenal medulla share a common embryological origin in the neural crest, and, not surprisingly, KO2 channels have been proposed to act as the oxygen sensor in chromaffin cells of the adrenal medulla of the rat (24) and sheep (20). Previous work in this laboratory (20) has shown that the adrenal chromaffin cells of the fetal sheep possess KO2 channels, and application of 5 mM Co2+ revealed that all of this oxygen-sensitive current is Ca2+ dependent. Ca2+-dependent KO2 channels, BK and SK, were found in rat adrenal chromaffin cells. Thompson and Nurse (25) reported that in neonatal rat chromaffin cells, BK channels account for the major portion of the hypoxic suppression of outward current; however, block of BK channels by iberiotoxin failed to induce membrane depolarization and did not affect the depolarization caused by hypoxia. Studies on the adult rat chromaffin cells implicated SK channels as the oxygen sensor (10). Apamin, a selective inhibitor of SK channels, blocked all of the oxygen-sensitive K+ current in these cells and caused depolarization of the cell membrane. The effects of hypoxia and apamin were not additive.
In the present work, we have found that oxygen-sensitive BK and SK channels are both present in the adult and fetal sheep adrenal chromaffin cells. In the presence of high concentrations of apamin (200 nM) that would block all SK channels, some oxygen-sensitive current remained, indicating that SK are not the only KO2 channels in these cells. The fact that this remaining oxygen-sensitive current was Ca2+ dependent and was completely blocked by application of 1 mM TEA indicates that the channel mediating this current is the BK channel. Complete block of all oxygen-sensitive currents by simultaneous application of apamin and TEA strongly suggests that SK and BK channels account for all of the oxygen-sensitive K+ current in sheep adrenal chromaffin cells.
Of the two subtypes of apamin-sensitive SK channels, SK2 has a higher affinity for apamin (IC50 = 60 pM) than does SK3 (IC50 = 1 nM) (8, 9). In the study on the adult rat adrenal chromaffin cells in which the presence of an oxygen-sensitive SK current was reported (10), the very high concentrations of apamin (400 nM) used did not allow identification of the particular channel subtype involved. In the fetal sheep adrenal chromaffin cells, 1 nM apamin produced a maximal effect, indicating that SK2 is most likely to be responsible for the apamin-sensitive current. This conclusion is supported by the analysis of mRNA in rat adrenal chromaffin cells where the mRNA for SK2, but not for SK3, has been found (9).
Oxygen sensitivity of the K+ currents by itself does not prove that they are involved in initiating the hypoxic response; for hypoxia to stimulate catecholamine secretion, closure of KO2 channels must depolarize the cells and open voltage-dependent Ca2+ channels. In adult sheep chromaffin cells, hypoxia caused a significant shift in Erev to more positive potentials. Similar results were seen with the application of either apamin or curare, indicating that the closure of SK channels is capable of depolarizing the cell membrane and initiating the hypoxic response. In contrast, TEA did not shift membrane potential of the sheep adrenal chromaffin cell, suggesting that BK channels are closed at resting potential in these cells and are not involved in the membrane depolarization caused by hypoxia.
These results were supported by measurements of [Ca2+]i in both the fetal and adult adrenal chromaffin cells. Because the increase in [Ca2+]i produced by the SK blockers apamin and curare was the same as that produced by hypoxia, and because the effects on [Ca2+]i of either of these blockers in conjunction with hypoxia were not additive, closure of SK channels alone could explain the change in membrane potential resulting from hypoxia. Although TEA itself did not induce Ca2+ influx, the combination of TEA and apamin resulted in a much greater increase in [Ca2+]i than that seen when apamin was applied alone. This observation, presumably, was a result of the prolongation of action potential duration when TEA-sensitive channels were blocked. This has been seen to occur in dorsal vagal neurones of the rat where blockade of SK channels causes increased action potential firing frequency, whereas BK channel block results in increased action potential duration (17).
The reduction in Ca2+-dependent K+ current during exposure to hypoxic conditions could result from a hypoxia-evoked decline in Ca2+ influx, while on the other hand, direct increase of the Ca2+ currents by hypoxia would explain catecholamine release. Although there is some evidence from the carotid body for a reduction in voltage-dependent Ca2+ currents as a result of hypoxia (12), more recent work has shown an increase in such currents in this tissue (22). In the present experiments, voltage-dependent Ca2+ currents were not affected by hypoxia, suggesting that the reduction in Ca2+-dependent K+ current was not secondary to a reduced [Ca2+]i. At the same time, this observation ruled out the possibility that the hypoxic response in these cells is initiated by the increase of the amplitude of Ca2+ currents, the leftward shift, or their voltage dependence.
In summary, our results show that the direct response to hypoxia in chromaffin cells isolated from sheep adrenal medulla, characterized by membrane depolarization and a subsequent influx of Ca2+ through voltage-dependent Ca2+ channels, is initiated by closure of SK2 channels. BK channels in these cells are also oxygen sensitive, but their likely role is to modulate the action potentials that result from closure of SK2 channels.
Involvement of KCa channels in depolarization of cell membrane due to hypoxia seems paradoxical, because increase in Ca2+ influx should activate more KCa channels and cause cell hyperpolarization. However, these events are separated in time, with membrane depolarization, Ca2+ influx, and catecholamine release happening first; a subsequent increase in Ca2+-dependent K+ currents due to an increase in [Ca2+ ]i may provide an important negative feedback mechanism that protects chromaffin cells from Ca2+ overload in long periods of hypoxia.
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ACKNOWLEDGEMENTS |
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We thank Dr. Michael Adams for the time and assistance given in developing the techniques for isolation and culture of fetal adrenal chromaffin cells.
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FOOTNOTES |
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This work was supported by the Australian Research Council.
Address for reprint requests and other correspondence: M. Roberts, Dept. of Physiology, Univ. of Adelaide, Adelaide 5005, Australia (E-mail: michael.roberts{at}adelaide.edu.au).
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.
Received 31 October 2000; accepted in final form 2 July 2001.
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