Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106
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ABSTRACT |
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Summers, Beth A.,
Jeffrey L. Overholt, and
Nanduri R. Prabhakar.
Augmentation of L-Type Calcium Current by Hypoxia in Rabbit
Carotid Body Glomus Cells: Evidence for a PKC-Sensitive Pathway.
J. Neurophysiol. 84: 1636-1644, 2000.
Previous studies have suggested that voltage-gated
Ca2+ influx in glomus cells plays a critical role
in sensory transduction at the carotid body chemoreceptors. The purpose
of the present study was to determine the effects of hypoxia on the
Ca2+ current in glomus cells and to elucidate the
underlying mechanism(s). Experiments were performed on freshly
dissociated glomus cells from rabbit carotid bodies.
Ca2+ current was monitored using the whole cell
configuration of the patch-clamp technique, with
Ba2+ as the charge carrier. Hypoxia
(pO2 = 40 mmHg) augmented the Ca2+ current by 24 ± 3% (n = 42, at 0 mV) in a voltage-independent manner. This effect was seen in
a CO2/HCO3-, but not
in a HEPES-buffered extracellular solution at pH 7.4 (n = 6). When the pH of a HEPES-buffered extracellular solution was
lowered from 7.4 to 7.0, hypoxia augmented the
Ca2+ current by 20 ± 5% (n = 4, at 0 mV). Nisoldipine, an L-type Ca2+
channel blocker (2 µM, n = 6), prevented, whereas,
-conotoxin MVIIC (2 µM, n = 6), an inhibitor of N
and P/Q type Ca2+ channels, did not prevent
augmentation of the Ca2+ current by hypoxia,
implying that low oxygen affects L-type Ca2+
channels in glomus cells. Protein kinase C (PKC) inhibitors, staurosporine (100 nM, n = 6) and bisindolylmaleimide
(2 µM, n = 8, at 0 mV), prevented, whereas, a protein
kinase A inhibitor (4 nM PKAi, n = 10) did not prevent
the hypoxia-induced increase of the Ca2+ current.
Phorbol 12-myristate 13-acetate (PMA, 100 nM), a PKC activator,
augmented the Ca2+ current by 20 ± 3%
(n = 8, at 0 mV). In glomus cells treated with PMA
overnight (100 nM), hypoxia did not augment the
Ca2+ current (
3 + 4%, n = 5, at 0 mV). Immunocytochemical analysis revealed PKC
-like
immunoreactivity in the cytosol of the glomus cells. Following hypoxia
(6% O2 for 5 min), PKC
-like immunoreactivity translocated to the plasma membrane in 87 ± 3% of the cells,
indicating PKC activation. These results demonstrate that hypoxia
augments Ca2+ current through L-type
Ca2+ channels via a PKC-sensitive mechanism.
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INTRODUCTION |
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The carotid bodies are the
principal sensory organs that detect changes in arterial oxygen.
Hypoxia increases the sensory discharge of the carotid bodies, and the
ensuing reflexes are crucial for maintaining homeostasis during
hypoxemia. Currently, it is believed that glomus cells, which are in
synaptic apposition with sensory nerve endings, are the initial sites
of sensory transduction. Several lines of evidence indicate that the
transduction mechanism(s) for hypoxia involve
Ca2+ influx through voltage-gated channels. It
has been reported that cytosolic Ca2+
([Ca2+]i) increases in
glomus cells in response to hypoxia (Biscoe and Duchen
1990; Bright et al. 1996
; Buckler and
Vaughn-Jones 1994
; Urena et al. 1994
) via
activation of voltage-gated Ca2+channels
(Buckler and Vaughn-Jones 1994
; Gonzalez et al.
1994
; Lopez-Barneo et al. 1993
). Blockade of
voltage-gated Ca2+ channels prevents augmentation
of sensory discharge by hypoxia (Shirahata and Fitzgerald
1991b
), implying that voltage-gated Ca2+
flux is required for the transduction of the hypoxic stimulus at the
carotid body. Therefore it is of considerable importance to understand
whether and how hypoxia regulates Ca2+ current in
glomus cells.
Many investigators, however, have focused on the effects of hypoxia on
K+ channels (see Gonzalez et al.
1994 for references) and have found that the
K+ current is inhibited by hypoxia in glomus
cells. These studies suggested an importance of
K+ channel closure in producing depolarization
resulting in voltage-gated Ca2+ influx as the
initial steps in triggering transduction of a hypoxic stimulus at the
carotid body. On the other hand, relatively little information is
available on the effects of hypoxia on other ionic conductances in
glomus cells. Although hypoxia has been found to increase
[Ca2+]i, interestingly,
several investigations have found that hypoxia has either no effect on
(Hescheler et al. 1989
; Lopez-Barneo et al.
1988
; Lopez-Lopez et al. 1989
; Peers
1990
) or inhibits (Montoro et al. 1996
)
Ca2+ current in glomus cells. The apparent
discrepancy between the effects of hypoxia on
[Ca2+]i and
Ca2+ channel activity prompted us to re-examine
the effects of low oxygen on Ca2+ current in
glomus cells.
It has been shown that bicarbonate
(CO2/HCO3)-buffered
solutions significantly improve the response of the in vitro carotid body preparation to hypoxia when compared with responses in a HEPES-buffered solution (Iturriaga and Lahiri 1991
;
Shirahata and Fitzgerald 1991a
). This could in
part be due to the finding that the intracellular pH in glomus cells
bathed in a HEPES buffer is more alkaline than in bicarbonate buffer
(Buckler et al. 1991a
). All of the previous studies that
examined the effects of hypoxia on the Ca2+
current in glomus cells employed a HEPES-based buffer in the medium
(Hescheler et al. 1989
; Lopez-Barneo et al.
1988
; Lopez-Lopez et al. 1989
; Montoro et
al. 1996
; Peers 1990
). In the present study, we
tested the idea that the effect of hypoxia on the
Ca2+ current might be occluded in a HEPES buffer,
which turns the intracellular milieu alkaline. It is possible that
hypoxia augments Ca2+ current in glomus cells in
a physiologically relevant
CO2/HCO3
-buffered
extracellular medium. Therefore we examined the effects of hypoxia on
the macroscopic Ca2+ current in glomus cells
isolated from rabbit carotid bodies using a
CO2/HCO3
-buffered
extracellular solution and found under these conditions that low oxygen
does indeed augment Ca2+ current.
Rabbit glomus cells express multiple types of voltage-gated
Ca2+ channels including L, P/Q, and N types of
channels (Overholt and Prabhakar 1997). In addition,
~27% of the total macroscopic Ca2+ current in
rabbit glomus cells is conducted by a channel that is resistant to
specific pharmacological blockers of N, P/Q, and L-type
Ca2+ channels, which we termed the
"resistant" current (Overholt and Prabhakar 1997
).
Whether hypoxia preferentially affects one or more of these channels in
glomus cells, however, has not been examined. Protein phosphorylation
elicited by protein kinase C (PKC) (Hartzell 1988
) and
cAMP-dependent protein kinase (PKA) (Gao et al. 1997
)
has been shown to modulate Ca2+ channel activity
in a wide variety of cell types. In the carotid body, hypoxia
stimulates phospholipase C activity (Pokorski and Stroznajder
1993
), which is linked to PKC activation. There is also
evidence that cAMP levels are increased during hypoxia, suggesting a
role for PKA in hypoxic chemotransduction (Wang et al.
1989
). In the second part of the study, we identified the types
of Ca2+ channels that are affected by hypoxia and
examined whether PKC and/or PKA are involved in the modulation of
Ca2+ current by low oxygen. Our results
demonstrate that hypoxia preferentially augments L-type
Ca2+ current, and the effects of low oxygen are
associated with PKC, but not PKA activation.
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METHODS |
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General procedures
Experiments were performed on glomus cells freshly isolated from
the carotid bodies of adult rabbits killed with
CO2. Individual glomus cells were dissociated
enzymatically as described previously (Overholt and Prabhakar
1997). Briefly, carotid bodies were incubated at 37°C in a
media containing trypsin (type II, 2 mg/ml, Sigma) and collagenase
(type IV, 2 mg/ml, Sigma). The composition of the incubation medium was
(in mM) 140 NaCl, 5 KCl, 10 HEPES, and 5 glucose, pH 7.2. The tissue
was triturated with a fire-polished, glass pasteur pipette every 10 min. After 30 min of incubation, cells were pelleted after
centrifugation at 1,200 rpm for 5 min. Dissociated cells were
resuspended in a 50/50 mixture of Dulbecco's modified Eagles medium
(DMEM) and HAM F12 supplemented with penicillin-streptomycin (GIBCO-BRL), insulin, transferrin, selenium (ITS, Sigma), and 10%
heat-inactivated fetal bovine serum. Cells were maintained at 37°C in
a CO2 incubator and were used within 36 h.
All experiments were performed at room temperature. Glomus cells were
identified using electrophysiological characterization as described
previously (Summers et al. 1999
).
Isolation of Ca2+ current
Ca2+ current was monitored using the whole
cell configuration of the patch-clamp technique (Hamill et al.
1981). Pipettes were made from borosilicate glass capillary
tubing and had resistances of 2-3 M. Currents were recorded using
an Axopatch 200B voltage-clamp amplifier, filtered at 5 kHz, and
sampled at a frequency of 28.6 kHz using an IBM-compatible computer
with a Digidata 1200 interface and pCLAMP software (Axon Instruments).
Currents were not leak subtracted. Current-voltage (I-V)
relations were elicited from a holding potential of
80 mV using 25-ms
steps (5 s between steps) to test potentials over a range of
50 to
+70 mV in 10-mV increments. Current at each potential was measured as
the average over a 2.5-ms span at the end of the 25-ms step.
Ca2+ current was isolated by using
K+- and Na+-free intra- and
extracellular solutions. The intracellular solution had the following composition (in mM): 115 CsCl, 20 TEA-Cl, 5 MgATP, 0.2 TrisGTP, 5 EGTA,
10 phosphocreatine, and 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and the pH was adjusted to 7.2 with CsOH. The
HEPES-buffered extracellular solution had an osmolarity of 300 mOsm and
contained (in mM) 140 NMGCl, 5.4 CsCl, 10 BaCl2,
10 HEPES, and 11 glucose, and the pH was adjusted to 7.4 with CsOH. The
CO2/HCO3-buffered
extracellular solution had an osmolarity of 296 mOsm and contained (in
mM) 120 NMGCl, 4.8 CsCl, 10 BaCl2, 25 NaHCO3, 1.2 NaH2PO4, and 11 glucose,
and the pH was adjusted to 7.4 by continuously bubbling with 5%
CO2. The extracellular solution was changed using
a fast-flow apparatus consisting of a linear array of borosilicate
glass tubes (Overholt and Prabhakar 1997
). In these
experiments, we used Ba2+ as the charge carrier.
For simplicity, Ba2+ current conducted by
Ca2+ channels will be referred to as
Ca2+ current. To observe
Na+ current to identify a glomus cell, cells were
first superfused with an extracellular solution containing
Na+, having the following composition (in mM):
140 NaCl, 5.4 KCl, 2.5 CaCl2, 0.5 MgCl2, 5.5 HEPES, and 11 glucose, and the pH was adjusted to 7.4 with NaOH.
Rundown of Ca2+ current and the effects of drugs were monitored using a wash protocol (25-ms step to 0 mV, 10 s between steps). The effects of drug agents were compensated for rundown using a linear regression of the current decrease during the wash protocol in the absence of test compounds. Rundown was negligible compared with drug effects over the same time period (e.g., 0.03 ± 0.3% per minute, mean ± SE, n = 4). Cells in which rundown was excessive or did not appear linear were excluded from the analysis. For comparison of I-V relations, Ca2+ current at each potential was normalized to the maximum value recorded during the control I-V relation in individual cells (usually 0 mV).
Tissue collection for histological procedures
Adult rabbits were anesthetized with a cocktail containing Ketamine, Rompun, and Acepromazine Maleate (1 ml/kg im). Tracheal intubation was performed, and the animals were ventilated with either 100% O2 (normoxia) or 6% O2, 94% N2 (hypoxia) for 5 min. Animals were then perfused transcardially with fixative, and tissues were removed and postfixed overnight. The fixative used was a 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4. Tissue samples were cryoprotected in 30% sucrose in PBS, infiltrated with a 1:1 mixture of 30% sucrose-PBS and OCT embedding medium (Tissue-Tek OCT-4583, Baxter Scientific), embedded in OCT, frozen over dry ice, and cut in a cyrostat (Leica, 10-µm sections). There are three animals in the normoxic group and three animals in the hypoxic group. For each specific antibody, 2 slides with 12 tissue sections on each slide were stained for each animal in the group. On each tissue section, the glomus cells were then counted to arrive at a percentage of cells that formed ringlets under hypoxic conditions versus diffuse staining under normoxic conditions.
Immunofluorescence labeling with PKC isoforms of carotid body tissue
The indirect immunofluorescence technique was used to detect the
presence of and to localize the cellular distribution of PKC isoforms
before and after exposure to hypoxia. Fixed tissues were washed with
0.1 M PBS containing 0.2% Triton-X (PBS-Tx) for 30 min, and then
blocked with 20% goat serum, 2% bovine serum albumin (BSA) in PBS-Tx
for 2 h. Subsequently, tissues were incubated with monoclonal,
isoform-specific PKC antibodies or monoclonal TH antibody (DiaSorin) at
the appropriate dilution in 2% goat serum in PBS-Tx for 24 h at
4°C. Monoclonal antibodies to seven individual PKC isoforms (,
,
,
,
,
, and
) were used for immunofluorescence
protocols (Transduction Laboratories). After incubation with primary
antibody, tissues were thoroughly washed with PBS-Tx and incubated with
Texas Red goat anti-mouse immunoglobin G (secondary antibody) diluted
1:1,000 in 2% goat serum in PBS-Tx for 2 h at room temperature.
An immunocytochemical control for antibody specificity was performed by
incubating the tissues with secondary antibody only. After a thorough
wash in PBS-Tx, the tissues were mounted on microscope slides with
Immuno-mount (Shandon). The tissue sections were viewed and imaged with
a Nikon Eclipse E600 epifluorescent microscope equipped with a
Diagnostic Instrument Spot camera and software.
Solutions and drugs
CO2/HCO3-buffered
extracellular solutions were made normoxic or hypoxic by continuously
bubbling with either 21% O2, 5%
CO2, 74% N2 or 1%
O2, 5% CO2, 94%
N2, respectively. HEPES-buffered extracellular
solutions were made normoxic or hypoxic by continuously bubbling the
solution with either 21% O2 79%
N2 or 1% O2 99%
N2, respectively. The pO2
was routinely monitored with a blood gas analyzer (Laboratory
Instruments) and found to be between 35 ± 5 mmHg
(n = 5) for hypoxic solutions and 148 ± 3 mmHg
(n = 5) for normoxic solutions. Stock solutions of
staurosporine (Calbiochem), bisindolylmaleimide I (Calbiochem), and
phorbol 12-myristate 13-acetate (PMA, Calbiochem) were prepared in
dimethylsulfonoxide (DMSO). Nisoldipine (Miles Laboratories) was
prepared as a stock solution in polyethylene glycol (PEG, MW = 400, Sigma). PKA inhibitor amide, myristoylated (Calbiochem) and
-conotoxin MVIIC (Alomone Labs, Jerusalem, Israel) stock
solutions were prepared in sterilized, deionized water. Experiments
were done in the dark when light-sensitive reagents were used (e.g.,
PMA, nisoldipine). The final concentrations of either DMSO or PEG were
0.1%. In control experiments (n = 4), the DMSO or PEG
vehicle alone (i.e., without drug) did not effect the
Ca2+ current.
Data analysis
All values are presented as means ± SE. Statistical significance was determined by a paired t-test or a one-way ANOVA, with Tukey's post hoc test where appropriate. P values <0.05 were considered significant.
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RESULTS |
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Hypoxia augments Ca2+ current in glomus cells
An example illustrating the effects of hypoxia
(pO2 of medium 40 mmHg) on the
Ca2+ current and the time course of the response
in a
CO2/HCO3
-buffered
extracellular solution recorded from a glomus cell is shown in Fig.
1, A and B. It is
obvious from these traces that hypoxia reversibly augmented the
Ca2+ current. Figure 1B shows the time
course for changes in Ca2+ current elicited at 0 mV from a holding potential of
80 mV. The effect of hypoxia began
within tens of seconds, plateaued within 1 min, and returned to control
levels within 1 min after terminating the hypoxic challenge. To assess
whether hypoxia affected the I-V relationship of the
Ca2+ current, the effects of hypoxia were tested
over a broad range of membrane potentials. Figure 1C shows
the average (n = 42), normalized I-V
relations before and during exposure to hypoxia. Hypoxia increased the
magnitude of the peak current equally over the range of potentials
tested, suggesting that the effect is voltage independent. On average,
the Ca2+ current was augmented by 24 ± 3%
at 0 mV (n = 42, P < 0.05, paired t-test) when Ba2+ is used as the
charge carrier. Similar results were obtained when
Ca2+ was used as the charge carrier. With 2.5 mM
Ca2+ as the charge carrier, hypoxia augmented the
current by 16 ± 4% at 0 mV (n = 4, P < 0.05, paired-t-test, data not shown).
Since the effects of hypoxia were qualitatively and quantitatively
similar (P > 0.05, ANOVA), subsequent experiments used
Ba2+ as the charge carrier to enhance the
magnitude of the current. These experiments demonstrate that hypoxia
augments Ca2+ current in glomus cells in a
CO2/HCO3
-buffered
extracellular solution.
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Comparison of the effects of hypoxia on the Ca2+
current in a CO2/HCO3- versus
HEPES-buffered extracellular solution
Previous studies have examined the effect of hypoxia on
Ca2+ current in glomus cells using a
HEPES-buffered extracellular solution (Hescheler et al.
1989; Lopez-Barneo et al. 1988
;
Lopez-Lopez et al. 1989
; Montoro et al.
1996
; Peers 1990
). For comparison, we tested the
effect of hypoxia on the Ca2+ current in a
HEPES-buffered extracellular solution (pH 7.4). We found that hypoxia
has no effect on the Ca2+ current under these
conditions (n = 6). These results are shown in Fig.
2, A and B, which
show the current traces and the time course, respectively, when a
glomus cell was exposed to hypoxia in a HEPES-buffered extracellular
solution. These results demonstrate that hypoxia does not affect
Ca2+ current in a HEPES-buffered extracellular
solution (pH 7.4).
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It has been reported that switching from a HEPES- to a
CO2/HCO3-buffered
extracellular solution causes intracellular acidification in rat glomus
cells (Buckler et al. 1991
). Therefore we tested whether
a change in pH could be responsible for the lack of
Ca2+ current augmentation by hypoxia when using a
HEPES-buffered extracellular solution. For this purpose, we adjusted
the pH of the HEPES-buffered extracellular solution to 7.0 with 1 N
HCl. The current traces shown in Fig. 2C clearly show that
hypoxia augments the Ca2+ current at pH 7.0 in a
HEPES-buffered external solution. The time course of the response shown
in Fig. 2D resembles that seen in a
CO2/HCO3
-buffered
extracellular solution (see Fig. 1). On average, hypoxia significantly
augmented the Ca2+ current by 20 ± 5% at 0 mV (n = 4, P < 0.05, paired
t-test), which is similar to the augmentation seen in a
CO2/HCO3
-buffered
extracellular solution (P > 0.05, ANOVA). These
results demonstrate that hypoxia can augment Ca2+
current in a HEPES-buffered extracellular solution when the pH is more
acidic. Furthermore, these observations suggest that the lack of
augmentation by hypoxia in a HEPES-buffered extracellular solution is
not due to the presence of HEPES itself, rather secondary to changes in
pHi. Since hypoxia augmented
Ca2+ current in glomus cells in a
CO2/HCO3
-buffered
extracellular solution, a more relevant physiological buffer, the
remainder of the study was performed with this buffer.
Nisoldipine prevents hypoxia-induced augmentation of the Ca2+ current
We have previously reported that Ca2+
current in rabbit glomus cells is conducted by four different types of
voltage-dependent Ca2+ channels including L, P/Q,
N, and resistant channels (Overholt and Prabhakar 1997).
Having characterized the conditions under which hypoxia augments the
Ca2+ current, we then asked whether hypoxia
affects a particular Ca2+ channel type. We first
tested the effect of nisoldipine (2 µM NISO), an L-type
Ca2+ channel blocker, on the hypoxia-induced
augmentation of the Ca2+ current. Figure
3A shows the effect of hypoxia
on the Ca2+current elicited by a step to 0 mV in
the presence of NISO. As expected, NISO by itself blocked a portion of
the basal Ca2+ current (28 ± 7%). However,
in the presence of NISO, hypoxia had little effect on the
Ca2+ current as can be seen in the current traces
shown in Fig. 3A. The time course in Fig. 3B
shows that hypoxia augments the Ca2+ current
prior to application of NISO, but this effect is negligible in the
presence of NISO. On average, hypoxia augmentation of the Ca2+ current was negligible (1 ± 6%,
n = 6; P > 0.05, paired
t-test) in the presence of nisoldipine. In another series of
experiments (n = 6), we examined the effects of
-conotoxin MVIIC (MVIIC), a selective inhibitor of N- and P/Q-type
Ca2+ channels, on the hypoxia-induced
augmentation of the Ca2+ current. An example
depicting the effect of hypoxia on the Ca2+
current in the presence of 2 µM MVIIC is shown in Fig. 3C.
As expected, MVIIC by itself blocked a portion of the basal
Ca2+ current (17 ± 7%). However, in the
presence of MVIIC, hypoxia still augmented the
Ca2+ current, and the time course of the response
resembled that of controls without MVIIC (Figs. 3D and
1B). Average data showed no significant difference in the
magnitude of the response to hypoxia in the presence and absence of
MVIIC (Fig. 7, hypoxia alone = 24 ± 3% vs. MVIIC + hypoxia = 26 ± 10%; P > 0.05, ANOVA). These results suggest that hypoxia selectively augments the
Ca2+ current conducted by L-type
Ca2+ channels, not by N- or
P/Q-Ca2+ channel types.
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PKC, but not cAMP-dependent kinase (PKA), inhibitors prevent the augmentation of the Ca2+ current by hypoxia
Hypoxia has been shown to increase phospholipase C (PLC)
activity (Pokorski and Stroznajder 1993) and cAMP levels
(Wang et al. 1989
) in glomus cells. To test whether PKC
and/or PKA are involved with Ca2+ current
augmentation by hypoxia, we examined the effects of hypoxia in the
presence of either PKC or PKA inhibitors. PKC inhibitors, staurosporine
(STRO; 100 nM; n = 6) and bisindolylmaleimide (BIM; 2 µM; n = 8), prevented the augmentation of the
Ca2+ current by hypoxia. As evidenced by the
current traces and the time courses presented in Fig.
4, A-D, STRO and BIM
augmented basal Ca2+ current by 18 ± 7%
and 18 ± 6%, respectively. More importantly, in the presence of
these inhibitors, hypoxia had no significant effect on the
Ca2+ current (see also Fig. 7). These results
suggest that PKC may be involved in the augmentation of the
Ca2+ current by hypoxia. Next, we tested the
effects of hypoxia on the Ca2+ current in the
presence of a cell-permeable form of a PKA inhibitor (4 nM PKAi).
Figure 5, A and B,
illustrates the effect of PKAi, and hypoxia in the presence of PKAi on
the current traces and the time course, respectively. PKAi also
augmented the basal Ca2+ current by 14 ± 6%. However, in contrast to the PKC inhibitors, PKAi did not prevent
further augmentation of Ca2+ current by hypoxia.
In the presence of PKAi, hypoxia significantly augmented the
Ca2+ current by 22 ± 4% (Fig. 7,
n = 10, at 0 mV), implying that PKA is not associated
with augmentation of the Ca2+ current by hypoxia.
|
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A phorbol ester mimics the effects of hypoxia on the Ca2+ current in glomus cells
The results described above indicate that activation of PKC is
associated with augmentation of Ca2+ current by
hypoxia. To further establish the role of PKC in the stimulatory effect
of hypoxia, we tested whether phorbol 12-myristate 13-acetate (100 nM
PMA), a PKC activator, could mimic the effect of low oxygen on the
Ca2+ current in glomus cells. Figure
6, A and B,
illustrates the effect of PMA on the current traces and the time
course, respectively. PMA, like hypoxia, augmented the basal
Ca2+ current by 20 ± 3% at 0 mV
(n = 8). When cells were challenged with PMA and
hypoxia together, their effects on the Ca2+
current were not additive (n = 3, 29 ± 5%, see
Fig. 7). The possible involvement of PKC
in the regulation of Ca2+ channels in glomus
cells was further tested with long-term exposure of cells to PMA, which
is known to deplete PKC (Zhong et al. 1999). For this
purpose, freshly dissociated glomus cells from rabbit carotid bodies
were split into two populations. One population of cells was left
untreated, and the other was treated with 100 nM PMA overnight. We then
compared the effect of hypoxia on the Ca2+
current in these two populations. In the untreated group, hypoxia increased Ca2+ current by 28 ± 7%, whereas
the cells treated overnight with PMA did not respond to hypoxia (see
Fig. 7,
3 ± 4%, n = 5, at 0 mV,
P < 0.05, ANOVA). A summary of the average percent
augmentation of the Ca2+ current under the
various conditions are summarized in Fig. 7. Taken together, these
results suggest that PKC is involved in the hypoxia-induced
augmentation of the Ca2+ current in glomus cells.
|
|
Hypoxia causes PKC to translocate from the cytosol to the
membrane in glomus cells
Immunocytochemical analysis was performed to assess the effects of
hypoxia on PKC isoforms in glomus cells. Since BIM is a specific
inhibitor of ,
,
,
, and
isoforms of PKC, we tested whether these isoforms are present in glomus cells, and if so, which
isoforms are activated by hypoxia. Sections from carotid bodies were
stained for
,
,
,
, and
isoforms of PKC using monoclonal antibodies conjugated to Texas red as the fluoroprobe. As
shown in Fig. 8C, only
PKC
-like immunoreactivity could be seen in glomus cells. To further
identify glomus cells, we stained carotid body sections with monoclonal
anti-tyrosine hydroxylase (TH), a well-established marker of glomus
cells (Gonzalez et al. 1994
). We found a similar pattern
of anti-TH and anti-PKC
immunoreactivity in glomus cells in the
normoxic tissues (Fig. 8, C and D). Figure 8E shows the control for antibody specificity in which the
tissues were incubated with secondary antibody only (anti-mouse goat
IgG). There was no immunoreactivity with omission of the primary
antibody (negative control). On the other hand, PKC
-like
immunoreactivity was exclusively localized to the vascular cells of the
carotid artery (arrows, Fig. 8B). These results suggest that
the PKC
isoform of PKC is found in rabbit glomus cells, whereas the
PKC
isoform of PKC is found in the vasculature. In one of the three animals, PKC
-like immunoreactivity was found in the carotid body vasculature (Fig. 8A).
|
To test whether PKC is activated by low oxygen, anesthetized animals
were exposed to hypoxia (6% O2 for 5 min,
n = 3). Subsequently, animals were perfused, carotid
bodies were removed, sectioned, and stained for PKC. As shown in
Fig. 9B, PKC
-like
immunoreactivity localized more toward the cell membrane of glomus
cells forming ringlet-like structures in the carotid bodies exposed to
hypoxia (arrows, Fig. 9B) compared with normoxic controls
(Fig. 9A). In all three animal preparations tested for the
hypoxic group, 87 ± 3% of the cells (n = 2,040 cells) under hypoxia formed ringlets of anti-PKC
like
imunoreactivity in glomus cell clusters throughout the carotid body
tissue (n = 30 sections), while the normoxic animal
group (100% O2, for 5 min, n = 3) showed no evidence of ringlet formation, but rather diffuse staining
in the cytosol of the glomus cell clusters (n = 2,550 cells). These results suggest that the PKC
isoform of PKC
translocates from the cytosol to the plasma membrane during hypoxia,
suggesting that PKC
is activated during hypoxia.
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DISCUSSION |
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Previous studies have shown that
[Ca2+]i increases in
response to hypoxia, and this response depends on
Ca2+ entry through voltage-activated
Ca2+ channels. However, several investigators
have reported that hypoxia either has no effect on, or inhibits
Ca2+ current (see references in
INTRODUCTION). Therefore in this study, we re-examined the
effects of low oxygen on the Ca2+ current. Our
results demonstrate that hypoxia augments Ca2+
current, and this effect is primarily confined to L-type
Ca2+ channels in glomus cells isolated from
rabbit carotid bodies. The effects of hypoxia are seen in a
CO2/HCO3-buffered
extracellular solution. Furthermore, the effect of hypoxia on the
Ca2+ current appears to be associated with
activation of a PKC-sensitive pathway.
Effect of hypoxia on the Ca2+ current in a
CO2/HCO3-buffered versus a HEPES-buffered
extracellular solution
It can be seen from our results that hypoxia reversibly augments
the Ca2+ current recorded in a
CO2/HCO3-buffered
extracellular solution. The onset of the effects were rapid (occurring
within tens of seconds after application of hypoxia) and reversible
(Fig. 1). However, the present results differ from those reported by
others who found that hypoxia either had no effect on (Hescheler
et al. 1989
; Lopez-Barneo et al. 1988
) or inhibited Ca2+ current (Montoro et al.
1996
). This discrepancy is not due to species-related
differences because rabbit glomus cells were utilized in all studies.
It is possible that the discrepancy between the studies is due to the
use of different experimental conditions. For instance, previous
studies applied hypoxia in a HEPES-buffered extracellular solution,
whereas we applied hypoxia in a
CO2/HCO3
-buffered
extracellular solution. It has been reported that without CO2/HCO3
,
tissues and cells respond differently, or even oppositely to those in
the presence of
CO2/HCO3
(Thomas 1989
). Also, in the carotid body, catecholamine
secretion is enhanced by the presence of bicarbonate as compared with
bicarbonate-free solution for the same hypoxic stimulus
(Panisello and Donnelly 1998
). Furthermore,
several investigators have reported that the presence of
CO2/HCO3
significantly improved the response to hypoxia in the in vitro carotid
body preparation as opposed to responses in HEPES (Iturriaga and
Lahiri 1991
; Shirahata and Fitzgerald 1991
).
Therefore it is possible that the presence
CO2/HCO3
in the
extracellular medium also improves the responsiveness of
Ca2+ channels to hypoxia.
Consistent with other investigators (Hescheler et al.
1989; Lopez-Barneo et al. 1988
; Peers
1990
), we also found no effect of hypoxia on
Ca2+ current when using a HEPES-buffered
extracellular solution at pH 7.4 (Fig. 2, A and
B). However, we did not observe inhibition of the
Ca2+ current in a HEPES-buffered medium as
reported by Montoro et al. (1996)
. This could possibly
be explained by the severity of hypoxia used in our study compared with
Montoro et al. (1996)
. In the present study, we applied
a moderate level of hypoxia (40 mmHg), whereas Montoro and
colleagues (1996)
applied a more severe level of hypoxia
(10-20 mmHg).
How might
CO2/HCO3 improve
the effects of hypoxia on the Ca2+ current? It
has been reported that the intracellular pH (pHi) is significantly higher in cells exposed to a HEPES-buffered media (7.8) than in bicarbonate-buffered media (7.2) (Buckler et al. 1991
; Thomas 1989
). Therefore it is possible
that the lack of an effect of hypoxia in a HEPES-buffered extracellular
solution could be due to a more alkaline intracellular pH in cells
under this condition. This idea is supported by our finding that
hypoxia is able to augment Ca2+ current in a more
acidic HEPES-buffered extracellular solution (pH 7.0, Fig. 2,
C and D). Most importantly, these observations suggest that the lack of augmentation by hypoxia in a HEPES-buffered extracellular solution is not due to the presence of HEPES itself, rather it is secondary to changes in pHi. These
secondary changes in pHi could influence
Ca2+ channel activity and thus mask the effect of
hypoxia on the Ca2+ current in glomus cells under
HEPES- buffered conditions at pH 7.4. In addition, secondary changes in
pHi could also influence PKC enzyme activity. For
example, the association of the catalytic and regulatory domains of PKC
is affected by changing pH (McFadden et al. 1989
).
Hypoxia augmentation is primarily confined to L-type Ca2+ channels
Since rabbit glomus cells express multiple types of
Ca2+ channels (Overholt and Prabhakar
1997), we examined whether the effect of hypoxia was confined
preferentially to one type of Ca2+ channel in
glomus cells. Our data indicate that the effects of hypoxia are
confined to L-type Ca2+ current in glomus cells
(Fig. 3, A and B), not N- and P/Q-type Ca2+ currents (Fig. 3, C and
D). Sensitivity of the L-type Ca2+
current to hypoxia resembles that reported for L-type
Ca2+ current in smooth muscle
(Franco-Obregon et al. 1995
). In a variety of cell
types, including glomus cells, the L-type Ca2+
channel, in particular, has been found to be affected by a variety of
gaseous molecules. This is supported by their sensitivity to O2 as well as another gaseous molecule, nitric
oxide (Campbell et al. 1996
; Franco-Obregon et
al. 1995
; Summers et al. 1999
). Most
importantly, some studies have suggested that the L-type current is
involved in the hypoxia-induced neurotransmitter release from glomus
cells (Gomez-Nino et al. 1994
; Obeso et al.
1992
). Taken together, these data suggest that augmentation of
the L-type Ca2+ current in glomus cells by low
oxygen may play a functional role by enhancing neurotransmitter release
during hypoxia at the carotid body.
Evidence for the involvement of PKC in the hypoxic-induced augmentation of the Ca2+ current
Several observations from the present study provide evidence for
the involvement of PKC in hypoxia-induced augmentation of the
Ca2+ current in rabbit glomus cells. First,
although kinase inhibitors (i.e., PKC and PKA inhibitors), in general,
augmented basal Ca2+ current, only PKC inhibitors
prevented hypoxia-induced augmentation of the
Ca2+ current (Figs. 4 and 5). However, one caveat
with using the whole cell configuration of the patch-clamp technique is
that the intracellular milieu becomes dialyzed, allowing escape of
second messengers, and therefore the possible involvement of PKA cannot
be ruled out totally. Second, PMA, an activator of PKC, mimicked the
effects of hypoxia (Fig. 6). Third, hypoxia had no effect on
Ca2+ current in glomus cells following overnight
treatment with PMA, which is known to deplete PKC (Zhong et al.
1999). A role for PKC in modulation of the
Ca2+ current by hypoxia was further supported by
the observation that glomus cells express PKC
isoform of PKC, and
that hypoxia translocates PKC
from the cytosol to the membrane
(ringlet formation), an event associated with PKC activation (Fig. 9)
(Wieloch and Cardell 1993
). These observations
are consistent with translocation of PKC by hypoxia reported elsewhere
in neural tissues (Wieloch and Cardell 1993
) and
activation of PKC isoforms in the pulmonary vasculature by low oxygen
(Weissmann et al. 1999
). In the present study, we found
evidence for PKC
isoform of PKC in glomus cells of the rabbit
carotid body. However, the present results differ from those reported
by another study that found the presence of the PKC
isoform in
glomus cells of the normoxic cat carotid body (Faff et al.
1999
). This difference could be due to the presence of
different PKC isoforms in glomus cells from different species. Nonetheless, our results support the idea that activation of PKC is
associated with modulation of Ca2+ channels by
hypoxia. Our study, however, only provides a beginning step toward the
characterization of what isoforms of PKC might be present in rabbit
carotid body tissue, and whether hypoxia affects PKC isoforms. Further
studies are needed to define the mechanisms by which hypoxia activates
the different isoforms of PKC, and how hypoxia modulates the
phosphorylation states of the Ca2+ channels in
glomus cells.
In summary, we have shown that hypoxia augments
Ca2+ current in a
CO2/HCO3-buffered
extracellular solution and this augmentation is primarily confined to
L-type Ca2+ current and seems to be coupled to
the activation of PKC.
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ACKNOWLEDGMENTS |
---|
The authors thank Drs. S. W. Jones and R. D. Harvey for constructive suggestions during this study. We are also grateful to Dr. Harvey for providing nisoldipine for the experiments. J. L. Overholt is a Parker B. Francis Fellow in Pulmonary Research.
This work was supported by National Heart, Lung, and Blood Institute Grant HL-25830. B. A. Summers was supported by Training Grant T32HL-07653.
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FOOTNOTES |
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Address for reprint requests: N. R. Prabhakar, Dept. of Physiology and Biophysics, School of Medicine, 10900 Euclid Ave., Case Western Reserve University, Cleveland, OH 44106-4970 (E-mail: nrp{at}po.cwru.edu).
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 3 January 2000; accepted in final form 25 May 2000.
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REFERENCES |
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