Cellular mechanisms involved in carotid body inhibition
produced by atrial natriuretic peptide
L.
He,
B.
Dinger, and
S.
Fidone
Department of Physiology, University of Utah School of Medicine,
Salt Lake City, Utah 84108
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ABSTRACT |
Atrial natriuretic peptide (ANP) and its analog,
atriopeptin III (APIII), inhibit carotid body chemoreceptor nerve
activity evoked by hypoxia. In the present study, we have examined the hypothesis that the inhibitory effects of ANP and APIII are mediated by
cyclic GMP and protein kinase G (PKG) via the phosphorylation and/or
dephosphorylation of K+ and Ca2+ channel
proteins that are involved in regulating the response of carotid body
chemosensory type I cells to low-O2 stimuli. In freshly
dissociated rabbit type I cells, we examined the effects of a PKG
inhibitor, KT-5823, and an inhibitor of protein phosphatase 2A (PP2A),
okadaic acid (OA), on K+ and Ca2+ currents. We
also investigated the effects of these specific inhibitors on
intracellular Ca2+ concentration and carotid sinus nerve
(CSN) activity under normoxic and hypoxic conditions. Voltage-dependent
K+ currents were depressed by hypoxia, and this effect was
significantly reduced by 100 nM APIII. The effect of APIII on this
current was reversed in the presence of either 1 µM KT-5823 or 100 nM
OA. Likewise, these drugs retarded the depression of voltage-gated Ca2+ currents induced by APIII. Furthermore, APIII
depressed hypoxia-evoked elevations of intracellular Ca2+,
an effect that was also reversed by OA and KT-5823. Finally, CSN
activity evoked by hypoxia was decreased in the presence of 100 nM
APIII, and was partially restored when APIII was presented along with
100 nM OA. These results suggest that ANP initiates a cascade of events
involving PKG and PP2A, which culminates in the dephosphorylation of
K+ and Ca2+ channel proteins in the
chemosensory type I cells.
chemoreceptor inhibition; cell currents; protein kinase G; protein
phosphatase 2A; atrial natriuretic peptide
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INTRODUCTION |
THE CAROTID BODY, an arterial chemoreceptor organ
located at the bifurcation of the common carotid artery, is excited by
hypoxia, hypercapnia, and acidosis. The parenchyma of the organ
consists of groups or lobules of specialized type I cells that lie
interspersed among a network of capillaries and sinusoids. Contemporary
views suggest that chemosensory activity is initiated in type I cells that activate synaptically apposed carotid sinus nerve (CSN) afferent terminals (11, 12). Although early investigations of the carotid body
envisioned a classical chemical synapse between type I cells and
afferent terminals involving the action of a single excitatory transmitter agent, more recent studies have revealed a neurochemically complex synaptic apparatus (11, 12, 27). The available evidence suggests that type I cells synthesize and release multiple excitatory and inhibitory neurotransmitter agents, and moreover, that the CSN
afferent fibers themselves contain substances capable of potently influencing the activity of type I cells (1, 27, 32). It is likely,
therefore, that in at least some physiological conditions, carotid body
output measured in terms of chemoreceptor nerve activity is the product
of multiple excitatory and inhibitory influences acting simultaneously.
Two potent inhibitory agents recently found in the carotid body, nitric
oxide (NO) and atrial natriuretic peptide (ANP), have been postulated
to participate as modulators of chemosensory activity (see Ref. 1). NO
is synthesized in a specialized subpopulation of inhibitory afferent
fibers that appear to mediate the efferent inhibition of chemoreceptor
activity, a phenomenon that was first documented some 30 years ago by
Neil and O'Regan (24) and Fidone and Sato (14). ANP, initially
discovered in cardiac myocytes, is also present in type I cells, and
available data indicate that these cells express ANP receptors as well
(see Ref. 1). Aside from the fact that both NO and ANP elevate cyclic
GMP (cGMP) in type I cells (36, 37), and that exogenous cGMP inhibits
hypoxia-evoked CSN activity (31), virtually nothing is known about the
cellular mechanisms that mediate the chemosensory inhibition produced
by these agents. In the present study, we have examined the hypothesis that the inhibitory effects of ANP are mediated by the primary target
for cGMP, namely, protein kinase G (PKG), in a mechanism that alters
the phosphorylation status of voltage-activated Ca2+- and
O2-sensitive K+ channel proteins. In freshly
dissociated cultured type I cells, we examined the effects of a
specific PKG inhibitor, KT-5823 (see Refs. 20 and 21), and an inhibitor
of protein phosphatase 2A (PP2A), okadaic acid (OA) (see Ref. 8), on
K+ and Ca2+ currents. We also investigated the
effects of these specific inhibitors on intracellular Ca2+
concentration ([Ca2+]i) and CSN
activity under normoxic and hypoxic conditions. Our results suggest
that ANP initiates a cascade of events that culminates in the
dephosphorylation of K+ and Ca2+ channel proteins.
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METHODS |
Dissociation of carotid body cells.
Young New Zealand White rabbits (1.4-2.0 kg) were anesthetized
with pentobarbital sodium (35 mg/kg iv), tracheostomized, and artificially ventilated. The carotid arteries, including the
bifurcations, were surgically exposed, removed, and placed into
ice-cold modified Tyrode's solution containing (in mM): 112 NaCl, 4.7 KCl, 2.2 CaCl2, 1.1 MgCl2, 42 sodium glutamate,
5.6 glucose, and 5 HEPES buffer (pH 7.43 at 37°C) and equilibrated
with 100% O2. The carotid bodies were dissected free of
surrounding connective tissue and transferred to Ham's F-12 medium
(Ca2+- and Mg2+-free) containing 0.2%
collagenase and 0.2% trypsin. Each carotid body was cut into 6-12
pieces and incubated for 40 min in a CO2 incubator (5%
CO2-95% air) at 36.5°C. Tissue fragments were rinsed (2 × 10 min, room temperature) in Hanks' balanced
salt solution (Ca2+- and Mg2+-free) and then
transferred to poly-L-lysine-coated glass coverslips, where
they were triturated in a small volume of Ham's F-12 medium, plus 10%
FCS and 5 µg/ml insulin. The coverslips containing dissociated type I
cells were placed in the CO2 incubator for at least 2 h before use.
Perforated whole cell recordings.
Coverslips containing type I cells were placed in a 0.3-ml flow chamber
mounted on the stage of a Zeiss phase-contrast inverted microscope.
Cells were bathed in modified Tyrode's solution delivered at 0.5 ml/min via a peristaltic pump. Bath temperature was maintained at
35-36.5°C. The bath was grounded via an Ag-AgCl electrode. Patch pipettes were fabricated from borosilicate glass tubing (outer
diameter = 1.5 mm; internal diameter = 0.75 mm; Sutter Instrument) in a Flaming/Brown micropipette puller, model P-87 (Sutter
Instrument). Pipette resistance varied between 2 and 10 M
. Stock
solutions of nystatin (Sigma Chemical) were prepared by ultrasonication
in DMSO at a concentration of 5 mg/100 µl for 30 s. Final pipette
concentrations of nystatin were 125-200 µg/ml. Maximal whole
cell currents were recorded 5-25 min after exposure to the
nystatin solution in the cell-attached mode. For K+ current
(IK) measurements, bath solutions contained 135 mM choline-Cl, 5 mM KCl, 50 µM CdCl2, 1.0 mM
CaCl2, 1.0 mM MgCl2, 5.6 mM glucose, and 10 mM
HEPES buffer, pH 7.43 at 37°C. The pipette solution contained (in
addition to nystatin) 145 mM K-glutamate, 15 mM KCl, 2 mM MgCl, and 20 mM HEPES, pH 7.2 at 37°C. IK was evoked by step
voltage changes from a holding potential of
70 mV.
Ca2+ currents were measured in a bath solution containing
140 mM choline-Cl, 10 mM CaCl2, and 10 mM HEPES, pH 7.43 at
37°C. In addition to nystatin, the pipette solution contained 120 mM CsCl, 25 mM tetraethylammonium chloride, 0.5 mM CaCl2,
5.5 mM EGTA, and 30 mM HEPES, pH 7.2 at 37°C. From a holding
potential of
70 mV, ICa was evoked by
applying ramp voltages of 200 ms duration between
40 and +80 mV.
Under control conditions, cells were superfused in a solution
equilibrated with air (PO2 ~128
Torr). Hypoxic solutions were equilibrated with air and contained 0.5 or 1.0 mM sodium dithionite, resulting in
PO2 values of ~58 and ~32 Torr, respectively. These PO2 values
obtained using an oxygen scavenger are in the range measured internally
in the intact carotid body during moderate hypoxia (5, 6). Bubbling of
superfusates occurred in a reservoir separated from the superfusion
chamber by the shortest possible lengths of peristaltic pump tubing;
nonetheless, final PO2 values were
undoubtedly influenced by exchange with ambient air. "Normoxic,"
therefore, refers to superfusion with air-equilibrated media, and
"hypoxia" or "hypoxic" indicates treatment of the
superfusate with air plus sodium dithionite.
Patch-clamp data analysis.
Whole cell currents were recorded with an Axopatch 200A patch-clamp
amplifier and a CV 201A headstage (Axon Instruments). Records were simultaneously displayed on an oscilloscope and digitized with a DigiData 1200 computer interface for analysis using pCLAMP version 5.0 software (Axon Instruments). The series resistance was
typically 40 M
for perforated whole cell recordings and was not
compensated in these experiments. Junction potentials, which varied
from 2 to 4 mV, were canceled at the beginning of the experiment. Current-voltage (I-V) relations were plotted after subtraction of any capacitance and leakage currents.
Intracellular Ca2+
measurements.
Freshly dissociated type I cells attached to coverslips were incubated
in F-12 medium containing 0.5 µM fura 2-AM for 10-15 min in a
CO2 incubator at 36.5°C. Coverslips were placed in a flow chamber where they were superfused with modified Tyrode solution at 0.75 to 1.0 ml/min. The temperature was maintained at
35-36.5°C. Drugs were delivered via a 500-µl sampling loop;
the bath exchange rate was 30-40 s. The chamber was mounted on the
stage of a Zeiss inverted microscope incorporated into a
Zeiss/Attofluor workstation equipped with an excitation wavelength
selector (filter changer) and an intensified charge-coupled
device camera system. Fura 2 fluorescent emission was
measured at 520 nm in response to alternating excitation wavelengths of
334 and 380 nm. Data were collected and analyzed using Attofluor
Ratiovision software (version 6.0). The 334/380 ratio was used to
calculate intracellular Ca2+ in accord with the expression
|
(1)
|
where
Ro is the observed fluorescence ratio, Rmin is
the ratio at 0 Ca2+, Rmax is the ratio at a
saturating concentration of Ca2+, Kd is
the dissociation constant for Ca2+ with fura 2, and
is
the 380 nm fluorescence at 0 Ca2+, divided by the 380 nm
fluorescence at the saturating Ca2+ concentration. The
Kd was assigned 145 nM in accord with the value
published by Molecular Probes (Eugene, OR) (16).
Typically, fluorescence observations were obtained from isolated type I
cells. However, some data were also obtained from multiple cells
aggregated into clusters. In these instances, data from each cell was
analyzed separately. We did not observe any consistent difference in
the basal or stimulus-evoked responses from isolated vs. clustered
cells, in contrast to a report showing an absence of Ca2+
responses in clustered rat glomus cells (4). Also, cells selected for
analysis in the present study displayed morphology typical of type I
cells, and they responded to the low-O2 stimulus with at
least a doubling of the basal
[Ca2+]i.
Electrophysiological recording of CSN activity.
Under pentobarbital anesthesia (35 mg/kg), carotid bodies along with
their attached nerves were removed from New Zealand White rabbits and
placed in a lucite chamber containing 100% O2-equilibrated modified Tyrode solution at 4°C. With the aid of a dissecting microscope, each carotid body was carefully cleaned of surrounding connective tissue. The preparation was then placed in a conventional superfusion/flow chamber where the carotid body was continuously superfused (up to 4 h) with modified Tyrode solution maintained at
37°C and equilibrated with a selected gas mixture. The CSN was
drawn through a tiny hole in a glass coverslip into an adjoining chamber containing mineral oil, where it was positioned on two platinum
wire electrodes for differential recording of chemoreceptor activity.
Neural activity was led to an alternate current-coupled preamplifier, filtered, and transferred to a window discriminator and a
frequency-to-voltage converter. Signals were processed by an AD/DA
converter for display of frequency histograms on a PC monitor. In these
intact superfused organs, basal neural activity was established in
solutions equilibrated with 100% O2. Chemoreceptor responses were evoked in solutions equilibrated with 20%
O2, a moderate hypoxic stimulus that evokes submaximal
activity in the superfused carotid body/CSN preparation (13).
Statistical comparisons.
Multiple observations obtained from identical experimental conditions
were combined to calculate means and SE of the mean. Between-group
comparisons were made using Student's t-test (two tailed) or
ANOVA where appropriate.
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RESULTS |
Figure 1 shows the effects of
hypoxia and the ANP analog, atriopeptin III (APIII), on
voltage-activated outward K+ current. A typical result is
presented (left) that shows currents evoked by a voltage step
from a holding potential of
70 mV to +60 mV. The four numbered
traces illustrate 1) the current evoked in normoxic media,
2) depression of the sustained current by hypoxic superfusion
media, 3) the effect of 100 nM APIII on the hypoxia-evoked depression, and 4) recovery of the current after a return to
normoxic media. The voltage sensitivity of this current is plotted
under the same four experimental conditions (right). In
agreement with previous reports by others (22, 23), the outward current
is activated at voltages of
20 mV and above, and furthermore, it is noticeably depressed by a hypoxic challenge (22). However, we also
show for the first time that the effects of hypoxia are substantially
mitigated in the presence of 100 nM APIII. The data presented in the
inset summarize the effects of APIII in seven cells, indicating
that this agent significantly (P < 0.001) retards the
depression of the outward current by hypoxia. We did not
observe any increase in the outward current when APIII was present in normoxic media equilibrated with air (data not shown). This latter result is in accord with our earlier reports that show that APIII does
not alter basal CSN activity (31).

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Fig. 1.
Atrial natriuretic peptide (ANP) analog, atriopeptin III (APIII),
retards hypoxia-induced depression of K+ current in type I
cells. Current evoked by a voltage step from a holding potential of
70 mV to +60 mV (left). Each trace represents current
recorded during different experimental conditions: 1) control,
current recorded in superfusate equilibrated with air, 2)
hypoxia, cells were exposed for 40 to 60 s to superfusate equilibrated
with air and containing 1 mM sodium dithionite, 3) after a 1- to 2-min wash in control solution, cells were exposed to hypoxic media
containing 100 nM APIII for 40 to 60 s before reevaluation of current,
and 4) recovery, current recorded after a 1- to 2-min wash with
air-equilibrated, drug-free solution. Corresponding current-voltage
relationships for 4 experimental conditions (right);
inset summarizes peak currents recorded at +40 mV from 7 cells,
indicating that when cells are hypoxic, outward current is
significantly larger (+++ P < 0.001) in
presence of 100 nM APIII (data expressed as a percentage of combined
mean peak control and recovery currents). *** P < 0.001 vs. control current recorded in normoxic media.
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In similar experiments, we examined the effects of agents capable of
interfering with intracellular signaling cascades on the ability of
APIII to retard the hypoxia-evoked depression of the K+
current. Figure 2 presents data from a
different cell, where the depression of the outward current by hypoxia
was reduced by 100 nM APIII (compare traces and plots 1,
2, and 3 in Fig. 2). When APIII was introduced into the
superfusate along with the specific PKG inhibitor, KT-5823 (1 µM),
the hypoxia-induced depression of the current was partially restored
(Fig. 2, trace and plot 4). These effects were
reproducible in 11 cells (see inset) where treatment with
hypoxia in the presence of APIII plus KT-5823 resulted in a
significantly greater depression (P < 0.001) of the current than in hypoxic cells treated with APIII alone.

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Fig. 2.
Protein kinase G inhibitor, KT-5823, partially reverses retardation of
K+ current induced by APIII. Superfusion times and other
details same as in Fig. 1. Inset summarizes peak currents
recorded at +40 mV from 11 cells, and indicates that treatment with
hypoxia in presence of 100 nM APIII plus 1 µM KT-5823 (condition
4) resulted in a significantly greater depression
(  P < 0.001) of current than
when cells were treated with APIII alone (condition 3).
*** P < 0.001 vs. control current recorded in normoxia;
+++ P < 0.001 vs. current recorded in hypoxia
without APIII.
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Figure 3 demonstrates the effects of the
PP2A inhibitor, OA, on the ability of APIII to retard the
hypoxia-induced depression of the K+ current. Again, the
data demonstrate that the ANP analog potently inhibits the current
depression evoked by hypoxia, yet, in the presence of 100 nM OA, the
depression is almost fully restored. Observations from five cells
(inset) indicate that in the presence of APIII, the current is
significantly larger than with hypoxia alone (P < 0.001) or
with hypoxia plus APIII and OA (P < 0.001).

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Fig. 3.
Effect of protein phosphatase 2A inhibitor, okadaic acid (OA), on
retardation of K+ current induced by APIII. Details same as
in Fig. 1. Inset summarizes data from 5 cells: hypoxia
significantly depressed current (*** P < 0.001), and 100 nM APIII retarded hypoxia-induced depression of current
(+++ P < 0.001). Presence of 100 nM OA
significantly reversed effects of APIII
(  P < 0.001). Peak current values
obtained at +40 mV for statistical comparisons.
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Voltage-gated Ca2+ channels in rabbit type I cells are not
sensitive to hypoxia in the physiological range of
PO2 (see Refs. 22 and 23). In the
present experiments, Ca2+ currents were evoked in normoxic
media. Voltage ramps (200 ms duration) applied between
40 and
+80 mV evoked inward currents that activated near 0 mV and peaked near
+20 mV. These I-V characteristics are similar to
Ca2+ currents in rabbit type I cells previously documented
by others (29). The superimposed current traces in Fig.
4 show that 100 nM APIII depressed the
voltage-activated inward current, and further, when 1 µM KT-5823 was
introduced into the bath along with 100 nM APIII, the evoked current
was nearly normal. The inset summarizes data from five cells,
showing that APIII depresses the inward current by 32 ± 8%
(means ± SE; P < 0.01). In the
presence of KT-5823 plus APIII, the current was restored to >90 ± 1% of the current observed in the absence of drugs (P = 0.01 vs. APIII alone). In some experiments the activation voltage appeared
to be elevated in the presence of APIII; this phenomenon was not
analyzed further. In a similar manner, the PP2A inhibitor, OA (100 nM),
reversed the APIII-induced inhibition, which amounted to a 27 ± 3%
reduction of the Ca2+ current (Fig.
5; P < 0.001). In 11 cells, OA
restored the current to 91 ± 3% of the control value (P < 0.001 vs. APIII alone; Fig. 5, inset).

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Fig. 4.
APIII inhibits voltage-dependent Ca2+ current in type I
cells, and this effect is partially reversed by KT-5823. Four
superimposed traces are shown in which inward current was evoked by 200 ms voltage ramps from 40 mV to +80 mV (holding potential = 70 mV). Superfusion conditions and solution changes same as in
Fig. 1. Inset summarizes data from 5 cells; depression of
inward current by 100 nM APIII was partially restored in presence of 1 µM KT-5823 (** and ++ indicate P < 0.01 vs.
control current and P < 0.01 vs. current recorded in presence
of APIII alone; data expressed as a percentage of combined mean peak
current recorded in control and recovery conditions).
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Fig. 5.
Effects of APIII and OA on voltage-dependent Ca2+ currents
in type I cells. Details same as in Figs. 1 and 4. Inset
summarizes data from 11 cells in which 100 nM OA partially restored
inward current in presence of 100 nM APIII (*** P < 0.001 compared with control currents recorded without APIII;
+++ P < 0.001 compared with currents recorded
with APIII alone).
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The effects of OA on the Ca2+ influx in type I cells were
confirmed in experiments that measured changes in intracellular
Ca2+ levels in response to hypoxic stimulation. The record
of [Ca2+]i presented in Fig.
6 demonstrates, in sequence, the response of an isolated type I cell to 1) hypoxia, 2) hypoxia
plus 100 nM APIII, 3) hypoxia plus APIII and 100 nM OA,
4) a second exposure to hypoxia plus APIII, and finally,
5) hypoxia. The record illustrates the inhibition
of the Ca2+ response in the presence of APIII, and the
reversal of this effect by OA. In 12 similarly treated cells, we
observed that 100 nM APIII reduced the peak response by 39 ± 7%
(P < 0.001; see Fig. 6, inset) and that with the
addition of 100 nM OA, the response was restored to 97 ± 3% (P < 0.001 vs. APIII alone) of the control Ca2+ levels evoked by hypoxia. In similar experiments, the
effects of 100 nM APIII on the hypoxia-evoked Ca2+ response
were reversed in the presence of 1.0 µM KT-5823 (Fig. 7). APIII depressed the hypoxic response by
43 ± 3.6%, but in the presence of KT-5823 plus APIII, the peak
Ca2+ levels were restored to 95 ± 4% of normal
(P < 0.001 vs. APIII alone).

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Fig. 6.
OA blocks APIII-induced inhibition of intracellular Ca2+
responses evoked by a hypoxic challenge in type I cells. Basal
[Ca2+]i levels (49.5 ± 9.2 nM;
means ± SE) were measured in modified Tyrode solution (see
METHODS) equilibrated with air; hypoxic solutions were
equilibrated with air and contained 0.5 mM sodium dithionite.
Inset summarizes data from 12 cells expressed as a percentage
of mean control responses; *** and +++ indicate P < 0.001 vs. control hypoxia and P < 0.001 vs. hypoxic
responses recorded in presence of APIII alone, respectively.
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Fig. 7.
KT-5823 prevents APIII-mediated inhibition of intracellular
Ca2+ responses in type I cells. Basal
[Ca2+]i levels were 37.9 ± 5.8 nM
(means ± SE). Details same as in Fig. 6; *** and +++
indicate P < 0.001 vs. control hypoxia and hypoxia plus
APIII, respectively.
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The effects of KT-5823 and OA, examined at the cellular level on
isolated type I cells, predict that these agents should reverse chemoreceptor inhibition produced by APIII. To test this hypothesis, we
recorded CSN activity in vitro and evaluated the ability of OA to
reverse chemoreceptor inhibition in the presence of APIII. The
integrated nerve discharges (3 superimposed traces) presented in Fig.
8 show a control response to hypoxia, and
the substantial inhibition produced by 100 nM APIII, confirming our
previous demonstrations of the potent nature of this agent (31). As we
have also shown previously, the peptide did not alter basal discharge
activity established in normoxic media (32). The introduction of 100 nM
OA into the superfusate likewise did not alter the basal discharge activity. However, OA partially restored the response to hypoxia nearer
to the value observed in the absence of drugs. In six similar experiments (Fig. 8, inset), 100 nM APIII inhibited the
response to hypoxia by 44 ± 6% (P < 0.001), and
this inhibition was reduced to 26 ± 7% in the presence of 100 nM OA
(P < 0.01 vs. APIII alone).

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Fig. 8.
OA reverses inhibition of hypoxia-evoked carotid sinus nerve (CSN)
activity induced by APIII. Three superimposed traces of integrated
nerve activity from 1 experiment are presented. Responses were evoked
by superfusion solution equilibrated with 20% O2. APIII
(100 nM) and OA (100 nM) did not alter basal nerve activity established
in media equilibrated with 100% O2. Summary data from 6 experiments expressed as a percentage of control hypoxic response in
absence of drugs (inset); *** and ++ indicate
P < 0.001 vs. control hypoxic response and P < 0.01 vs. response in the presence of APIII alone,
respectively.
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DISCUSSION |
ANP and its analog, APIII, are potent inhibitors of hypoxia-evoked
activity in the rabbit carotid body (30, 31). ANP has been
immunocytochemically localized to type I chemosensory cells (30), and
these cells also express ANP receptors that are known to incorporate
guanylate cyclase (7, 30). Exposure to submicromolar concentrations of
ANP/APIII generates high levels of cGMP in type I cells (31, 37), and
cell-permeant cGMP analogs likewise inhibit stimulus-evoked CSN
activity (31). The present findings that low concentrations of APIII
retard the hypoxia-induced depression of K+ currents, and
reduce the magnitude of voltage-activated Ca2+ currents and
intracellular Ca2+ levels, suggest that APIII and ANP
potently modulate the excitation of type I cells by hypoxia. The
involvement of cGMP in the inhibitory mechanism is further suggested by
the finding that the specific PKG antagonist, KT-5823, reverses the
effects of APIII on the voltage-dependent currents and intracellular
free Ca2+ concentration. Thus it appears that the
inhibitory actions of APIII in the carotid body are mediated by the
cGMP-dependent activation of PKG.
Although a role for PKG is strongly implicated by these data, the
mechanism of kinase modulation of K+ and Ca2+
channels appears to be indirect. If PKG had phosphorylated the channels
directly, we would have expected to find that phosphatase inhibition
enhanced the effects of APIII. That the process instead involves
dephosphorylation of these proteins is indicated by our results with
the PP2A inhibitor, OA, which, like KT-5823, retards the effects of the
ANP analog. Moreover, the parallel reduction in
chemoreceptor discharge induced by APIII is likewise reversed by
exposure to OA. On average, however, the reversal of the APIII inhibition of nerve activity by OA was incomplete, whereas at the
cellular level the PP2A inhibitor restored
[Ca2+]i levels to 97% of control.
This apparent discrepancy may indicate that APIII acts via additional
mechanisms or sites to inhibit chemoreceptor activity (e.g., direct
action on CSN endings). Alternatively, in the intact superfused organ,
APIII and OA may not gain equal access to type I cells.
Previous studies have shown that the activity of specific types of
K+ channels is downregulated by phosphorylation (19, 28)
and that dephosphorylation by PP2A reverses this effect (19). Likewise, numerous data indicate that dihydropyridine-sensitive Ca2+
channels, similar to those found in type I cells, can be modulated by
the action of cAMP-dependent protein kinase (PKA) and PP2A. In the case
of these channels, however, phosphorylation appears to enhance their
activity (2, 3, 18, 25). Findings similar to ours have been reported by
White et al. (38), who used pituitary-derived GH4C1 cells to show that ANP enhances
voltage-sensitive outward K+ currents and inhibits inward
Ca2+ currents. Based on these and other data, White and
colleagues concluded that ANP, via cGMP, activates PKG, which in turn
phosphorylates PP2A or its regulatory subunit, thus initiating the
dephosphorylation step (38). Although other possible mechanisms cannot
be ruled out, the current results are consonant with this hypothesis.
In any case, our data strongly favor a role for second
messenger-mediated modulation of specific cell currents in type I
cells. Hypoxic excitation of the carotid body is known to increase cAMP in these cells (9, 26, 30), and previous studies have shown that the
presence of ATP and the catalytic subunit of PKA in patch pipettes
retards the "rundown" of Ca2+ currents in type I
cells (10, 17), suggesting that channel protein function during
excitation is modulated by specific kinases. In contrast, inhibition of
the carotid body by "efferent" fibers in the CSN has been shown
to involve NO, which like ANP, elevates cGMP in type I cells (35, 36).
Consequently, modulation of type I cell activity may involve
antagonistic signaling pathways controlled by either cAMP or cGMP. In
contrast to our findings, Hatton and Peers recently reported that
cell-permeant analogs of cyclic nucleotides, including specific
activators of PKA and PKG, fail to alter the properties of voltage- and
hypoxia-sensitive currents in rat type I cells (15).
However, these data were collected using the whole cell configuration
of the patch-clamp technique (not the nystatin perforated-patch
technique employed here), where intracellular constituents are dialyzed
against the patch-pipette solution.
In summary, our findings suggest that chemoreceptor inhibition produced
by APIII is initiated by the formation of cGMP, which activates PKG,
and in turn, PP2A. During hypoxic stimulation in the presence of APIII,
dephosphorylation of specific proteins appears to enhance
K+ currents and depress Ca2+ influx via
Ca2+ channels. Consequently, the state of ion channel
phosphorylation may be an important determinant of type I cell
excitability and chemoreceptor output. Type I cell excitability could
be influenced via feedback loops involving autoreceptors coupled to
adenylate and guanylate cyclases and multiple secretory agents released from these cells and CSN terminals.
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ACKNOWLEDGEMENTS |
This work was supported by National Institute of Neurological
Disorders and Stroke Grants NS-12636 and NS-07938.
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FOOTNOTES |
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: S. J. Fidone,
Dept. of Physiology, Univ. of Utah School of Medicine, 410 Chipeta Way,
Research Park, Salt Lake City, UT 84108 (E-mail:
toni.gillett{at}m.cc.utah.edu).
Received 13 August 1999; accepted in final form 15 November 1999.
 |
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