Am J Physiol Cell Physiol 284: C378–C388, 2003.
First published October 9, 2002; 10.1152/ajpcell.00260.2002.
Metabolic inhibition with cyanide induces calcium
release in pulmonary artery myocytes and Xenopus oocytes
YONG-XIAO WANG,1 YUN-MIN ZHENG,1
ISKANDAR ABDULLAEV,1 AND MICHAEL I. KOTLIKOFF2
1
Center for Cardiovascular Sciences, Albany Medical College, Albany 12208; and
2
Department of Biomedical Sciences, Cornell University, Ithaca, New York 14853
Submitted 4 June 2002; accepted in final form 30 September 2002
Address for reprint requests and other correspondence: Y.-X.
Wang, Center for Cardiovascular Sciences (MC-8), Albany Medical
College, 47 New Scotland Ave., Albany, NY 12208 (E-mail:
[email protected]).
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.
sarcoplasmic reticulum; mitochondria
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0363-6143/03 $5.00 Copyright © 2003 the American Physiological Society
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HYPOXIA SELECTIVELY CONSTRICTS pulmonary arterial vessels, which serves as an important regulatory mechanism in the maintenance of arterial oxygenation. Sustained hypoxic pulmonary vasoconstriction (HPV),
however, is a key factor in the development of pulmonary hypertension. Although the mechanism responsible for HPV is not certain, there is increasing evidence
supporting the idea that an increase in intracellular
Ca2⫹ concentration ([Ca2⫹]i) in smooth muscle cells
plays a crucial role in the development of HPV. The
hypoxic increase in [Ca2⫹]i is associated with Ca2⫹
release from intracellular stores and influx of Ca2⫹
through voltage-dependent Ca2⫹ channels (6, 27, 36,
38). Moreover, Ca2⫹ release may be an initial step for
hypoxia-induced [Ca2⫹]i increase (13, 29, 30).
Inhibitors of oxidative phosphorylation and glycosis
have been widely used to mimic hypoxia in an effort to
define mechanisms of HPV. Metabolic inhibition by
cyanide (CN) and other agents results in a significant
vasoconstrictor response in isolated rat lungs and pulmonary artery muscle strips (2, 25, 26, 37), and this
vasoconstriction is associated with an increase in
[Ca2⫹]i in pulmonary arterial smooth muscle cells
(PASMCs). Yuan et al. (41) have shown that inhibition
of glycosis with deoxyglucose results in a reduction in
voltage-dependent K⫹ currents and membrane depolarization in cultured rat PASMCs, suggesting influx of
Ca2⫹ through voltage-dependent Ca2⫹ channels. In
contrast to these data, Archer et al. (2) have reported
that metabolic inhibition with CN results in pulmonary vasoconstriction but does not affect voltage-dependent K⫹ currents in freshly dissociated rat pulmonary artery myocytes. Direct measurements of
spatially averaged whole cell [Ca2⫹]i in cultured rat
PASMCs have demonstrated that metabolic inhibition
with deoxyglucose induces an increase in [Ca2⫹]i,
which occurs due to Ca2⫹ release from intracellular
stores and influx of Ca2⫹ through voltage-dependent
Ca2⫹ channels (4). We have recently shown that metabolic inhibition with CN induces an increase in
[Ca2⫹]i and associated Ca2⫹-activated Cl⫺ currents
[ICl(Ca)] in freshly dissociated rat PASMCs (30). The
[Ca2⫹]i increase is greatly inhibited after the depletion
of sarcoplasmic reticulum (SR) Ca2⫹ with caffeine,
although slightly attenuated by prior application of the
voltage-dependent Ca2⫹ channels blocker nisoldipine.
Taken together, Ca2⫹ release from intracellular stores
is likely to make an important contribution to an increase [Ca2⫹]i after metabolic inhibition in PASMCs.
However, recent studies have indicated that metabolic
inhibition with CN may constitute distinct cellular
effects from hypoxia per se. For example, hypoxia hyperpolarizes, whereas CN depolarizes, cultured Drosophila neurons (14), possibly by affecting Na⫹ and/or
K⫹ currents. These findings, together with the well-
Wang, Yong-Xiao, Yun-Min Zheng, Iskandar Abdullaev, and Michael I. Kotlikoff. Metabolic inhibition with
cyanide induces calcium release in pulmonary artery myocytes and Xenopus oocytes. Am J Physiol Cell Physiol 284:
C378–C388, 2003. First published October 9, 2002; 10.1152/
ajpcell.00260.2002.—We examined the effects of metabolic
inhibition on intracellular Ca2⫹ release in single pulmonary
arterial smooth muscle cells (PASMCs). Severe metabolic
inhibition with cyanide (CN, 10 mM) increased intracellular
calcium concentration ([Ca2⫹]i) and activated Ca2⫹-activated
Cl⫺ currents [ICl(Ca)] in PASMCs, responses that were
greatly inhibited by BAPTA-AM or caffeine. Mild metabolic
inhibition with CN (1 mM) increased spontaneous transient
inward currents and Ca2⫹ sparks in PASMCs. In Xenopus
oocytes, CN also induced Ca2⫹ release and activated ICl(Ca),
and these responses were inhibited by thapsigargin and
cyclopiazonic acid to deplete sarcoplasmic reticulum (SR)
Ca2⫹, whereas neither heparin nor anti-inositol 1,4,5trisphosphate receptor (IP3R) antibodies affected CN responses. In both PASMCs and oocytes, CN-evoked Ca2⫹
release was inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP) and oligomycin or CCCP and thapsigargin.
Whereas hypoxic stimuli resulted in Ca2⫹ release in pulmonary but not mesenteric artery myocytes, CN induced release
in both cell types. We conclude that metabolic inhibition with
CN increases [Ca2⫹]i in both pulmonary and systemic artery
myocytes by stimulating Ca2⫹ release from the SR and mitochondria.
CA2⫹ RELEASE BY METABOLIC INHIBITION WITH CYANIDE
established finding that hypoxia exposure induces
Ca2⫹ release in myocytes from pulmonary but not
systemic arteries (11, 12, 28), led us to question whether
Ca2⫹ release from intracellular stores after metabolic
inhibition is a unique response in PASMCs. Here, we
sought to examine the cellular mechanisms underlying
CN-induced Ca2⫹ release in freshly isolated PASMCs
and Xenopus oocytes to determine whether CN-induced Ca2⫹ release is a response unique to PASMCs
and to compare CN and hypoxia-induced Ca2⫹ release
in pulmonary and systemic arterial myocytes.
METHODS
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tained (in mM) 115 NaCl, 2.8 KCl, 1.8 CaCl2, 1.8 MgCl2, and
10 HEPES (pH 7.2). To test the ion selectivity of CN-activated currents, 92% of extracellular NaCl was replaced with
an equal concentration of NaI, Na-isethionate, Tris 䡠 Cl, or
NMDGCl.
Measurement of whole cell [Ca2⫹]i. Measurements of spatially averaged Ca2⫹ fluorescence in pulmonary and mesentery artery myocytes were made by a dual excitation wavelength fluorescence method as described previously (33),
using the IonOptix fluorescence photometric system (Milton,
MA). Cells were loaded with 4 ␮M fura 2-AM (Molecular
Probes, OR) for 30 min at 35°C. Experiments were initiated
after 20 min of perfusion to wash out extracellular fura 2-AM
and to allow the conversion of intracellular dye into its
nonester form. The dye was excited at 340 and 380 nm
wavelengths (Xenon 75 W arc lamp), and the emission fluorescence at 510 nm was detected by a photomultiplier tube.
Photobleaching was minimized by the use of neutral density
filters and by shuttering excitation light between sampling
periods. Background fluorescence was determined by removing the cell from the field after the experiment.
Confocal laser scanning microscopic imaging of Ca2⫹
sparks. Localized Ca2⫹ release events (Ca2⫹ sparks) were
measured as described previously (5) using a high-speed
confocal laser scanning microscopic system (Zeiss LSM510,
Germany) coupled to an inverted microscope (Zeiss Axert
300). Single myocytes were loaded with fluo-4-AM (5 ␮M)
(Molecular Probes) for 30 min at 35°C. After 20 min of bath
perfusion to wash out extracellular fluo-4-AM and to allow
the conversion of intracellular dye into its nonester form, the
dye fluorescence was excited with 488 nm light emitted from
a Krypton/Argon laser and detected by a confocal laser scanning head. High bandwidth time profiles of fluorescence intensity were obtained using line scanning mode.
Hypoxia. Hypoxic responses were achieved by switching
the perfusing solution from a bath solution equilibrated with
20% O2 and 5% CO2 and balanced with N2 (normoxic) to a
solution equilibrated with 5% CO2 and balanced with various
O2/N2 mixtures, as described previously (32). The oxygen
tension of the solution was continuously monitored by means
of an oxygen electrode (OXEL-1, WPI). The bath PO2 was
ⱖ140 and 10–20 Torr in the normoxic and hypoxic solutions,
respectively. Under normoxic and hypoxic conditions, pH
values were the same (7.4). To avoid atmospheric O2 reequilibration with the hypoxic bath solution, mineral oil was
placed on top of the recording chamber.
Reagents. CN (Mallinckrodt, Phillipsburg, NJ) was freshly
prepared just before experiments and applied to individual
cells through a puffer pipette connected to a Picospritzer
pressure ejection device (Parker Instrumentation, Fairfield,
NJ). Caffeine, CCCP, heparin, norepinephrine, nystatin, and
oligomycin were obtained from Sigma (St. Louis, MO); fura
2-AM and fluo-4-AM were from Molecular Probes; and antibody against inositol 1,4,5-trisphosphate receptors (antiIP3R antibody), cyclopiazonic acid, and thapsigargin were
from Calbiochem (La Jolla, CA).
Statistics. Data were expressed as means ⫾ SE of n cells
investigated. Student’s t-test was used for determining the
significance of differences between two groups, whereas oneway ANOVA was used for multiple comparisons. P ⬍ 0.05
was accepted as the level of statistical significance.
RESULTS
Metabolic inhibition with CN induces an increase in
[Ca2⫹]i and activates ICl(Ca) in PASMC cells. Freshly
isolated PASMCs were loaded with fura 2-AM and
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Cell preparation. Single smooth muscle cells of rat resistance (external diameter ⬍300 mm) pulmonary arteries were
isolated as described previously (32). Briefly, female or male
Sprague-Dawley rats were euthanized by intraperitoneal injection of sodium pentobarbital (150 mg/kg) under approved
animal care and use protocols. The heart and lungs were
rapidly removed en bloc and placed in normal physiological
saline solution (PSS). After the connective tissues and endothelium were removed, resistance pulmonary arteries were
cut into small pieces (1 ⫻ 10 mm). The tissue was incubated
in nominally Ca2⫹-free PSS (1.5 ml) containing 2 mg papain
(Worthington) and 0.2 mg dithioerythritol (Sigma) for 20 min
(37°C), then in nominally Ca2⫹-free PSS containing 0.5 mg
type H collagenase (Sigma), 1.0 mg type F collagenase (Sigma), and 100 ␮M Ca2⫹ for 10–15 min (37°C), and finally in
ice cold nominally Ca2⫹-free PSS for 10–15 min. Single cells
were harvested by gentle trituration and then stored on ice
for use up to 8 h.
Freshly isolated rat mesenteric artery smooth muscle cells
were prepared using the same procedure as described above.
Xenopus oocytes were prepared as described previously
(31). Adult female Xenopus laevis were euthanized by the
anesthetic aminobenzoic acid ethyl ester. Ovarian fragments
were gently teased out with forceps, and oocytes were defoliculated in Ca2⫹-free Barth’s medium containing collagenase (20 mg/ml) at a temperature of 19°C. Stage V or VI
Xenopus oocytes were selected for experiments.
Membrane current recording. Whole cell membrane currents in single PASMCs were measured by the nystatinperforated patch-clamp technique (34) using a patch-clamp
amplifier (EPC-9; Heka Electronics, Germany). When filled
with intracellular solution, patch pipettes for the perforated
patch-clamp experiments had a resistance of 2–3 M⍀. When
electrical access was detected, cells were clamped at a holding potential of ⫺55 mV. Membrane capacitance and series
resistance were continuously monitored and compensated,
and experiments were initiated after a decrease in the access
resistance to below 40 M⍀. Voltage-command protocols were
generated by the EPC-9 system (Heka Electronics, Germany). Data were recorded on a Macintosh computer and
VHS tape for off-line analysis.
Whole cell membrane currents in Xenopus oocytes were
measured by the two-electrode voltage-clamp technique (31).
Recording electrodes had a resistance of 0.5–1.5 M⍀. After
impalement, oocytes were clamped at ⫺60 mV and continuously perfused with bath solution (19°C). Current and voltage signals were recorded on a computer for off-line analysis.
The bath solution used for pulmonary artery myocytes was
(in mM) 125 NaCl, 5 KCl, 1 MgSO4, 10 HEPES, 1.8 CaCl2,
and 10 glucose (pH 7.4). The pipette solution contained (in
mM) 130 CsCl, 5 MgCl2, 3 EGTA, 1 CaCl2, and 10 HEPES
(pH 7.3). The standard oocyte extracellular solution con-
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other myocytes tested (Fig. 1B). Prior application of the
ryanodine receptor (RyR) activator caffeine markedly
decreased, but did not abolish, CN-induced Ca2⫹ and
current responses, consistent with a previous study
(30). As shown at right in Fig. 1A, application of CN
only induced a small increase in [Ca2⫹]i and ICl(Ca) in a
cell pretreated with caffeine (10 mM) for 5 min. The
effects of caffeine on CN Ca2⫹ and current responses
are summarized in Fig. 1B. The CN-induced [Ca2⫹]i
increase and ICl(Ca) were reduced by 72 and 77%, respectively. These results suggest that RyRs may be
potential targets for metabolic inhibition.
In agreement with this notion, exposure of voltageclamped myocytes to a lower concentration of CN (1
mM) increased the frequency and amplitude of spontaneous transient inward currents (STICs). An example
of these experiments is shown at left in Fig. 2A. In a
total of six cells tested, the frequency and amplitude of
STICs were increased from 0.27 ⫾ 0.04 to 0.51 ⫾ 0.06
Hz and from 43 ⫾ 10 to 71 ⫾ 22 pA (P ⬍ 0.05),
respectively. Moreover, in electrically quiescent cells,
CN (1 mM) induced transient inward currents that
showed similar characteristics to STICs (n ⫽ 5). In
both cases, no significant changes in global [Ca2⫹]i
were observed. By contrast, using a high-speed confocal imaging system (Zeiss LSM510), we have found
that exposure of CN (1 mM) significantly increased the
amplitude and frequency of localized Ca2⫹ release
events (Ca2⫹ sparks) in PASMCs (Fig. 2B). Previous
studies have shown that STICs occur due to the simultaneous opening of many Ca2⫹-activated Cl⫺ channels,
which are caused by Ca2⫹ sparks generated by the
opening of RyRs in airway, cerebral artery, and blad-
Fig. 1. Cyanide (CN) triggers Ca2⫹ release and activates Ca2⫹-activated Cl⫺
currents [ICl(Ca)] in pulmonary artery
smooth muscle cells (PASMCs). A: application of CN (10 mM) induced a sustained intracellular calcium concentration ([Ca2⫹]i) increase (top) and ICl(Ca)
(bottom) in a control cell (left), failed to
induce either Ca2⫹ or current response
in another cell preincubated with
BAPTA-AM (50 ␮M) for 30 min (middle), and induced a much smaller
[Ca2⫹]i increase and ICl(Ca) in a third
cell pretreated with caffeine (10 mM)
for 5 min (right). All cells were loaded
with fura 2-AM and voltage clamped at
⫺55 mV using the perforated patchclamp method. Cesium intracellular
solution was used to block potassium
currents. Nisoldipine was added in the
bath solution to block voltage-dependent Ca2⫹ channels. B: graphs summarize the effects of BAPTA and caffeine
on CN-induced Ca2⫹ and current responses. Numbers in parenthesis indicate the number of cells tested. *P ⬍
0.05 vs. CN alone (control).
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voltage clamped at ⫺55 mV using the perforated patchclamp technique. The intracellular solution contained
Cs⫹ ions in place of K⫹ ions to block outward potassium currents, and myocytes were preexposed to nisoldipine (5 ␮M) for 5 min before CN exposure to prevent
potential influx of Ca2⫹ through voltage-dependent
Ca2⫹ channels during metabolic inhibition (30). Under
these conditions, application of CN (10 mM) induced an
increase in spatially averaged whole cell [Ca2⫹]i and an
inward current that mirrored the kinetics of the rise in
[Ca2⫹]i. A typical example of these experiments is
shown at left in Fig. 1A, in which both Ca2⫹ and
current signals activated over a period of several seconds and decayed over tens of seconds. The current,
previously shown to be a Ca2⫹-activated Cl⫺ current
[ICl(Ca)] (30), activated with a slight delay after the rise
in [Ca2⫹]i and was sustained as long as CN exposure
was maintained, unlike the more rapidly inactivating
[Ca2⫹]i transient observed after Ca2⫹ release by engagement of G protein-coupled receptors (34). In a total
of six myocytes tested, [Ca2⫹]i was increased from a
resting level of 114 ⫾ 27 nM to a peak of 749 ⫾ 33 nM,
whereas ICl(Ca) had a mean amplitude of 551 ⫾ 42 pA
(Fig. 1B), similar to the values reported in our previous
study (30).
CN-induced Ca2⫹ release may occur through ryanodine receptors in pulmonary artery myocytes. The CNinduced [Ca2⫹]i increase and ICl(Ca) were blocked by
buffering intracellular Ca2⫹ with BAPTA. As shown at
middle in Fig. 1A, in a pulmonary artery myocyte
pretreated with BAPTA-AM (50 ␮M) for 30 min, application of CN failed to induce either an increase in
[Ca2⫹]i or ICl(Ca). We obtained similar results in four
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der smooth muscle cells (5, 23, 42). Therefore, metabolic inhibition after CN exposure may result in the
opening of RyRs, thereby inducing Ca2⫹ release from
the SR.
RyRs and IP3Rs are functionally coupled to the same
SR in PASMCs. Experiments indicate a substantial
functional overlap between SR Ca2⫹ stores expressing
RyRs and IP3Rs (1, 15, 16, 20, 35), suggesting that
IP3Rs might also be involved in the Ca2⫹ response after
metabolic inhibition. To test this hypothesis, experiments were performed in which pulmonary artery
myocytes were first exposed to the ␣-adrenergic receptor agonist norepinephrine and then to caffeine. As
shown in Fig. 3A, norepinephrine (300 ␮M) induced an
increase in [Ca2⫹]i and ICl(Ca) in a cell. In the continued
presence of norepinephrine, however, application of
caffeine (10 mM) failed to evoke further [Ca2⫹]i increase or ICl(Ca) in the same cell. After washout of
norepinephrine, caffeine induced typical Ca2⫹ and current responses. Similar observations were obtained in
six similar experiments. Consistent with this result,
after the depletion of SR Ca2⫹ with caffeine (10 mM),
norepinephrine (300 ␮M) was no longer able to induce
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an increase in [Ca2⫹]i, whereas, after washout of caffeine, norepinephrine triggered a normal Ca2⫹ release
(Fig. 3B). We observed similar results in a total of
seven cells. These experiments indicate that IP3Rs and
RyRs are functionally coupled to the same SR in pulmonary artery myocytes, complicating the interpretation of the role of RyRs in CN-induced Ca2⫹ release.
CN induces the SR Ca2⫹ release through IP3Rs in
Xenopus oocytes. Because IP3Rs, but not RyRs, are
expressed in the SR of Xenopus oocytes (24), we sought
to use these cells as a simplified system to define the
role of IP3Rs in Ca2⫹ release after metabolic inhibition.
To determine whether CN induced Ca2⫹ release in
oocytes, cells were voltage clamped at ⫺60 mV using
the two-electrode voltage-clamp technique and bathed
in nominally Ca2⫹-free solution to prevent Ca2⫹ influx.
Under these conditions, exposure of Xenopus oocytes to
CN (10 mM) induced a sustained current similar to
that observed in pulmonary artery myocytes (Fig. 4).
The CN current was blocked in oocytes preloaded with
a Ca2⫹ buffer; as shown in Fig. 4A, incubation of
oocytes with BAPTA-AM (50 ␮M) for 4 h almost completely blocked CN-induced currents in 6 cells tested.
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Fig. 2. CN at lower concentration increases spontaneous transient inward currents (STICs) and localized
Ca2⫹ release events (Ca2⫹ sparks) in pulmonary artery
myocytes. A: mild metabolic inhibition by exposure to 1
mM CN increased the frequency and amplitude of
STICs (left) and induced transient inward currents
(right) in PASMCs. In both cases, no measurable
changes in spatially averaged whole cell [Ca2⫹]i were
observed. Cells were voltage clamped at ⫺55 mV using
the perforated patch-clamp technique and dialyzed
with cesium intracellular solution. B: application of a
low concentration of CN (1 mM) increased the amplitude and frequency of Ca2⫹ sparks in pulmonary artery
myocytes. At top, original imagings were recorded before and after application of CN by line scan mode using
Zeiss LSM510 confocal laser scanning microscope. The
bottom graphs summarize the effects of CN on the
amplitude and frequency of Ca2⫹ sparks from 5 cells
tested. *P ⬍ 0.05 compared with control.
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Replacement of 92% external NaCl with NaI or Naisethionate (n ⫽ 6) shifted the reversal potential,
whereas similar replacement with NMGCl or Tris 䡠 Cl
(n ⫽ 5) did not, indicating anion selectivity of the
CN-induced current (Fig. 4C). Moreover, niflumic acid,
a chloride channel blocker, blocked CN-induced currents (Fig. 4B). Collectively, these results indicate that
CN induces Ca2⫹ release from intracellular stores,
activating ICl(Ca).
Using ICl(Ca) as an indicator of Ca2⫹ release, we next
sought to determine whether the CN-induced Ca2⫹
release was associated with release of Ca2⫹ from the
endoplasmic reticulum (ER) of Xenopus oocytes. As an
example shown in Fig. 5A, depletion of ER Ca2⫹ with
thapsigargin (1 ␮M) for 4 h markedly inhibited CNinduced current. The effect of thapsigargin on CNinduced currents is summarized in Fig. 5B. The mean
current amplitude was 1.84 ⫾ 0.25 ␮A in six control
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Fig. 3. Ryanodine receptors (RyRs) and inositol 1,3,5-trisphosphate
receptors (IP3Rs) are functionally coupled to the same sarcoplasmic
reticulum in pulmonary artery myocytes. A: norepinephrine (300
␮M) induces an increase in [Ca2⫹]i and ICl(Ca) in a myocyte. In the
continued presence of norepinephrine, however, application of caffeine (10 mM) failed to induce a further Ca2⫹ release and ICl(Ca).
After washout of norepinephrine, caffeine-induced Ca2⫹ and current
responses were restored. The cell was voltage clamped at ⫺55 mV,
and cesium intracellular solution was used. B: application of caffeine
(10 mM) caused an increase [Ca2⫹]i and blocked subsequent norepinephrine (300 ␮M)-induced Ca2⫹ response in a non-voltage-clamped
cell. After washout of caffeine, norepinephrine evoked a normal Ca2⫹
release in the same cell.
cells and 0.69 ⫾ 0.16 ␮A in six cells treated with
thapsigargin (P ⬍ 0.05). Similar inhibition of CN-induced currents was observed in experiments in which
oocytes were incubated with both thapsigargin and
cyclopiazonic acid (10 ␮M for 4 h) (Fig. 5B). These data
indicate that metabolic inhibition results in the release
of Ca2⫹ from ER Ca2⫹ stores in Xenopus oocytes. The
expression of a single type of ER Ca2⫹ release channel
in this system suggests that CN exposure may result in
the gating of IP3R calcium release channels.
Next, we sought to examine whether inhibition of
IP3Rs with heparin, a prototypical IP3R antagonist,
could block CN-induced Ca2⫹ release in oocytes. Figure
6A shows an example of these experiments in which
preinjection of heparin at a final concentration of 10
mg/ml for 10 min, previously shown to block IP3-mediated Ca2⫹ release in oocytes (31), did not affect CNinduced current. As summarized in Fig. 6B, the mean
amplitude of CN currents was 1.32 ⫾ 0.28 ␮A in a total
of eight cells tested, which was not significantly different from that in control oocytes (1.40 ⫾ 0.31 ␮A, n ⫽
18). Because dialysis of anti-IP3R antibody (5 ␮g/ml)
through the patch pipette prevents neurotransmitterinduced Ca2⫹ release in tracheal smooth muscle cells
(21), we sought to further test whether inhibition of
IP3Rs by preinjection of this antibody was able to block
CN response in oocytes. Similar to heparin, preinjection of anti-IP3R antibody (5 ␮g/ml) for 10 min did not
produce any significant effect on CN-induced currents
(Fig. 6B). Therefore, metabolic inhibition with CN may
directly affect the channel domain, rather than the
binding domain of IP3Rs on the ER of Xenopus oocytes.
CN induces Ca2⫹ release from mitochondria in Xenopus oocytes and pulmonary artery myocytes. As shown
in Fig. 5, depletion of ER Ca2⫹ did not completely block
the rise in [Ca2⫹]i after exposure to cyanine in Xenopus
oocytes recorded in Ca2⫹-free bath solution. We reasoned that a second source of intracellular Ca2⫹, possibly arising from the mitochondria, might also contribute to the CN response. To test this possibility,
oocytes were first exposed to CCCP, which disrupts
mitochondrial transmembrane potential and depletes
mitochondrial Ca2⫹, and then to CN. As shown in Fig.
7, CN induced a smaller ICl(Ca) in oocytes pretreated
with CCCP (50 ␮M) for 10 min than in control oocytes.
To ensure the complete depletion of mitochondrial
Ca2⫹, oocytes were exposed to CCCP and oligomycin
(10 ␮M for 10 min), which inhibits mitochondrial ATP
synthesis, was simultaneously applied to oocytes. Under these conditions, similar inhibition of CN-induced
Ca2⫹ release was observed; the mean current was
2.44 ⫾ 0.28 ␮A in control oocytes (n ⫽ 6), 1.46 ⫾ 0.21
␮A in CCCP-pretreated oocytes (n ⫽ 6), and 1.48 ⫾
0.13 ␮A in CCCP/oligomycin-pretreated oocytes (n ⫽
7). Thus mitochondrial Ca2⫹ release likely contributes
to CN-induced intracellular Ca2⫹ rise.
To further examine the contributions of ER and
mitochondrial Ca2⫹ release in the CN-induced Ca2⫹
response in oocytes, cells were exposed to both thapsigargin and CCCP to deplete ER and mitochondrial
Ca2⫹ and then to CN. Figure 8A shows an example of
CA2⫹ RELEASE BY METABOLIC INHIBITION WITH CYANIDE
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such an experiment; application of CN failed to induce
any chloride current in an oocyte pretreated with thapsigargin (1 ␮M) for 4 h and CCCP (50 ␮M) for 10 min,
indicating no Ca2⫹ release. Similar results were observed from a total of six oocytes tested (Fig. 8B).
Collectively, these data suggest that CN-induced Ca2⫹
release results from both ER and mitochondria in Xenopus oocytes.
We conducted similar experiments in PASMCs to
explore whether mitochondrial Ca2⫹ also contributes
to CN-induced Ca2⫹ release. Fura 2-AM-loaded cells
were treated with CCCP (50 ␮M) for 5 min to deplete
mitochondrial Ca2⫹. Nisoldipine (5 ␮M) was added to
the bath solution to prevent potential influx of Ca2⫹
through voltage-dependent Ca2⫹ channels during exposure of CN. An example of these experiments is
shown in Fig. 9A. Application of CN induced a smaller
[Ca2⫹]i rise than that in control cells. Similar results
were obtained from eight other identical experiments.
The mean values of CN-induced [Ca2⫹]i rise were
617 ⫾ 31 nM in eight control cells and 439 ⫾ 24 nM in
nine CCCP-treated cells (P ⬍ 0.05) (Fig. 9B), suggesting that mitochondrial Ca2⫹ release contributes to CNinduced Ca2⫹ release in pulmonary artery myocytes.
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Moreover, as with Xenopus oocytes, combined exposure
to thapsigargin (1 ␮M) and CCCP (50 ␮M) for 5 min
completely ablated CN-induced increases in [Ca2⫹]i.
These data suggest that metabolic inhibition with CN
releases Ca2⫹ from the SR and mitochondria of pulmonary artery myocytes.
CN, unlike hypoxia, induces Ca2⫹ release in systemic
(mesenteric) artery smooth muscle cells. Although CN
has been widely used to examine hypoxic cellular responses, this metabolic inhibitor may produce different
effects than hypoxia (14). Thus we sought to examine
whether CN exposure also induced Ca2⫹ release in
freshly isolated systemic (mesenteric) artery smooth
muscle cells. As an example of these experiments
shown in Fig. 10A, application of CN (10 mM) induced
an increase in [Ca2⫹]i in mesenteric artery myocytes.
In a total of seven cells tested, the mean [Ca2⫹]i increase was 658 ⫾ 28 nM. By contrast, hypoxia exposure did not induce an increase in [Ca2⫹]i in mesenteric
artery smooth muscle cells. Figure 10B shows a typical
example of these experiments. However, in the same
cell, application of norepinephrine evoked a typical
Ca2⫹ response. Similar results were obtained from five
other myocytes. As shown in Fig. 10C, hypoxia induced
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Fig. 4. CN induces intracellular Ca2⫹ release and
ICl(Ca) in Xenopus oocytes. A: recording traces show
CN-induced currents obtained from a control cell (left)
and an oocyte preincubated with BAPTA-AM (50 ␮M)
for 4 h (right). Both oocytes were voltage clamped at
⫺60 mV using the two-electrode voltage-clamp method.
Ca2⫹-free bath solution was used to block potential
influx of Ca2⫹. B: graphs summarize the effects of
BAPTA and niflumic acid on CN currents. *P ⬍ 0.05 vs.
CN alone. C: replacement of external NaCl (115 mM)
with NaI or Na-isethionate (9/106 mM) (left), but not
with NMGCl or Tris 䡠 Cl (9/106 mM) (right), significantly shifted the reversal potentials of CN-activated
currents. Oocytes were voltage clamped at ⫺60 mV.
After full activation of CN currents, ramp pulses from
⫺80 to 60 mV were applied.
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CA2⫹ RELEASE BY METABOLIC INHIBITION WITH CYANIDE
an increase in [Ca2⫹]i in PASMCs. These results are
consistent with previous reports that hypoxic [Ca2⫹]i
increase has not been observed in myocytes from other
types of systemic arteries such as celiac, cerebral, cor-
Fig. 6. Inhibition of IP3Rs with heparin and anti-IP3R antibody does
not block CN-induced ICl(Ca) in Xenopus oocytes. A: original recordings show CN-induced ICl(Ca) in a control oocyte (left) and an oocyte
preinjected with heparin (10 mg/ml) for 10 min (right). B: graphs
summarize the effects of heparin and anti-IP3R antibody (5 ␮g/ml, 10
min) on CN currents.
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Fig. 7. Depletion of mitochondrial Ca2⫹ with carbonyl CN m-chlorophenylhydrazone (CCCP) and oligomycin (Oli) inhibits CN-induced ICl(Ca) in
Xenopus oocytes. A: current traces were recorded in a control oocyte
(left) and an oocyte preincubated with CCCP (50 ␮M) for 10 min (right).
B: graphs summarize effects of CCCP (50 ␮M) alone and CCCP plus
oligomycin (10 ␮M, 10 min) on CN-induced currents. *P ⬍ 0.05 vs. control.
onary, and femoral artery (11, 12, 28). Therefore, metabolic inhibition with CN induces Ca2⫹ responses in
both pulmonary and systemic (mesenteric) artery
smooth muscle cells, whereas hypoxia-induced Ca2⫹
Fig. 8. Depletion of both the sarcoplasmic reticulum and mitochondrial Ca2⫹ with thapsigargin and CCCP completely blocks CNinduced ICl(Ca) in Xenopus oocytes. A: current recordings show CNinduced ICl(Ca) in a control oocyte (left) and an oocyte preincubated
with thapsigargin (1 ␮M, 4 h) and CCCP (50 ␮M, 10 min) (right). B:
summary of the effects of thapsigargin and CCCP on CN currents.
*P ⬍ 0.05 vs. control.
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Fig. 5. Depletion of sarcoplasmic reticulum Ca2⫹ with thapsigargin
(Tha) and cyclopiazonic acid (CPA) blocks CN-induced ICl(Ca) in
Xenopus oocytes. A: traces show CN currents in a control oocyte (left)
and an oocyte preincubated with thapsigargin (1 ␮M) for 4 h (right).
B: summary of the effects of thapsigargin (1 ␮M, 4 h) alone and
thapsigargin plus cyclopiazonic acid (10 ␮M, 4 h) on CN currents.
*P ⬍ 0.05 vs. control.
CA2⫹ RELEASE BY METABOLIC INHIBITION WITH CYANIDE
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release is unique to PASMCs but not systemic artery
myocytes.
DISCUSSION
Inhibition of cellular metabolism by exposure to CN
or glucose analogs results in an increase in [Ca2⫹]i in
PASMCs in a manner similar to that observed for
hypoxic exposure. Although intracellular Ca2⫹ release
appears to make an important contribution to the increase in [Ca2⫹]i during metabolic inhibition (4, 30, 41),
the underlying mechanisms are not fully understood.
In an effort to more fully understand this process, we
have exposed single cells to CN and attempted to
identify the sources and mechanisms of intracellular
Ca2⫹ release. Application of low concentrations of CN
increased the frequency and amplitude of spontaneous
inward currents or induced typical STICs in PASMCs,
suggesting the gating of RyRs, whose activity is known
to underlie these currents (5, 23, 42). These findings
are similar to previous studies of hypoxic Ca2⫹ release
that occurs through RyRs (7–9, 13, 17, 22, 28, 39).
Because the activity of voltage-dependent channels is
an important mechanism for the refilling of intracellular Ca2⫹ stores, it is possible that activation of depolarizing Ca2⫹-activated Cl⫺ currents due to Ca2⫹ release during metabolic inhibition and hypoxia may not
only cause Ca2⫹ influx through voltage-dependent
Ca2⫹ channels but may also induce more Ca2⫹ to be
released from the SR.
Based on the finding that the [Ca2⫹]i rise during
exposure of 2-deoxy-D-glucose is blocked by the SR
Ca2⫹ pump inhibitor cyclopiazonic acid in cultured
PASMCs, it has been suggested that SR Ca2⫹ release
after metabolic inhibition is mediated by IP3Rs (4).
However, our data indicate that the functional coupling of IP3Rs and RyRs to SR Ca2⫹ stores overlaps
Fig. 10. CN, unlike hypoxia, also induces
Ca2⫹ release in systemic (mesenteric) artery
smooth muscle cells. A: CN (10 mM) induced
an increase [Ca2⫹]i in a freshly isolated mesenteric artery smooth muscle cell. B: hypoxia
exposure failed to induce [Ca2⫹]i increase in a
mesenteric artery myocyte. However, application of norepinephrine (NE, 100 ␮M)
evoked a typical Ca2⫹ response in the same
cell. C: in a pulmonary artery myocyte, hypoxia induced an increase in [Ca2⫹]i.
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Fig. 9. Mitochondrial Ca2⫹ contributes to CN
Ca2⫹ release in PASMCs. A: application of
CN (10 mM) induced an increase [Ca2⫹]i in a
control myocyte (left) and a smaller increase
in [Ca2⫹]i in another cell pretreated with
CCCP (50 ␮M) for 5 min (middle) and failed
to induce any Ca2⫹ response in a third myocyte pretreated with thapsigargin (1 ␮M) and
CCCP (50 ␮M) for 5 min (right). B: graphs
summarize the effects of CCCP alone and
CCCP plus thapsigargin on CN-induced [Ca2⫹]i
increase. *P ⬍ 0.05 vs. control.
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CA2⫹ RELEASE BY METABOLIC INHIBITION WITH CYANIDE
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Ca2⫹ mobilization (3, 10). These findings, together with
the fact that CN inhibits mitochondrial cytochrome c
oxidase, suggest that CN effects may occur through
complicated signaling pathways rather than through
the direct inhibition of ATP production. Consistent
with this view, recent studies have shown that the
mitochondrial proximal electron transport chain (ETC)
inhibitors such as rotenone and myxothiazol inhibit
generation of reactive oxygen species (ROS) and block
HPV, whereas the distal ETC inhibitors antimycin A
and CN increase generation of ROS and mimic/potentiate HPV in PASMCs (2, 19, 37).
Metabolic inhibitors such as CN have been widely
used to examine hypoxic cellular responses in a variety
of cell types, including PASMCs. Indeed, metabolic
inhibition after CN exposure mimics hypoxia in many
aspects of cellular responses. For example, CN, similar
to hypoxia, induces an increase in [Ca2⫹]i in freshly
isolated PASMCs (30) and vasoconstriction in isolated
lungs, pulmonary artery strips, and PASMCs (2, 25,
26, 37). However, we found that CN exposure resulted
in Ca2⫹ release in both pulmonary and systemic (mesenteric) artery myocytes (Figs. 1 and 10A). Consistent
with our findings, a recent study has shown that hypoxia hyperpolarizes, whereas CN depolarizes, membrane potential, possibly by affecting inward Na⫹ and
outward K⫹ currents in Drosophila neurons (14). In
contrast to CN, hypoxia induces an increase [Ca2⫹]i
only in pulmonary but not systemic artery smooth
muscle cells (Fig. 10B). Similarly, hypoxic [Ca2⫹]i increase has not been seen in smooth muscle cells from
celiac, cerebral, coronary, or femoral arteries (11, 12,
28). Furthermore, hypoxia has been shown to inhibit
outward K⫹ currents in PASMCs but not in mesenteric
artery myocytes (40). Collectively, these data indicate
that metabolic inhibition with CN may produce different cellular effects from hypoxia.
It has been suggested that mitochondria are likely
implicated in O2 sensing, by which hypoxia increases
generation of ROS through the ETC, mediating HPV
(19, 37). However, other reports have shown that metabolic inhibition with CN also increases the generation
of ROS in isolated lungs and cultured PASMCs (2, 37).
In addition, our data indicate that CN, unlike hypoxia,
induces Ca2⫹ release in both pulmonary and systemic
(mesenteric) artery smooth muscle cells. Therefore,
additional experiments are needed to further verify the
view that mitochondria serve as the oxygen sensor and
ROS as the hypoxic signaling molecule in PASMCs.
In summary, the present study has demonstrated
that metabolic inhibition with a high concentration of
CN results in intracellular Ca2⫹ release and activation
of ICl(Ca) in both PASMCs and Xenopus oocytes. Exposure to a low concentration of CN induces transient
inward Cl⫺ currents and increases the frequency and
amplitude of STICs, as well as increases the amplitude
and frequency of localized Ca2⫹ release events (Ca2⫹
sparks). Ca2⫹ release after metabolic inhibition results
from both SR and mitochondria, although the former
makes a greater contribution. Both RyRs and IP3Rs
may be targets for metabolic inhibition and hypoxia.
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substantially, because stimulation of ␣-adrenergic receptors with norepinephrine failed to induce Ca2⫹ release in cells pretreated with caffeine to deplete SR
Ca2⫹ and vice versa (Fig. 3). Similar findings have been
obtained in other smooth muscle cells (1, 15, 16, 20,
35). To further address this question, we sought to use
Xenopus oocytes as a simplified model system to determine the role of IP3Rs in CN-induced Ca2⫹ release,
because Xenopus oocytes express IP3Rs but not RyRs
(24). Similar to PASMCs, exposure of Xenopus oocytes
to CN activated ICl(Ca), a commonly used assay system
for Ca2⫹ release in oocytes (Fig. 4). CN-induced Ca2⫹
release in oocytes was greatly inhibited by the prior
depletion of SR Ca2⫹ with thapsigargin and/or cyclopiazonic acid in the absence of extracellular Ca2⫹ (Fig.
5). Taken together, these results further indicate that
Ca2⫹ release after metabolic inhibition is associated
with IP3Rs, which is similar to the findings obtained in
cerebellar Purkinje cells (18). However, it is worth
noting that inhibition of IP3Rs by preinjection of heparin, a prototypical IP3R antagonist that has been
shown to block IP3-mediated Ca2⫹ release in oocytes
(31), did not affect CN-induced Ca2⫹ release (Fig. 6).
Similarly, preinjection of anti-IP3R antibody that inhibits Ca2⫹ release after stimulation of muscarinic
receptors in tracheal smooth muscle cells (21) was also
without effect on CN response. The data suggest that
Ca2⫹ release after metabolic inhibition with CN may
occur by directly affecting the channel domain of IP3R
Ca2⫹ release channels.
In the absence of extracellular Ca2⫹ influx, the depletion of SR Ca2⫹ with the SR Ca2⫹ pump inhibitors
thapsigargin and cyclopiazonic acid cannot completely
prevent Ca2⫹ release after metabolic inhibition in Xenopus oocytes (Fig. 5). This result, together with the
view that mitochondria play an important role in maintaining intracellular Ca2⫹ homeostasis under physiological and pathophysiological conditions, suggests
that mitochondrial Ca2⫹ release may be involved in the
CN-induced Ca2⫹ response. We tested this possibility
by examining whether the depletion of mitochondrial
Ca2⫹ could inhibit CN response. As shown in Fig. 7,
pretreatment of Xenopus oocytes with CCCP and oligomycin significantly blocked CN-induced Ca2⫹ release. Similarly, CCCP also blocked CN response in
pulmonary artery myocytes (Fig. 9). Moreover, after
depletion of both SR and mitochondrial Ca2⫹ with
thapsigargin and CCCP, application of CN failed to
induce Ca2⫹ release in both Xenopus oocytes (Fig. 8)
and PASMCs (Fig. 9). These experiments suggest that
mitochondrial Ca2⫹ release makes a contribution, although smaller than that of the SR, to a rise of [Ca2⫹]i
during metabolic inhibition.
CCCP collapses mitochondrial membrane potential,
and oligomycin inhibits mitochondrial ATP synthesis
by direct blockade of the F0F1 ATPase. Despite uncoupling and inhibition of phosphorylation, Ca2⫹ release
by CN was only seen partially inhibited. Similar observations have been made in dorsal root ganglia and
adrenal chromaffin cells, in which, in the presence of
CCCP and oligomycin, CN is still able to cause cytosolic
CA2⫹ RELEASE BY METABOLIC INHIBITION WITH CYANIDE
Unlike hypoxia, CN also induces Ca2⫹ release in systemic (mesenteric) artery myocytes.
19.
This work was supported by American Heart Association, the
Pennsylvania Thoracic Association, and National Heart, Lung, and
Blood Institute Grants R01-HL-64043 (to Y.-X. Wang) and R01-HL45239 (to M. I. Kotlikoff).
20.
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