1 Center for Cardiovascular Sciences, Albany Medical College, Albany 12208; and 2 Department of Biomedical Sciences, Cornell University, Ithaca, New York 14853
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ABSTRACT |
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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
1,4,5-trisphosphate 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.
sarcoplasmic reticulum; mitochondria
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INTRODUCTION |
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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-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.
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METHODS |
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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 µM 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
nystatin-perforated 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.
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 µM 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 µM) (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 140 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 (anti-IP3R 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 one-way ANOVA was used for multiple comparisons. P < 0.05 was accepted as the level of statistical significance.
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RESULTS |
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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 voltage clamped
at 55 mV using the perforated patch-clamp technique. The
intracellular solution contained Cs+ ions in place of
K+ ions to block outward potassium currents, and myocytes
were preexposed to nisoldipine (5 µM) 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).
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CN-induced Ca2+ release may occur through ryanodine receptors in pulmonary artery myocytes. The CN-induced [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 µM) for 30 min, application of CN failed to induce either an increase in [Ca2+]i or ICl(Ca). We obtained similar results in four 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 voltage-clamped 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
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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 µM)
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 µM) was no longer able to
induce 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.
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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 µM) for 4 h almost completely blocked CN-induced currents in 6 cells tested.
Replacement of 92% external NaCl with NaI or Na-isethionate
(n = 6) shifted the reversal potential, whereas similar
replacement with NMGCl or Tris · 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).
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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 µM) for 10 min than in control oocytes. To ensure the complete depletion of
mitochondrial Ca2+, oocytes were exposed to CCCP and
oligomycin (10 µM 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 µA in control
oocytes (n = 6), 1.46 ± 0.21 µA in
CCCP-pretreated oocytes (n = 6), and 1.48 ± 0.13 µA in CCCP/oligomycin-pretreated oocytes (n = 7).
Thus mitochondrial Ca2+ release likely contributes to
CN-induced intracellular Ca2+ rise.
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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 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, coronary, 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+ release is
unique to PASMCs but not systemic artery myocytes.
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DISCUSSION |
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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 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+ 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. Unlike hypoxia, CN also
induces Ca2+ release in systemic (mesenteric) artery myocytes.
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ACKNOWLEDGEMENTS |
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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-HL-45239 (to M. I. Kotlikoff).
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FOOTNOTES |
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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: wangy{at}mail.amc.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.
First published October 9, 2002;10.1152/ajpcell.00260.2002
Received 4 June 2002; accepted in final form 30 September 2002.
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