© 1999 American Heart Association, Inc.
Original Contribution |
Mechanism of Nitric Oxide–Induced Vasodilatation
Refilling of Intracellular Stores by Sarcoplasmic Reticulum Ca2+ ATPase and Inhibition of Store-Operated Ca2+ Influx
From the Vascular Biology Unit, Whitaker Cardiovascular Institute, Evans Department of Clinical Research, Department of Medicine, Boston University Medical Center, Boston, Mass.
Correspondence to Richard A. Cohen, MD, Director, Vascular Biology Unit, R408, Boston University School of Medicine, 80 E Concord St, Boston, MA 02118. E-mail racohen{at}med-med1.bu.edu
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Abstract |
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Abstract—The precise mechanisms by which nitric oxide (NO) decreases free [Ca2+]i, inhibits Ca2+ influx, and relaxes vascular smooth muscle are poorly understood. In rabbit and mouse aorta, agonist-induced contractions and increases in [Ca2+]i were resistant to nifedipine, suggesting Ca2+ entry through non–L-type Ca2+ channels. Relaxations to NO were inhibited by thapsigargin (TG) or cyclopiazonic acid (CPA) indicating the involvement of sarcoplasmic reticulum ATPase (SERCA). Studies of the effect of NO on [Ca2+]i and the rate of Mn2+ influx with fura-2 fluorometry in rabbit aortic smooth muscle cells in primary culture were designed to test how SERCA is involved in mediating the response to NO. When cells were stimulated with angiotensin II (AII), NO accelerated the removal of Ca2+ from the cytoplasm, decreased [Ca2+]i, and inhibited Ca2+ and Mn2+ influx. Inhibition of SERCA abolished all the effects of NO. In contrast, inhibition of the Na+/Ca2+exchanger or the plasma membrane Ca2+ ATPase had no influence on the ability of NO to decrease [Ca2+]i. NO maximally decreased [Ca2+]i within 5 s, whereas significant inhibition of AII-induced Ca2+ and Mn2+ influx required more than 15 s. The inhibition of cation influx strictly depended on [Ca2+]o and functional SERCA, suggesting that during the delay before NO inhibits Ca2+ influx, the influx of Ca2+ and the uptake into intracellular stores are required. In the absence of [Ca2+]o, NO diminished the AII-induced [Ca2+]i transient by a SERCA-dependent mechanism and increased the amount of Ca2+ in the stores subsequently released by ionomycin. The present study indicates that the initial rapid decrease in [Ca2+]i caused by NO in vascular smooth muscle is accounted for by the uptake of Ca2+ by SERCA into intracellular stores. It is proposed that the refilling of the stores inhibits store-operated Ca2+ influx through non–L-type Ca2+ conducting ion channels and that this maintains the decrease in [Ca2+]i and NO-induced relaxation.
Key Words: nitric oxide • Ca2+ ATPase • Ca2+ • Ca2+ stores • vascular smooth muscle
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Introduction |
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The endothelial cell production of nitric oxide (NO) accounts for endothelium-dependent vasodilatation.1 Despite a growing understanding of the mechanisms by which NO is released from the endothelium, the precise molecular mechanisms by which NO relaxes vascular smooth muscle are not fully understood. A variety of mechanisms have been proposed by which NO participates in the regulation of smooth muscle free [Ca2+]i, which is the primary determinant of contractile tone.2 Among them, several different mechanisms for NO-induced inhibition of Ca2+ influx through L-type Ca2+ channels have been proposed, including their inhibition by cGMP-dependent mechanisms3 or by membrane hyperpolarization due to direct4 or indirect cGMP-dependent5 6 activation of Ca2+-dependent K+ channels. NO also has been proposed to reduce [Ca2+]i by inhibiting the agonist-induced release of Ca2+ from intracellular stores7 or by increasing Ca2+ removal from the cytoplasm by accelerating the plasma membrane Ca2+ ATPase8 or the Na+/Ca2+ exchanger.9 The sarcoplasmic(endo)plasmic reticulum Ca2+ ATPase (SERCA), which may be accelerated by the NO-induced rise in cGMP,10 is also thought to be involved.11 Thus, several potential Ca2+ regulatory targets have been proposed for NO in vascular smooth muscle, but their relative importance in decreasing [Ca2+]i and their individual contribution to smooth muscle relaxation is still unclear.
Supporting previous observations,12 the present study indicates that agonist-induced increases in [Ca2+]i and contractions of rabbit and mouse aortic smooth muscle cells are highly resistant to inhibitors of L-type Ca2+ channels, indicating that Ca2+ influx through different channels can mediate the responses. In a variety of cell types, including smooth muscle, Ca2+ entry is thought to be initiated by agonist-induced Ca2+ release and depletion of [Ca2+]i stores.13 Activation of store-operated Ca2+ entry is known to be triggered not only by receptor-dependent agonists but also by store depletion that occurs after inhibiting Ca2+ uptake into the stores by thapsigargin (TG) or cyclopiazonic acid (CPA).13 14 15
Increases in [Ca2+]i and contractions stimulated by agonists including phenylephrine, angiotensin II (AII), or 5-hydroxytryptamine (5-HT) are shown in the present study to be highly sensitive to NO, but those caused by inhibitors of SERCA are demonstrated to be resistant to NO. This important finding led us to further investigate the effect of NO on [Ca2+]i stores and store-operated Ca2+ entry. Our results suggest a simple model whereby NO initiates its response by accelerating sequestration of Ca2+ into intracellular stores via SERCA, thereby rapidly decreasing [Ca2+]i. The resulting refilling of Ca2+ stores would indirectly inhibit store-operated Ca2+ influx, thereby maintaining the decrease in [Ca2+]i and mediating smooth muscle cell relaxation. This coordinated effect on [Ca2+]i homeostasis represents a novel mechanism for NO-induced vasodilatation.
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Materials and Methods |
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Relaxation of Intact Rabbit and Mouse Aorta
Rings of New Zealand White rabbit descending thoracic aorta were cleaned of adherent fat and connective tissue and were suspended in organ chambers filled with physiological salt solution (PSS) for tension measurements as previously described.16 Rings (3 mm long) of the thoracic aorta of mice (8 to 11 weeks of age) were suspended on horizontal wire myographs (Kent Scientific). After equilibration at the optimal resting tension (mouse aorta, 1.5 g; rabbit aorta, 6 g), the smooth muscle was contracted with phenylephrine hydrochloride, and after achieving a stable contraction, NO was added in increasing concentrations. When needed, the contractile tone caused by SERCA inhibitors was supplemented with phenylephrine to match the tone achieved by phenylephrine in the absence of the inhibitors.
[Ca2+]i Measurement in Intact Mouse
Aorta
The mouse aorta was slid over a Teflon filament to fix it and to
destroy the endothelial layer. In a Petri dish
containing ice-cold PSS (in mmol/L: NaCl 140, KCl 2.8,
MgCl2 1, CaCl2 2, glucose
11, and HEPES 10 [pH 7.4]), the aorta was cleaned of fat and
connective tissue and cut into 2-mm-long rings. Each ring was
transferred into an experimental chamber (
TC3 dish, Bioptechs, Inc)
between 2 small plaques of neutral silicon grease. Here the ring was
cut open, fixed by a piece of woven nylon mesh (Spectra/Mesh nylon,
Spectrum) and stored in filtered PSS solution at 4°C until use, but
not longer than 4 hours. Fura-2 loading of the strip was done in
10-5 mol/L fura-2 AM in PSS with 0.02% Pluronic
F-127 (Molecular Probes) for 1 hour at 37°C. Strips were then washed
in prewarmed PSS for 15 minutes at 37°C, and the chamber was mounted
into a TC3 temperature control system (Bioptechs, Inc) on the stage
of the microscope (Olympus, x20 fluorescence objective). The
solution was warmed to 37°C. The microscope was focused on the
elastin and collagen structure of the tissue beneath the nylon mesh. NO
was applied as a 50-µL drop of 1 µmol/L solution close to the
aortic strip. At the end of each measurement, digitonin and
MnCl2 were applied in several steps to avoid
contraction and dislocation of the strip, reaching final concentrations
of 10-4 mol/L and 2x10-2
mol/L, respectively. Fluorescence background was automatically
subtracted. Because of the well-known difficulties in calibrating
fura-2 in intact tissues and obtaining precise values,
[Ca2+]i changes are
reported as ratios after background subtraction.
Cell Culture
The thoracic aorta was removed from New Zealand White rabbits
and cleaned of adherent fat and connective tissue. Strips of rabbit
thoracic aorta were aseptically removed from the media, dispersed with
collagenase (4 mg/mL) and elastase (1 mg/mL), and grown
in medium M199 with 20% heat-inactivated FBS as previously
described.17 Cells were seeded onto 9x22 mm No. 1
glass coverslips for
[Ca2+]i studies. On
reaching confluence, cells were growth-arrested by placing them in M199
with 1% FBS for 3 to 4 days before the experiments. All experiments
were done on primary cultures that stain uniformly for smooth muscle
-actin.
[Ca2+]i Measurement in Cultured
Smooth Muscle
As described previously,17 cells in culture on
coverslips were loaded for 45 minutes with fura-2 AM (5 µmol/L)
in PSS at 37°C and washed for 15 minutes to allow complete ester
cleavage. The PSS was of the following composition (mmol/L): NaCl 119,
NaHEPES 20, KCl 4.6, CaCl2 1.2,
MgSO4 1.0,
Na2HPO4 0.15,
KH2PO4 0.4, and
NaHCO3 5.0, supplemented with 0.1%
albumin. Nominally Ca2+ free PSS was
composed of the same solution without CaCl2 or
EGTA added. Fura-2 fluorescence was measured in a
photomultiplier-based fluorometer at 37°C or in an imaging system at
22°C to 24°C (Figure 3
) (both from IonOptix Inc). Sampling
rate for the ratio of 510-nm emitted light from excitation at 340 and
380 nm was 2 Hz (photomultiplier system) or 5 Hz (imaging system).
[Ca2+]i in cultured cells
was estimated by previously published methods,17 and the
concentration is reported as nanomoles per liter (nmol/L).
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Measurement of Divalent Cation Influx Rate
Mn2+-induced quenching of fura-2
fluorescence was used to estimate rates of divalent cation
influx.17 18 Fluorescence of fura-2–loaded cells
was measured at the isosbestic point wavelength of 360 nm. Data are
expressed as the Mn2+ influx rates, which were
calculated from the slope of the linear decline in fura-2
fluorescence during the 30 s immediately after addition of
MnCl2 (100 µmol/L), and were normalized to
the level of fluorescence at the time of
Mn2+ addition. When the rate of
Mn2+ influx in the presence and absence of
[Ca2+]o was compared, the
rates were normalized to that measured under basal conditions.
Measurement of Inositol 1,4,5-Trisphosphate
Rabbit aortic smooth muscle cells were grown to confluence
in 35-mm culture dishes and made quiescent by reducing the supplemental
serum to 1% during the last 4 days before the experiment. Quiescent
cells were incubated for 1 hour in serum-free PSS and then stimulated
with 0.1 µmol/L AII for 15 s to 70 s. The
extracellular solution was removed, and 250 µL of cold 1 mol/L
trichloroacetic acid was added. The lysate was collected and
centrifuged for 1 minute at 12 000g at 4°C. The
trichloroacetic acid was extracted with a 3:1 solution of
1,1,2-trichloro-1,2,2-trifluoroethane-triotylamine, and the
remaining inositol 1,4,5-trisphosphate (IP3) was
quantified according to the methodology provided with the
IP3 (3H) radioreceptor
assay kit (Dupont NEN).
Materials
NO gas was obtained from Matheson. Saturated NO solution was
prepared at 4°C and diluted in deoxygenated sealed tubes
of aqueous solution as described.17 Responses of intact
rings and cultured cells to NO (10-10 to
10-6 mol/L) were entirely prevented by
deoxyhemoglobin (10-5 mol/L). Fura-2 AM was from
Molecular Probes. All other chemicals were from Sigma Chemical
Co.
Statistical Analysis
All data are expressed as mean±SE. Evaluation of statistics was
done by Student t test. Values of P<0.05
indicated significant differences and are designated in the figures by
an asterisk.
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Results |
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Effect of Nifedipine and Inhibitors of SERCA on NO-induced Relaxations and Decreases in [Ca2+]i in Intact Rabbit and Mouse Aorta
The role of L-type Ca2+ channels in agonist-induced contractions and NO-induced relaxations of rings of rabbit thoracic aorta denuded of endothelium was determined. Figure 1A
(left) shows that
phenylephrine (10-8 to
3x10-6 mol/L) caused concentration-dependent
contractions, and NO (10-9 to
10-5 mol/L) caused concentration-dependent
relaxations (Figure 1A, right), neither of which was
significantly affected by pretreatment of the aorta with
nifedipine (10-6 mol/L, 30 minutes;
n=5 to 6, P>0.5). This concentration of
nifedipine abolished contractions to potassium chloride
(30 mmol/L, not shown).
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7 g at a lower concentration of the
agonist, the values shown in the rectangle represent the
mean±SEM of the final tension before administration of NO regardless
of the concentration. Nifedipine also had no significant
effect on NO-induced relaxation (A, right). TG or CPA had no
consistent direct effect on tone in resting aortic rings, and
similar concentrations of phenylephrine were required to
contract the rings to equivalent levels of tone. In the absence or
presence of TG or CPA, respectively, the concentration of
phenylephrine (-log mol/L: 7.2±0.5, 7.5±0.6, and
7.2±0.7) and the contraction produced (g: 7.5±1.1, 7.4±0.7, and
6.2±0.9) were not significantly different. There was a significant
inhibition of the NO-induced relaxation caused by TG
(P<0.05) or CPA (P<0.05), designated by
asterisks.
The role of calcium uptake into intracellular stores in the
relaxations to NO was determined in rabbit aortic rings pretreated with
the SERCA inhibitors TG and CPA (Figure 1B
). TG
(10-4 mol/L) or CPA
(2x10-4 mol/L) caused small, transient, and
inconsistent contractions of resting rabbit thoracic aortic
rings denuded of endothelium. The concentration of
phenylephrine required to contract the rings in the
presence of TG or CPA was unchanged (Figure 1B). Relaxations
caused by NO (10-10 to
10-5 mol/L) were significantly inhibited by both
TG and CPA (P<0.05, n=5; Figure 1B).
In rings of mouse aorta contracted with phenylephrine, NO
caused rapid, transient, and concentration-dependent relaxations
(Figure 2A). In the mouse aorta, TG
consistently caused slowly developing contractions, whereas CPA
caused small and variable contractions (Figure 2B and 2C).
The tone induced in the presence of the inhibitors of
SERCA, with or without supplemental phenylephrine, was not
significantly affected by NO (Figure 2B through 2D).
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To determine whether in the above experiments contraction and
relaxation reflected changes in
[Ca2+]i, intact mouse
aortic strips were loaded with fura-2, and the effects of NO on
agonist- and TG-induced increases in
[Ca2+]i were measured.
5-HT, used in these experiments because it was the most potent agonist,
caused a rapid transient increase in
[Ca2+]i followed by a
plateau (Figure 2E). NO significantly decreased the plateau
level from a ratio of 0.045±0.011 to 0.014±0.010 (P<0.02,
n=9). Treatment of the mouse aorta with nifedipine 10
µmol/L did not significantly affect either the plateau (0.038±0.016)
or the decrease caused by NO (0.016±0.009, n=6). The NO-induced
decrease in [Ca2+]i was
86±26% in the absence and 64±14% in the presence of
nifedipine (P>0.1). TG caused a slow increase
in [Ca2+]i to a plateau
value of 0.104±0.016, and NO did not significantly affect this
response (0.099±0.013, P>0.1, n=4; Figure 2F).
SERCA Inhibitors Prevent the Decrease in
[Ca2+]i Caused by NO in Rabbit Aortic Smooth
Muscle Cells in Culture
The experiments discussed above showing that TG or CPA inhibited
decreases in [Ca2+]i and
relaxations to NO in intact aortic tissue suggest that calcium uptake
into intracellular stores by SERCA is involved in mediating the
response to NO. Therefore, additional experiments of the effect of TG
and CPA on [Ca2+]i
regulation by NO were performed in fura-2–loaded rabbit aortic smooth
muscle cells in primary culture. In the presence of
[Ca2+]o (1.2
mmol/L), TG (100 nmol/L) caused a rise in
[Ca2+]i similar to that
observed in intact mouse aorta. NO (10-6 mol/L)
had no effect on the TG-induced increase in
[Ca2+]i when it was
applied after [Ca2+]i
reached maximum level (Figure 3A).
If NO was added 1 minute before TG, the rise in
[Ca2+]i caused by the
SERCA inhibitor was slowed but reached a similar maximum
level (Figure 3B). If NO was added 1 minute after addition of
TG, it caused a small transient decrease in
[Ca2+]i (Figure 3C). Thus, in the presence of TG, there is a time-dependent
disappearance of the response to NO.
The effect of NO was studied further on AII-induced increases in
[Ca2+]i. In these
experiments, AII was used because, similar to phenylephrine
and 5-HT, it activates IP3-induced
Ca2+ release and influx, and it gave the most
consistent response in the smooth muscle cells in culture. The
AII-induced elevation of
[Ca2+]i was separated
into the transient rise due to the release of
Ca2+ from intracellular stores (shown to occur in
the nominal absence of
[Ca2+]o) and the
sustained elevation in
[Ca2+]i after the
addition and influx of
[Ca2+]o (Figure 4A). Similar to
phenylephrine-induced contractions and 5-HT induced
increases in [Ca2+]i in
the intact rabbit and mouse aorta, the Ca2+
influx–induced sustained elevation of
[Ca2+]i was insensitive
to nifedipine (5 µmol/L) but was sensitive to NO
(10-10 to 10-9 mol/L), as
well as to blocking Ca2+ influx by
Ni2+ (Figure 4A).
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NO (10-6 mol/L) applied during the
elevation of [Ca2+]i
caused by AII-induced Ca2+ influx (Figure 4B) produced a very rapid and sustained decrease in
[Ca2+]i. Because the
response to NO could result not only from inhibition of
Ca2+ influx but also from acceleration of
Ca2+ extrusion from the cytoplasm via the
Na+/Ca2+ exchanger and the
plasma membrane Ca2+ ATPase (PMCA) or by
sequestration of Ca2+ into the stores by SERCA,
the effects of inhibition of these systems were determined. Inhibition
of the Na+/Ca2+ exchanger
(by replacing extracellular Na+ with choline)
and/or PMCA (by elevating pHo to 8.8 and
Mg2+ concentration to 20
mmol/L8 ) increased the sustained elevation of
[Ca2+]i but did not
affect the NO-induced decrease in
[Ca2+]i (Figure 4C; Table). In contrast, a similar
Ca2+ elevation evoked by inhibition of SERCA with
TG or CPA (Figure 4D; Table) was completely insensitive
to NO (1 µmol/L). Blocking Ca2+ influx
with Ni2+ decreased
[Ca2+]i when either TG
(Figure 1D) or PMCA and
Na+/Ca2+ exchanger (Figure 1C) were inhibited (Table). However, when SERCA, PMCA,
and Na+/Ca2+ exchanger were
all inhibited (Figure 1E), blocking Ca2+
influx with Ni2+ was not followed by a decrease
in [Ca2+]i. This
indicates that these 3 mechanisms are together responsible for
eliminating Ca2+ from the cytoplasm and that they
were effectively inhibited. These studies indicate that SERCA activity,
but not that of the
Na+/Ca2+ exchanger or PMCA,
is required for NO to decrease
[Ca2+]i.
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NO-Induced Inhibition of Ca2+ and Mn2+
Influx Requires SERCA Activity and
[Ca2+]o
After stimulation with AII in the absence of
[Ca2+]o, treatment of
cells with NO (1 µmol/L) significantly inhibited the rise in
[Ca2+]i caused by the
addition of [Ca2+]o
(Figure 5A). The initial rise in
[Ca2+]i started similarly
in the absence or presence of NO, but in the presence of NO,
[Ca2+]i stopped
increasing and reached a stable plateau of 214±60 nmol/L after 21±4 s
(n=3), compared with a peak of 560±89 nmol/L (P<0.05, n=3)
reached in 18±2 s in the absence of NO. In contrast, NO did not affect
the rise in [Ca2+]i
caused by addition of Ca2+ in the presence of TG
(Figure 5B; 551±20 and 531±28 nmol/L, n=3, with or without NO,
respectively). These studies suggest that NO inhibits AII- but not
TG-induced Ca2+ influx.
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To study cation influx while excluding the influence of
mechanisms that remove Ca2+ from the cytoplasm,
AII-induced Mn2+ influx was measured. Figure 5C shows that AII increases the rate at which
Mn2+ quenched fura-2 fluorescence. NO
(1 µmol/L), applied 1 minute before Mn2+,
significantly inhibited cation influx caused by AII. Interestingly, in
the nominal absence of
[Ca2+]o, NO had no effect
(Figure 5E). TG (100 nmol/L) increased
Mn2+ influx to similar levels as AII, but NO had
no effect on Mn2+ influx in either the presence
or absence of [Ca2+]o
(Figure 5D and 5F).
Effect of NO on [Ca2+]i Precedes Its
Effect on Cation Influx
Having investigated the effects of NO on cation influx,
studies were conducted to determine the temporal relationship between
the inhibition of cation influx and the decrease in
[Ca2+]i caused by NO. The
effect of NO was compared with that of Ni2+,
which is known to directly block cation influx. When
[Ca2+]i was increased by
AII, NO (1 µmol/L) decreased
[Ca2+]i more rapidly than
did Ni2+ (Figure 6A and 6B). The exponential half-time, 
, was significantly shorter with
NO than with Ni2+ (1.4±0.1 versus 3.1±0.2 s,
respectively; P<0.001, n=4). In contrast, when
Mn2+ influx was measured in cells stimulated with
AII, the initial rate of cation influx was inhibited faster by
Ni2+ than by NO (Figure 6C and 6D). When
added simultaneously with, or at any time before the
addition of Mn2+, Ni2+
fully and immediately blocked AII-induced Mn2+
influx (Figure 6D), consistent with its blocking
cation-conducting channels directly. In contrast, when NO was added
simultaneously with Mn2+, the cation
influx (estimated over the next 30 s) was not significantly
different from that obtained with AII alone (Figure 6C). Only if
NO was added >15 s before the addition of Mn2+
was there sufficient time for NO to inhibit AII-induced
Mn2+ influx. This delay in the action of NO is
similar to that needed for inhibition of Ca2+
influx, observed in Figure 5A, and suggests why after addition
of [Ca2+]o,
[Ca2+]i begins to rise
normally, despite treatment with NO. These results indicate that a
considerable delay occurs before NO inhibits the influx of
extracellular cations and that the decrease in
[Ca2+]i caused by NO
occurs more rapidly than can be accounted for by a direct inhibition of
cation channels. This finding is consistent with the initial
rapid decrease in [Ca2+]i
by NO being primarily due to stimulation of
[Ca2+]i removal
mechanisms.
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NO Accelerates the Removal of Ca2+ From the Cytoplasm
via SERCA and Promotes Refilling of
[Ca2+]i Stores
Because the intracellular action of NO precedes its effect
on Ca2+ influx, the effect of NO on the rate of
removal of Ca2+ from the cytoplasm was further
studied. When AII-induced Ca2+ influx was stopped
by rapidly eliminating Ca2+ from the
extracellular solution,
[Ca2+]i decreased with a
half-time for exponential decay, , of 21±4.6 s (n=5; Figure 7A, left). The NO donor
S-nitroso-N-acetylpenicillamine (SNAP,
10-4 mol/L) added at the same time that
[Ca2+]o was eliminated
significantly increased the rate at which
[Ca2+]i declined,
indicating that NO accelerates the removal of
Ca2+ from the cytoplasm (=8.1±1.1 s, n=5,
P<0.04; Figure 7A, right). When
Ca2+ was readmitted to the extracellular solution
in the presence of SNAP,
[Ca2+]i initially rose
rapidly and then after a delay of 10 s to 15 s, reached a new
plateau that was lower than in the absence of SNAP (Figure 7A, right).
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Ca2+ influx stimulated by TG elevated
[Ca2+]i to a level
similar to that caused by AII. Elimination of
[Ca2+]o resulted in a
decline in [Ca2+]i at a
rate that was not significantly different from that observed in the
presence of AII (=27±1.5 s, n=4, P>0.3; Figure 7B, left). This suggests that in the absence of
Ca2+ influx,
[Ca2+]i removal
mechanisms other than SERCA are primarily responsible for decreasing
[Ca2+]i in the presence
of either AII or TG. However, inhibition of SERCA with TG completely
prevented the increase in the rate of
[Ca2+]i removal caused by
SNAP (=27±2.6 s, P>0.7; Figure 7B, right). This
result supports the concept that NO rapidly decreases
[Ca2+]i in the presence
of AII primarily by accelerating the removal of cytosolic
Ca2+ via SERCA.
To determine if NO accelerates the removal of cytoplasmic
Ca2+ by increasing its uptake into intracellular
stores, the effect of NO was determined on the
Ca2+ content of the stores. Ionomycin (10
µmol/L) alone or in combination with AII (0.1 µmol/L) was used
to release Ca2+ from the stores in the nominal
absence of [Ca2+]o.
Ionomycin alone caused a peak increase in
[Ca2+]i from a basal
value of 92±9 to 835±72 nmol/L (Figure 8A). Application of NO (1 µmol/L)
4 minutes before had no significant effect on the subsequent response
to ionomycin (-7±9% compared to without NO, not shown in figure).
AII (0.1 µmol/L) caused a peak transient increase in
[Ca2+]i to 591±33 nmol/L
and significantly reduced the release caused by the subsequent
application of ionomycin (442±22 nmol/L, n=6, P<0.003;
Figure 8A). When NO (1 µmol/L) was given 1 minute before
AII, the [Ca2+]i
transient caused by AII was significantly reduced (434±40 nmol/L,
P<0.007), whereas the subsequent ionomycin-induced release
was increased by 42±7% compared with that in the absence of NO
(631±47 nmol/L, P<0.003; Figure 8B). Even if NO was
added 20 s after AII (after the peak increase in
[Ca2+]i caused by the
agonist), the ionomycin-induced release was 26±9% greater than in the
absence of NO (564±63, P<0.04; Figure 8C),
consistent with an NO-induced augmentation of the refilling of
Ca2+ stores.
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Inhibition of the peak increase in
[Ca2+]i caused by AII
could be achieved by either inhibition of Ca2+
release from the stores7 19 or by accelerated
sequestration of Ca2+ into the stores. The role
of uptake by SERCA was determined by releasing
Ca2+ from the stores with AII in the absence
or presence of TG. In the absence of
[Ca2+]o, NO (1
µmol/L), added before AII, inhibited the transient increase in
[Ca2+]i caused by AII by
44±7% (Figure 9A and 9B). The peak
increase in [Ca2+]i was
528±57 nmol/L compared with 319±52 nmol/L in the presence of NO. Two
minutes after treatment with TG, AII caused a transient
Ca2+ increase to a level that was not
significantly different from that in the absence of TG (Figure 9C and 9D). However, NO failed to inhibit the AII response when
SERCA was inhibited by TG (Figure 9C and 9D). Similarly, NO
inhibited the Ca2+ influx-induced rise in
[Ca2+]i caused by
releasing the stores with AII but not with AII and TG. This suggests
that NO inhibits the peak increase in
[Ca2+]i caused by AII
primarily by accelerating the sequestration of
Ca2+ into the stores via SERCA, rather than by
interfering with IP3-induced
Ca2+ release.
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NO Does Not Affect IP3 Production Caused
by AII
The effect of NO on AII-induced IP3
production was measured by radioimmunoassay. Basal levels of
IP3 were 0.9±0.1 pmol/plate. During the first
45 s after AII (0.1 µmol/L), IP3
levels were significantly increased (3.1±0.5, 3.1±0.7, and 3.1±0.5
pmol/plate at 15, 30, and 45 s after AII, respectively, n=5) but
were not significantly different after 1 minute of treatment with NO
(3.4±0.6, 2.7±0.4, 2.2±0.6 pmol/plate, respectively). Furthermore,
IP3 levels 60 s and 70 s after AII were
2.3±0.4 and 2.1±0.3 pmol/plate, respectively. When NO was
administered at 60 s after AII, the level of
IP3 10 s later was not significantly
decreased compared with that in the absence of NO (2.0±0.4
pmol/plate). Thus, changes in AII-induced IP3
levels do not explain the effect of NO on
[Ca2+]i.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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According to the capacitative Ca2+ entry model, Ca2+ influx can be activated not only by emptying the stores with agonists but also by depletion of the stores after inhibition of SERCA with TG or CPA.13 14 15 An important role of SERCA in the physiological response to NO in agonist-stimulated smooth muscle cells is suggested by the present study in which these 2 structurally different, highly specific inhibitors of SERCA20 21 interfered with the actions of NO. The present study also demonstrates that the initial action of NO in agonist-stimulated smooth muscle cells is to rapidly sequester [Ca2+]i via SERCA and secondarily to inhibit Ca2+ influx. This temporal sequence suggests the proposal that NO regulates agonist-induced Ca2+ influx by a 2-step process: (1) NO initiates a rapid decrease in [Ca2+]i by sequestering cytoplasmic Ca2+ into intracellular stores via SERCA, and (2) the refilling of intracellular stores inhibits store-operated Ca2+ entry, which in turn maintains the decrease in [Ca2+]i and relaxation of vascular smooth muscle.
Several pieces of evidence support sequestration of
[Ca2+]i by SERCA as the
primary mechanism by which NO initiates the decrease in
[Ca2+]i. First, NO causes
[Ca2+]i to fall more
rapidly than can be explained by either directly inhibiting
Ca2+ influx with Ni2+
(Figure 6) or by removing
[Ca2+]o (Figure 7). Second, NO does not decrease
[Ca2+]i when SERCA is
inhibited by TG or CPA (Figures 2 through 4;
Table). The ability of the inhibitors of SERCA to
prevent the response to NO is time-dependent (Figure 3),
presumably indicating that the inhibitors require
sufficient time to completely block uptake of
Ca2+ into the stores. Third, studies in which
PMCA or Na+/Ca2+ exchanger
was inhibited (Figure 4) indicate that although these mechanisms
actively remove [Ca2+]i
from the cytoplasm when it is elevated by either AII or TG, they are
not essential for NO to decrease
[Ca2+]i.
The ability of NO to increase the content of
[Ca2+]i stores was made
evident by the increased size of the response to ionomycin after NO was
administered before the partial emptying of the stores caused by AII
(Figure 8). Ionomycin, at the concentrations used, is known to
permeabilize membranes to Ca2+,
to release all Ca2+ from stores, and to make
Ca2+ pumps and transporters
ineffective.22 In the absence of
[Ca2+]o, an increased
amount of Ca2+ released by ionomycin in the
presence of NO can only be explained by increased uptake from the
cytoplasm or decreased release from the stores. NO increased the
ionomycin-releasable store even when NO was administered after
Ca2+ release induced by AII was nearly complete,
suggesting that NO increased reuptake, rather than inhibited the
IP3-mediated release of
Ca2+ from the stores. Indeed, the effect of NO on
the AII-induced transient increase in
[Ca2+]i in the absence of
[Ca2+]o was entirely
prevented by pretreatment with TG (Figure 9). The latter
experiment suggests that NO has little or no direct effect on the
production of, or response to, IP3
initiated by AII in these cells. This finding is also supported by the
lack of an effect of NO on AII-stimulated IP3
levels. Thus, our results indicate that NO initiates a decrease in
[Ca2+]i primarily as a
result of its sequestration into intracellular stores by SERCA.
The hypothesis that the inhibition of Ca2+
influx by NO is secondary to its initial effects on sequestration of
Ca2+ into the stores by SERCA is based on several
observations. The inhibition by NO of AII-induced
Mn2+ influx was strictly dependent on
[Ca2+]o (Figure 5). This result suggests the apparent paradox that
Ca2+ influx is required for NO to inhibit
divalent cation influx. Indeed, when
[Ca2+]o was added after
AII in the presence of NO (Figures 5, 7, and 9),
[Ca2+]i initially rose
rapidly but then equilibrated at a lower level compared with control.
This requirement for Ca2+ influx for NO to
inhibit Ca2+ influx is also evident from the
delay observed in the NO-induced inhibition of
Mn2+ influx (Figure 6). Together with the
lack of effects of NO when SERCA is inhibited, these experiments
support a requirement for SERCA-dependent uptake of
Ca2+ into the stores for divalent cation influx
to be inhibited by NO.
Thus, our results can best be explained by proposing that NO indirectly inhibits capacitative or store-operated, agonist-activated Ca2+ influx in vascular smooth muscle cells by increasing Ca2+ in the stores. The observation that inhibitors of SERCA can prevent the actions of NO on [Ca2+]i was made previously in studies on platelets23 and HEK 293 cells,24 but the finding was interpreted as meaning that NO is not capable of regulating capacitative Ca2+ influx. However, the present findings suggest that functional SERCA is an essential requirement for NO to regulate store-operated Ca2+ influx. Although we have not measured a direct effect of NO on SERCA itself, others have shown that nitrovasodilators stimulate the ATPase activity of SERCA by a mechanism dependent on cGMP and protein kinase G.10 Furthermore, cGMP-dependent phosphorylation of phospholamban is induced by NO in vascular smooth muscle.10 25 Dissociation of phosphorylated phospholamban from SERCA would provide a molecular mechanism for NO to regulate SERCA activity and thus to regulate store-operated Ca2+ influx. cGMP-independent effects of NO on SERCA via redox-sensitive thiols are potentially possible, similar to those described recently for K+ channels4 and the ryanodine receptor.26 Indeed, the effects of NO on vascular smooth muscle [Ca2+]i and relaxation persist despite effective inhibition of cGMP and protein kinase G.27
Ca2+ entry in vascular smooth muscle is known to occur via 2 distinct pathways, only one of which is sensitive to inhibitors of L-type Ca2+channels.12 In the present study, nifedipine had little effect on agonist-induced increases in [Ca2+]i and contractions of rabbit or mouse aortic smooth muscle, indicating that non–L-type Ca2+ channels are responsible for the majority of Ca2+ entry in these conduit arteries. Thus, the mechanism proposed in the present study can explain how NO regulates the portion of Ca2+ influx and contractions induced by agonists and growth factors that occurs independently of L-type Ca2+ channels. The voltage-dependent activation of smooth muscle L-type Ca2+ channels also can be regulated by NO either directly3 or indirectly by hyperpolarization mediated by K+ channels.4 5 6 Thus, the proposal that NO regulates agonist-activated, store-operated Ca2+ influx widens the potential mechanisms by which NO causes vasodilatation.
|
Acknowledgments |
|---|
This study was supported by NIH grants HL31607, HL54150, and HL55620.
Received September 25, 1998; accepted November 3, 1998.
|
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B. Thyagarajan, M. Poteser, C. Romanin, H. Kahr, M. X. Zhu, and K. Groschner Expression of Trp3 Determines Sensitivity of Capacitative Ca2+ Entry to Nitric Oxide and Mitochondrial Ca2+ Handling. EVIDENCE FOR A ROLE OF Trp3 AS A SUBUNIT OF CAPACITATIVE Ca2+ ENTRY CHANNELS J. Biol. Chem., December 14, 2001; 276(51): 48149 - 48158. [Abstract] [Full Text] [PDF]
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Q. Zhang, B. Molino, L. Yan, T. Haim, Y. Vaks, P. M. Scholz, and H. R. Weiss Nitric oxide and cGMP protein kinase activity in aged ventricular myocytes Am J Physiol Heart Circ Physiol, December 1, 2001; 281(6): H2304 - H2309. [Abstract] [Full Text] [PDF]
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T. Adachi, R. Matsui, R. M. Weisbrod, S. Najibi, and R. A. Cohen Reduced Sarco/Endoplasmic Reticulum Ca2+ Uptake Activity Can Account for the Reduced Response to NO, but Not Sodium Nitroprusside, in Hypercholesterolemic Rabbit Aorta Circulation, August 28, 2001; 104(9): 1040 - 1045. [Abstract] [Full Text] [PDF]
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Z. Ungvari and A. Koller Signal Transduction in Smooth Muscle: Selected Contribution: NO released to flow reduces myogenic tone of skeletal muscle arterioles by decreasing smooth muscle Ca2+ sensitivity J Appl Physiol, July 1, 2001; 91(1): 522 - 527. [Abstract] [Full Text] [PDF]
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D. Xiao, W. J. Pearce, and L. Zhang Pregnancy enhances endothelium-dependent relaxation of ovine uterine artery: role of NO and intracellular Ca2+ Am J Physiol Heart Circ Physiol, July 1, 2001; 281(1): H183 - H190. [Abstract] [Full Text] [PDF]
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Y. Li, T. Adachi, V. M. Bolotina, C. Knowles, K. A. Ault, and R. A. Cohen Abnormal Platelet Function and Calcium Handling in Dahl Salt-Hypertensive Rats Hypertension, April 1, 2001; 37(4): 1129 - 1135. [Abstract] [Full Text] [PDF]
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M. Sausbier, R. Schubert, V. Voigt, C. Hirneiss, A. Pfeifer, M. Korth, T. Kleppisch, P. Ruth, and F. Hofmann Mechanisms of NO/cGMP-Dependent Vasorelaxation Circ. Res., October 27, 2000; 87(9): 825 - 830. [Abstract] [Full Text] [PDF]
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R. W. Jeremy and H. McCarron Effect of hypercholesterolemia on Ca2+-dependent K+ channel-mediated vasodilatation in vivo Am J Physiol Heart Circ Physiol, October 1, 2000; 279(4): H1600 - H1608. [Abstract] [Full Text] [PDF]
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Q.-K. Tran, K. Ohashi, and H. Watanabe Calcium signalling in endothelial cells Cardiovasc Res, October 1, 2000; 48(1): 13 - 22. [Abstract] [Full Text] [PDF]
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E. Thorin, M. Lucas, P. Cernacek, and J. Dupuis Role of ETA receptors in the regulation of vascular reactivity in rats with congestive heart failure Am J Physiol Heart Circ Physiol, August 1, 2000; 279(2): H844 - H851. [Abstract] [Full Text] [PDF]
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C. Mundina-Weilenmann, L. Vittone, G. Rinaldi, M. Said, G. C. de Cingolani, and A. Mattiazzi Endoplasmic reticulum contribution to the relaxant effect of cGMP- and cAMP-elevating agents in feline aorta Am J Physiol Heart Circ Physiol, June 1, 2000; 278(6): H1856 - H1865. [Abstract] [Full Text] [PDF]
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N. I. Gokina and J. A. Bevan Role of intracellular Ca2+ release in histamine-induced depolarization in rabbit middle cerebral artery Am J Physiol Heart Circ Physiol, June 1, 2000; 278(6): H2105 - H2114. [Abstract] [Full Text] [PDF]
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D. Wang, X. Yu, R. A. Cohen, and P. Brecher Distinct Effects of N-Acetylcysteine and Nitric Oxide on Angiotensin II-induced Epidermal Growth Factor Receptor Phosphorylation and Intracellular Ca2+ Levels J. Biol. Chem., April 14, 2000; 275(16): 12223 - 12230. [Abstract] [Full Text] [PDF]
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J. Geiger, A.-P. Zou, W. B. Campbell, and P.-L. Li Inhibition of cADP-Ribose Formation Produces Vasodilation in Bovine Coronary Arteries Hypertension, January 1, 2000; 35(1): 397 - 402. [Abstract] [Full Text] [PDF]
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F. M. Faraci and C. D. Sigmund Vascular Biology in Genetically Altered Mice : Smaller Vessels, Bigger Insight Circ. Res., December 3, 1999; 85(12): 1214 - 1225. [Full Text] [PDF]
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E. S. Trepakova, R. A. Cohen, and V. M. Bolotina Nitric Oxide Inhibits Capacitative Cation Influx in Human Platelets by Promoting Sarcoplasmic/Endoplasmic Reticulum Ca2+-ATPase–Dependent Refilling of Ca2+ Stores Circ. Res., February 5, 1999; 84(2): 201 - 209. [Abstract] [Full Text] [PDF]
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J. Chen, Y. Wang, Y. Wang, T. Nakajima, K. Iwasawa, H. Hikiji, M. Sunamoto, D.-K. Choi, Y. Yoshida, Y. Sakaki, et al. Autocrine Action and Its Underlying Mechanism of Nitric Oxide on Intracellular Ca2+ Homeostasis in Vascular Endothelial Cells J. Biol. Chem., September 8, 2000; 275(37): 28739 - 28749. [Abstract] [Full Text] [PDF]
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E. S. Trepakova, M. Gericke, Y. Hirakawa, R. M. Weisbrod, R. A. Cohen, and V. M. Bolotina Properties of a Native Cation Channel Activated by Ca2+ Store Depletion in Vascular Smooth Muscle Cells J. Biol. Chem., March 9, 2001; 276(11): 7782 - 7790. [Abstract] [Full Text] [PDF]
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A. Ammendola, A. Geiselhoringer, F. Hofmann, and J. Schlossmann Molecular Determinants of the Interaction between the Inositol 1,4,5-Trisphosphate Receptor-associated cGMP Kinase Substrate (IRAG) and cGMP Kinase Ibeta J. Biol. Chem., June 22, 2001; 276(26): 24153 - 24159. [Abstract] [Full Text] [PDF]
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