Departments of 1Physics and 3Biology, Portland State University, Portland, Oregon 97207; and 2Department of Physics, Eastern China Normal University, Shanghai 200062, China
Submitted 22 January 2002 ; accepted in final form 11 March 2003
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ABSTRACT |
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NADH-dependent superoxide production; ryanodine receptor; oxidative stress; sarcoplasmic reticulum
Through well-characterized electron exchange reactions, superoxide gives
rise to hydrogen peroxide and subsequently to hydroxyl radicals (in the
presence of iron) (37). The
cell protects itself from oxidative damage by rapidly converting superoxide to
hydrogen peroxide (via the action of SOD) and peroxide into water [utilizing
glutathione peroxidase (GPX) or catalase]. Nonenzymatic antioxidants such as
reduced glutathione (GSH), vitamins E and C, -carotene, and
-lipoic acid all protect muscle against oxidative stress.
The SR is a subcellular organelle that controls the contractile state of muscle by regulating the Ca2+ concentration in the cytosol. By hydrolysis of ATP, the SR actively accumulates Ca2+ into its lumen, which leads to muscle relaxation. Depolarization of the transverse tubule membrane results in the release of Ca2+ from the SR and muscle contraction. The Ca2+ release protein is pharmacologically characterized by its ability to bind the plant alkaloid ryanodine with high affinity and high specificity, and, hence, this protein is now known as the ryanodine receptor (RyR). [3H]ryanodine has been used to identify the Ca2+ release protein and is important in characterizing this receptor (14, 23). It has been repeatedly demonstrated that reagents that open the Ca2+ release channel increase equilibrium binding of ryanodine. The binding of ryanodine has become a functional probe to characterize the open vs. closed state of the Ca2+ release mechanism.
The SR Ca2+ release mechanism has been shown to be a potent target of oxidative modification. It is well established that oxidation of critical thiol groups activate the Ca2+ release mechanism, whereas addition of thiol-reducing agents closes down the Ca2+ channel (2, 20, 35). Oxidative modification of Ca2+ channel function has been observed at the level of skinned fibers, in Ca2+ flux measurements, in single channel measurements, and at the level of high-affinity ryanodine-binding measurements (1, 2, 20, 28, 35). It has recently been shown that the RyR is sensitive to the local redox potential (10, 36). More positive redox potentials sensitize the receptor to activation by Ca2+. Under mild oxidative stress, small changes to the cellular redox potential result in significant stimulation of the RyR. Redox reactions may play a critical role in controlling the kinetics of the Ca2+ release mechanism.
It has also been observed that ROS activate Ca2+
release from SR (3,
9,
31) and may act as redox
active signaling molecules to activate Ca2+ transport
(34). Although it is well
known that superoxide is a normal byproduct of mitochondrial electron
transport (8), which accounts
for 3% of total O2 consumption, and that
can be generated by xanthine
oxidase under ischemic-reperfusion conditions in cardiac muscle, no previous
study has shown that the SR membrane contains an enzymatic mechanism for
synthesizing
.In this
article, it is shown that skeletal muscle SR contains an NADH-dependent
oxidase that generates
,
which in turn activates the SR Ca2+ release
mechanism.
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EXPERIMENTAL PROCEDURES |
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SR was further fractionated on a discontinuous sucrose gradient (27). The following sucrose solutions (percent by weight) plus 10 mM HEPES, pH 7.0, were layered sequentially in a SW28 centrifuge tube (Beckman): 4 ml of 45%, 7 ml of 40%, 12 ml of 35%, 7 ml of 30%, and 4 ml of 27%. Thirty milligrams of unfractionated SR were layered on top of the gradient and then spun at 22,000 rpm overnight. The heavy fraction (HSR) and light fraction (LSR) were processed as previously described (27) and then stored in liquid N2.
The Ca2+ release protein, RyR1, was isolated
according to the method of Lai et al.
(17) with small modifications.
Sixty milligrams of SR were suspended in 10 ml of 0.2 M NaCl, 150 µM
CaCl2, 100 µM EGTA, 25 mM PIPES, 1.6%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 100 µM
dithiothreitol, 2 µg/ml leupeptin, 0.5 mM PMSF, and 3 mg/ml
phosphatidylcholine (PC), pH 7.1, and stirred on ice for 2 h. The solubilized
SR was then spun at 40,000 rpm for 60 min in a Beckman Ti50 rotor (100,000
g), and 10 mg of the supernatant were loaded onto 30 ml of a
520% linear sucrose gradient containing 0.2 M NaCl, 0.9% CHAPS, 4 mg/ml
PC, 150 µM CaCl2, 100 µM EGTA, 1 µg/ml leupeptin, 0.5 mM
PMSF, and 40 mM Tris, pH 7.1. The sucrose gradient was centrifuged at 25,000
rpm (Beckman SW28 rotor) for 18 h at 2°C. Seventeen fractions were pumped
off the gradient and assayed for ryanodine binding, NOX activity, and
NADH-dependent
production.
Samples were stored in liquid N2.
The initial rate of [3H]ryanodine binding was determined from time-dependent measurements at 3, 6, 9, and 12 min at 37°C (36). SR at 1.0 mg/ml was incubated at 37°C in binding buffer containing 250 mM KCl, 15 mM NaCl, 50 µM free Ca2+ (buffered with 50 µM EGTA), and 20 mM PIPES, pH 7.1. The time-dependent reaction was initiated by dilution into an equal volume of binding buffer containing 8 nM [3H]ryanodine, 50 µM free Ca2+, and various concentrations of NADH, NADPH, NADP, or NAD+. In all Ca2+-dependent measurements, Ca2+ was buffered with 50 µM EGTA to a free Ca2+ concentration as calculated by WinMaxc (6). The binding reaction was quenched by rapid filtration through Whatman GF/B filters mounted on a 48-well Brandel cell harvester. Filters were rinsed twice with binding buffer containing 50 µM Ca2+. Scintillation vials were filled with scintillation fluid, shaken overnight, and counted the next day.
NADH oxidase. The initial rate of NADH oxidase activity was measured at varying concentrations of NADH by monitoring the absorbance at 338 nm vs. time. The extinction coefficient of 6.25 mM1 · cm1 was used to convert from absorbance units to concentration of NADH. Experiments were carried out with either rabbit skeletal muscle or rabbit heart mitochondria at a protein concentration of 0.1 mg/ml at room temperature in 50 mM potassium phosphate and 10 mM KCl, pH 7.4 (phosphate buffer). The rate of NADH-dependent oxidase was normalized per milligram of protein.
Superoxide production.
was measured according to
the method of Azzi et al. (4),
using the reduction of acetylated ferricytochrome c (AFC) as a
measure of
production. The
initial rate of AFC reduction was determined by monitoring the absorbance
difference at 550540 nm as a function of time and using the extinction
coefficient of 16.8 mM1 ·
cm1. The reaction was initiated by addition of
the indicated concentration of NADH. The assay was carried out at 0.1 mg/ml of
either SR or mitochondria with 80 µg/ml AFC (Sigma Chemical) in 50 mM
potassium phosphate buffer at room temperature. The rate of production of
superoxide was determined by subtracting the rate of reduction of AFC in the
presence of SOD (300 U/ml) from the rate in the absence of SOD. Measurements
of NADH oxidase activity (338 nm) and cytochrome c reduction
(550540 nm) were simultaneously monitored using an HP 8452A diode array
spectrophotometer. A calibration curve was generated in which the absorbance
difference between 550 and 540 nm was plotted vs. the concentration of AFC
(not shown). The data were then corrected for the low concentration of AFC
used in these experiments (at 80 µg/ml, only 17% of the superoxide produced
was detected).
Measurement of mitochondrial copurification with SR. Mitochondria were isolated from rabbit heart ventricles (21) and stored in liquid N2. Polyacrylamide gels (10%) were run according to the method of Laemmli (16). Varying amounts of SR vesicles (10, 20, 40, and 80 µg), and mitochondria (1.25, 2.5, 5.0, and 10 µg) per lane were electrophoresed and stained with either Coomassie blue or transferred onto nitrocellulose paper (Schleicher and Schuell). The nitrocellulose paper was blocked with 5% powdered milk and washed with Tris-buffered saline (TBS). The transfer was then incubated overnight in 0.5 µg/ml anti-F1F0-ATPase mouse IgG (Molecular Probes, Eugene, OR; cat. no. A-21350). The transfer was washed with TBST (TBS with 0.05% Tween 20) and incubated for 30 min in alkaline phosphatase-linked anti-mouse secondary antibody (Sigma-A 3562) diluted 1:20,000. Color was visualized using 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT) as described by the manufacturer (Promega). The transfer was scanned with a Molecular Dynamics Typhoon 9200 imager. The integrated area of the F1F0-ATPase was plotted vs. the amount of added protein per lane. These plots were linear (r2 > 0.97), and their slopes were used to estimate the amount of mitochondria in our SR preparation (4.1 ± 1.3%, mean ± SD). This value was obtained from measurements made with three different SR preparations. Similar measurements were repeated with light and heavy SR vesicles to determine the amount of F1F0-ATPase associated with SR derived from the longitudinal and the terminal cisternae region of the SR.
Partially acetylated ferricytochrome c was purchased from Sigma, [3H]ryanodine was purchased from New England Nuclear (NEN), and SOD and catalase were purchased from CalBiochem. All other chemicals were obtained from Sigma Chemical.
Statistics. All figures show means ± SD. Student's t-test was used to determine whether the differences in mean values were statistically significant at a P < 0.05 or P < 0.01, as indicated.
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RESULTS |
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It has previously been shown that the hyperreactive thiols associated with
RyR1 have a well-defined redox potential that is sensitive to the open vs.
closed state of the channel. At 50 µM, Ca2+ the redox
potential of the receptor (Ered) is 157 mV
(36). Addition of channel
inhibitors results in a shift of Ered to more positive
values, whereas addition of channel activators shifts Ered
to more negative values. As shown in Fig.
2, addition of 1 mM NADH shifts the redox potential to 177
mV. This shift, induced by
,
occurs in spite of the significant amount of GSH present in the buffer that
sets the redox potential of the solution. Moreover, consistent with this
observation, addition of 50 µM xanthine and 6.25 nM xanthine oxidase
activates ryanodine binding (Fig.
1) and shifts Ered to 175 mV in the
absence of NADH (not shown).
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As shown in Fig. 3, NADH-dependent activation is strongly O2 dependent. Addition of SOD decreased the rate of receptor binding. A decrease in oxygen tension was even more effective in inhibiting the binding of ryanodine. Addition of the detergent CHAPS increased binding rates, in the absence of NADH, approximately fourfold. However, the NADH dependency of the rate of ryanodine binding was not altered by CHAPS. The degree of activation by NADH is similar between control and CHAPS-solubilized SR vesicles. As was shown in Fig. 1, the activation by NADH was significantly different from the control. Activation of the receptor was less evident in the presence of SOD and at low O2 tensions (Fig. 3).
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NADH oxidase activity and the rate of production of
were measured as described
in EXPERIMENTAL PROCEDURES with both SR and mitochondrial
preparations. As seen in Fig.
4, 72% of the SR NOX activity is inhibited by 200 nM antimycin A,
whereas 94% of the mitochondrial NOX activity is inhibited by the same
concentration of antimycin A (Fig.
5). Antimycin A and rotenone are potent inhibitors of electron
transport in mitochondria. The Ki for antimycin A
inhibition of SR NADH oxidase activity = 12.5 nM
(Fig. 4). The rate of
production by the SR (3.3
± 0.6 nmol · mg1 ·
min1) was not affected by antimycin A (3.3
± 0.4 nmol · mg1 ·
min1). Rotenone (40 nM) inhibited NOX activity in
a similar manner but also did not affect
production. Diphenylene
iodonium (DPI), an inhibitor of flavoprotein-containing oxidases in
mitochondria (18), also
inhibited NOX activity to the same level as did rotenone and antimycin A at
concentrations of DPI >10 µM. DPI also had no effect on the rate of
production of
(not
shown).
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These observations suggest that a large fraction of the NADH oxidase
activity is caused by mitochondria that copurify with the SR preparation, but
the generation of superoxide is not caused by the mitochondria. This was
further examined by isolating rabbit heart mitochondria
(21). Because the SR vesicles
used in all studies described in this paper were stored in liquid
N2, the isolated mitochondria were also rapidly frozen in liquid
N2. Freezing and thawing of pigeon heart mitochondria has been
shown to dramatically increase NADH oxidase activity by increasing the
permeability of the mitochondria to NADH
(24). As shown in
Fig. 5, the high NOX activity
of rabbit heart mitochondria is inhibited by 200 nM antimycin A to 6% of
control, whereas a negligible amount of
was produced by the
mitochondria (1.3 ± 1.8 nmol · mg1
· min1). SOD had no effect on the NOX
activity of the mitochondria.
If the antimycin A inhibitable component of SR NOX activity is caused by mitochondria that copurify with the SR, this should be evident by measuring the amount of mitochondrial proteins in our SR preparation. By assaying for the presence of the F1F0-ATPase from the inner mitochondrial membrane (see EXPERIMENTAL PROCEDURES), it was determined that 4.1 ± 1.3% of the unfractionated SR that was used in most of the experiments described (Figs. 1, 2, 3, 4) is mitochondrial in origin. Given the large NOX activity of the mitochondria (Fig. 5), mitochondria associated with the SR accounts for a significant contribution to the NOX activity of the isolated SR. This is also reflected in the difference in NOX activity of HSR and LSR. As shown in Fig. 5, the ratio of antimycin A inhibitable NOX activity in HSR compared with that in LSR = 2.5 ± 0.6, whereas the ratio of F1F0-ATPase in HSR compared with LSR = 2.8 ± 1.2. This latter result was obtained by comparing Western blots of HSR and LSR vesicles stained with an antibody to the F1F0-ATPase (repeated three times). There is a good correlation between the antimycin A-inhibitable component of NOX activity of the HSR vesicles and the increased presence of mitochondria. These observations further support the hypothesis that the antimycin A inhibitable component of NOX activity is caused by mitochondria associated with the SR. This result also suggests that the approximate threefold enhancement of mitochondria in the HSR is a natural consequence of its native association with the terminal cisternae of the SR.
NOX activity associated with the SR that is not inhibited by antimycin A
(5.7 ± 0.1 nmol · mg1 ·
min1) is coupled to the production of
(3.3 ± 0.6 nmol
· mg1 ·
min1). NADH plus AFC in the absence of SR showed
a negligible, time-dependent reduction of AFC and no oxidation of NADH.
Moreover, SR in the absence of NADH does not reduce cytochrome c;
was not produced in the
absence of SR or NADH.
The rate of reduction of AFC has been used to monitor the reduction of
in biological membranes
(4). AFC can be reduced by low
molecular weight reducing agents other than superoxide (i.e., GSH and
semiquinones). However, by measuring the difference in the rate of AFC
reduction, in the absence and presence of SOD (300 U/ml), the rate of
production was reproducibly
determined.
AFC measures production
on the outside of the SR vesicle. Control experiments were carried out at 1 mg
CHAPS/mg SR to solubilize the SR vesicles. The rate of reduction of AFC in the
presence of CHAPS was no different than that of experiments carried out in the
absence of CHAPS. This indicates that superoxide is exclusively produced on
the cytoplasmic face of the SR.
The rate of production
per milligram of SR is
3.3 ± 0.6 nmol ·
mg1 · min1.
This is six to seven times higher than the rate of
production previously
measured in the presence of 2 µM antimycin A and 3 mM succinate in
submitochondrial particles derived from rat heart measured at the same pH
(7). Moreover, unlike
mitochondria, the SR preparation showed no measurable
production in the presence
of 10 mM succinate, in the absence of NADH (not shown).
To identify which protein or proteins were responsible for the production of superoxide and the oxidation of NADH, a 520% sucrose gradient was prepared as described in EXPERIMENTAL PROCEDURES, and various fractions were eluted from the gradient. The highest density fractions were eluted first (fraction 1). As shown in Fig. 6, high-affinity [3H]ryanodine binding was localized in fractions 25. As observed in Fig. 1, with isolated SR vesicles, addition of 1 mM NADH to those fractions containing the RyR resulted in a twofold stimulation in receptor binding. Moreover, NADH oxidase activity also copurifies with those fractions rich in RyR1 (not shown).
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DISCUSSION |
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In this article, we show for the first time that skeletal muscle SR
contains an NADH-dependent oxidase that produces
, which in turn stimulates
the SR RyR1. This effect is inhibited by SOD and decreased O2
tension. Moreover, the
produced is not due to mitochondrial contamination. Unlike the mitochondria,
SR's production of
is not
activated by succinate, and it is not affected by either rotenone, antimycin
A, or DPI. It is important to recognize that the rate of
production per milligram of
protein in SR is higher than the corresponding rate measured in
submitochondrial particles derived from rat heart muscle
(7).
The EC50 for NADH-dependent activation of NOX activity is
40 µM, whereas the EC50 for reduction of AFC is <2 µM
(not shown). This large difference in sensitivity to NADH is due to the
presence of at least two NADH-dependent oxidases. The one that comprises
75% of the oxidase activity has a lower affinity for NADH and is
inhibitable by antimycin A, rotenone, and DPI. Given the high NOX activity of
mitochondria after freezing and thawing and that
4.1% of this SR
preparation is mitochondrial in origin, it appears that the majority of the SR
NOX activity originates from the associated mitochondria. The remaining NOX
activity (5.7 ± 0.1 nmole · mg1
· min1) is coupled to the production of
superoxide (3.3 ± 0.6 nmole · mg1
· min1).
The interaction between the SR/endoplasmic reticulum and the adjoining
mitochondria has recently received a great deal of attention
(11).
Ca2+ spikes associated with Ca2+
release from the SR are coupled to an increase in the internal mitochondrial
Ca2+ concentration
(22). It has also been shown
that 90% of the Ca2+ release units in cardiac
ventricular myocytes are in close proximity to adjoining mitochondria
(29). The observation in this
article that there is three times as much mitochondria associated with the HSR
than with the LSR suggests that these mitochondria may be functionally
associated with the SR and that they represent more than just a contamination
resulting from the SR preparation.
The NADH concentration, at which the initial rate of either NADH oxidation
or production is half
maximal, is in the low micromolar concentration range (not shown). The
concentration of NADH in resting human quadricep femoris muscle is
80
µM. This level increases approximately threefold after a brief isometric
contraction protocol (13). The
concentration of NADH in muscle is at least one order of magnitude higher than
the Km associated with the generation of
. The oxidation rate of NADH
and the production rate of
appears to be maximal and is therefore not controlled by changes in the
cellular NADH concentration. Whether or not the activity of this protein in
muscle is controlled by changes in O2 tension or other endogenous
modulators is not yet known.
The observation that NADH oxidase activity, ryanodine binding, and
activation of ryanodine-binding activity induced by NADH copurifies in sucrose
gradient fractions suggests that the RyR1 contains NADH oxidase activity. This
hypothesis is supported by a recent publication showing that NAD binds to
residues 41420 of RyR1 with an apparent dissociation constant,
Kd = 10 µM
(5). Alternatively, it is
possible that a small amount of contaminating protein that copurifies with
RyR1 is responsible for NADH oxidase activity and the production of
observed.
Superoxide generated by an endogenous NADH oxidase present in SR increases
RyR activity. It has previously been demonstrated that in a similar manner, an
increase in the local redox potential activates the RyR
(36) and single channel
activity of the reconstituted RyR1
(10). A more oxidizing
environment sensitizes the Ca2+ release mechanism to
activation. The purpose of having an NADH oxidase capable of generating
may well be to introduce a
low level of oxidative stress, which turns up the gain and increases the open
probability of the Ca2+ release channel. Under more
severe oxidizing conditions, during fatigue, high levels of ROS may close down
the Ca2+ release mechanism to prevent further rundown of
the muscle.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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