Download Ryanodine Inhibits the Release of Calcium from Intracellular Stores

730
Ryanodine Inhibits the Release of Calcium from
Intracellular Stores in Guinea Pig Aortic Smooth
Muscle
Katsuaki Ito, Sachiko Takakura, Kohichi Sato, and John L. Sutko
From the Department of Veterinary Pharmacology, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan, and the
Departments of Physiology and Internal Medicine (Cardiology Division), The University of Texas Health Science Center, Dallas, Texas
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SUMMARY. We have examined the effects of ryanodine, an inhibitor of the release of sarcoplasmic
reticulum calcium in cardiac muscle, on contractile tension and calcium-45 movement in aortic
smooth muscle of guinea pigs to learn whether this agent also modifies the release of stored
calcium in vascular smooth muscle. Ryanodine (3-100 /*M) suppressed the phasic contractions
induced by caffeine and norepinephrine in calcium-free medium and prevented the stimulation
of calcium-45 efflux by these agonists. Ryanodine did not significantly alter either the contractile
response or the increased cellular influx of calcium-45 caused by high potassium in more than 1
mM calcium, suggesting that this agent does not affect depolarization-induced calcium entry into
the cells. In a calcium-free, high potassium solution, the addition of calcium at concentrations of
1 mvi and less resulted in a contraction which appeared to depend largely on the release of
calcium from intracellular stores. This contraction was blocked by ryanodine. These data are
consistent with the hypothesis that ryanodine causes a diminished release of calcium from the
intracellular store in vascular smooth muscle, as it does in cardiac muscle. Moreover, our results
indicate that a calcium-induced calcium release may exist in smooth muscle, and that this release
is antagonized by ryanodine. (Circ Res 58: 730-734, 1986)
IT HAS been suggested that mobilization of calcium
from intracellular stores, presumably sarcoplasmic
reticulum (SR), in vascular smooth muscles, underlies the contractile effects of agents such as vasoactive amines and peptides 0ohansson and Somlyo,
1980). Nevertheless, the role of the SR in regulating intracellular calcium concentration in vascular
smooth muscle is not well understood, due to its
low density and distribution in the cell, and to
difficulties in isolating SR membranes from vascular
smooth muscles. Pharmacological interventions
which modify the release of intracellularly stored
calcium have been used to investigate the contributions made by intracellularly stored calcium to
smooth muscle contractions. These interventions include caffeine and local anesthetics, such as procaine
(Endo, 1977; Itoh et al., 1981; Saida, 1982). However, a limitation to the use of these agents is that
both caffeine and procaine decrease the influx of
calcium from the extracellular space (Jacobs and
Keatinge, 1974; Leijten and Van Breemen, 1984;
Spedding and Berg, 1985). Therefore, it is difficult
to differentiate the effect of these agents on the
calcium release from that on the calcium influx when
intact smooth muscle is used.
Ryanodine, a naturally occurring alkaloid, has
been demonstrated to alter specifically calcium
movements across SR membranes in cardiac and
skeletal muscles (Jenden and Fairhurst, 1969; Sutko
et al., 1979; Sutko and Willerson, 1980; Fabiato,
1985; Sutko et al., 1985). Moreover, this agent alters
the mechanical responses of certain smooth muscle
preparations in a manner that suggests that it might
produce similar effects in this tissue (Steinsland et
al., 1973). If ryanodine selectively modifies the function of the SR, it will be a powerfull tool for the
study of the role of the SR in vascular smooth muscle
contraction.
The present study was undertaken to learn how
ryanodine modifies the release of calcium from intracellular stores in guinea pig aortic smooth muscle.
The results suggest that this agent blocks the release
of intracellularly stored calcium caused by norepinephrine (NE) and caffeine, but does not affect
depolarization-dependent calcium entry from the
extracellular space.
Methods
Aortas were isolated from 250- to 400-g male guinea
pigs following a blow on the neck. For tension experiments, the helical strip of aorta was suspended in an organ
bath filled with 10 ml Tyrode's solution containing (mM):
NaCl, 136.8; KC1, 5.4; MgCl2, 0.5; NaHCO3, 11.9; and
dextrose, 5.5; gassed with 95% O2 and 5% CO2 (pH 7.4;
37°C). High potassium solution was prepared either by
hypertonically increasing KG to 60 mM or by isotonically
substituting KC1 for NaCl. Norepinephrine (Sigma) and
ryanodine (lot 704 RWP-1, S.P. Penick Corp.) were added
to the Tyrode's solution from 1 mM stock solution, while
caffeine (Nakarai Chemicals) was dissolved directly in the
lto et al./Effect of Ryanodine on Vascular Smooth Muscle
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buffer solution. The muscle strip was placed under 1 g of
resting tension, and its isometric tension was recorded.
The muscle was equilibrated for 30 minutes and then was
treated alternately with 60 mM KC1 and 1 fiM NE until
stable reproducible contractions were obtained.
For 45Ca flux experiments, the solutions were buffered
with tris(hydroxymethyl)aminomethane (Tris) and gassed
with 100% O2 to avoid precipitation in the medium due
to the presence of lanthanum. We had ensured, in preliminary experiments, that the results of tension experiments
in Tris buffer were identical with those in a bicarbonate
buffer. Uptake of 45Ca by aortic muscles was measured by
a cold lanthanum wash procedure (Karaki and Weiss,
1979). First, the muscles were depleted of calcium by 20
minutes of exposure to 10 /*M NE in calcium-free medium,
and then were transferred to calcium-free, potassiumdepolarizing solution containing KG, 142; MgCU, 0.5;
dextrose, 5.5; 5 mM Tris (pH 7.4). Fifteen minutes later,
1 mM calcium containing 45Ca (1 /iCi/ml) was added. After
incubation with 45Ca for 5 or 30 minutes, the muscles
were transferred to lanthunum solution (LaCl3, 80.8; dextrose, 11; and Tris, 5 mM; pH 6.8) cooled to 0.5°C. The
residual 45Ca content after 60 minutes of incubation in
cold lanthanum solution was determined as the net cellular 45Ca uptake.
For the measurement of 45Ca efflux, the muscles were
loaded with 45Ca during a 150-minute incubation period
in Tyrode's solution containing 45Ca (2 jiCi/ml; total calcium was 2.5 mM). The muscles then were placed in a
calcium-free medium, and the efflux of 45Ca was initiated.
The efflux medium was changed every 10 minutes. After
60-80 minutes, the muscles were exposed to 10 HM NE or
20 mM caffeine. The radioactivity in the efflux medium
was counted in a liquid scintillation counter. Data are
expressed as rate coefficient, which means the percent
loss of 45Ca content during each washing interval.
Data represent the mean ± SEM. Differences were considered to be significant at the level of P < 0.05 when
tested by Student's /-test.
Results
When 2.5 HIM calcium was present in the external
medium, pretreatment of aortic muscles with 30 or
100 HM ryanodine did not significantly affect the
rate of tension development and the peak amplitude
of the contraction elicited by 60 mM potassium (n =
8 for each concentration). On the other hand, 30 JUM
ryanodine slowed the development of responses to
1 fiM NE (the time for half-maximal response increased from 19 ± 3 seconds to 96 ± 17 seconds; P
< 0.05) and decreased its peak tension by 22 ± 8%
( P < 0 . 0 5 ; H = 8).
In calcium-free solutions containing 0.5 mM
EGTA, both caffeine (5-10 mM) and NE (1 I*M)
induced an initial phasic contraction which, in the
case of NE, was followed by a tonic one. Ryanodine
selectively suppressed the phasic contraction due to
both agents in a dose-dependent manner (Fig. 1).
The ryanodine-insensitive tonic contraction produced by NE was decreased by elevating the concentration of EGTA in the medium to 2 mM, supporting the view that this component was due to
entry of superficially bound calcium or to the release
731
Caffeine
Ryanodine Caffeine
7
T
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]0.2g
CCa-1,
2.5mM
0 mM
1
2.5mM
3
10
OmM
30
Ryanodine (uM)
FIGURE 1. Suppression of the phasic contractions induced by caffeine
and norepinephrine (NE) in calcium-free medium by ryanodine. After
a 15-minute incubation in calcium-free medium containing 0.5 mM
EGTA, aortic strips were contracted with either 10 niM caffeine or
1 HM NE. After the agonists from the organ bath were washed, the
muscles were equilibrated in 2.5 niM calcium for 30 minutes. The
external medium was then changed to the calcium-free medium
containing ryanodine. Panel A: abolition by 30 HM ryanodine of
caffeine-induced contraction. Panel B: suppression by 10 HM ryanodine of NE-induced phasic contraction. Panel C: concentration-dependence of the effects of ryanodine on the phasic (closed circle) and
tonic (open circle) contractions induced by 1 HM NE in calcium-free
medium containing 0.5 mM EGTA. The ordinate represents the relative amplitude of the contraction as compared to the maximum
contraction induced by 60 mM KCI in 2.5 niM calcium-Tyrode's
solution. Each point represents a mean of seven preparations with
SEM.
of calcium from intracellular site which can be readily depleted by extracellular EGTA (Wheeler and
Weiss, 1979; Karaki and Weiss, 1980a, 1980b).
NE (10 IIM) and caffeine (20 mM) increased the
45
Ca efflux from aortic muscles into calcium-free
medium, which effect is thought to result from an
acceleration of release of stored calcium by these
agents (Godfraind, 1976; Deth and Casteels, 1977;
Casteels and Droogmans, 1981). As shown in Figure
2, the presence of 30 fiM ryanodine prevented this
effect caused by NE. Similarly, ryanodine prevented
the increase of 45Ca efflux caused by 20 mM caffeine
(data not shown). Thus, the results shown in Figures
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Circulation Research/Vol. 58, No. 5, May 1986
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60
30
120
90
Time (min)
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FIGURE 2. Inhibition by ryanodine of the stimulation by 10 IXM NE of
the i5Ca efflux from the aortic muscles. After the muscles were loaded
with 4SCa for 150 minutes, the muscles ivere exposed to the efflux
medium which was changed every 10 minutes. After 60-80 minutes
muscles were exposed to 10 \IM NE. When present, 30 fiM ryanodine
was added 20 minutes prior to NE, as indicated by arroiv (filled
circle). Open circle: control. Each curve represents the average of four
preparations.
1 and 2 clearly indicate that ryanodine inhibits the
release of calcium from the intracellular stores sensitive to NE or caffeine.
In the next series of experiments, we examined
N.S.
200
T
T
150
o
c
N.S.
100
Q.
o
o
50
0
L
(mM)
5.4 142 142
+ Ry
5 min
5.A K2
• Ry
30 min
FIGURE 3. Cellular uptake of45Ca into aortic muscles in the presence
of 5.4 HIM or 142 mM potassium. After the intracellular calcium store
was depleted by 10 HMNE in calcium-free medium, the muscles were
exposed to calcium-free medium containing 5.4 HIM or 142 mM
potassium. Twenty minutes later, 1 HIM calcium with >5Ca was added.
The muscles were allowed to take up <5Ca for 5 or 30 minutes, and
were then transferred to the lanthanum solution at 0.5°C to measure
cellular *5Ca uptake. In the ryanodine-treated group (Ry), 30 HM
ryanodine was present in the calcium-free, 142 mM potassium solution. Each bar represents a mean of eight preparations with SEM. N.S.
= not significant.
the effects of ryanodine on the contraction of aortic
smooth muscle resulting from the reintroduction of
calcium to muscles previously incubated in calciumfree, high potassium (140 mM) solution. Ryanodine
(30 HM) significantly decreased the calcium-induced
contraction of the muscles by 73 ± 6%, 57 ± 6%,
and 42 ± 4% (n = 7) when the concentration of
calcium added was 0.1, 0.3, and 1.0 mM, respectively. However, ryanodine did not significantly inhibit the contraction when the added calcium exceeded 2 mM. The suppression of this contraction
could be due to inhibition by ryanodine of either
calcium influx into the cell or of calcium-induced
calcium release from the intracellular stores. To test
these possibilities, we first examined the effect of
ryanodine on the uptake of calcium by the muscles
(Fig. 3). After the muscles had been incubated in
calcium-free, isotonic potassium solution for 20 minutes, 1 mM calcium with 45Ca was added. High
potassium significantly increased 45Ca uptake into
the muscle cells. Ryanodine (30 /J.M) did not significantly affect the 45Ca uptake increased by high
potassium. The results suggest that ryanodine does
not affect depolarization-dependent calcium entry
from the external medium. This hypothesis is consistent with the fact that ryanodine did not affect
the contraction induced by 60 mM potassium in the
presence of 2.5 mM calcium.
To assess the second possibility, we examined the
dependency of the calcium-induced contraction on
the extent of calcium loading of the intracellular
store (Fig. 4). In one group of muscles, the intracellular store was filled with calcium by exposing the
muscles to a medium containing 60 mM KG and 2.5
mM calcium just before the introduction of calciumfree, high potassium solution. The ability of this
procedure to load intracellular calcium stores was
confirmed in separate experiments in which NEinduced phasic contractions obtained in calciumfree medium were shown to be augmented by this
procedure. In a second group, the intracellular calcium store was depleted of calcium by three successive exposures to 1 HM NE in calcium-free buffer.
Contractions that developed during the initial 5
minutes after the addition of 0.1 mM calcium were
compared, since the calcium store might be significantly replenished after longer times. Figure 4 shows
that both the rate of development and the amplitude
of the contractions induced by 0.1 mM calcium were
greater in the calcium-loaded than in the calciumdepleted muscles. Ryanodine suppressed the calcium-induced contraction developed for 5 minutes
after exposure to calcium in the calcium-loaded muscles, but not in those that had been depleted of
calcium. These results suggest that intracellularly
stored calcium makes a significant contribution to
the calcium-induced contraction elicited from depolarized muscles with intact calcium stores, and
that a calcium-induced calcium release mechanism
may be operable in this preparation.
lto et fl/./Effect of Ryanodine on Vascular Smooth Muscle
30
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2
3
Time (min)
FIGURE 4. Effects of ryanodine on calcium-induced contractions in
potassium-depolarized aortic smooth muscles. Upper panel: calciumdepleted muscles. The muscles were depleted of calcium by three
successive applications of NE in the calcium-free medium containing
0.5 HIM ECTA. Subsequently, the external medium was changed to
the calcium-free, potassium-depolarizing medium (142 niM KC1, no
ECTA). Lower panel: calcium-loaded muscles. Aortic muscles were
loaded with calcium by application of 60 IUM potassium and 2.5 niM
calcium just before changing to a calcium-free, high-potassium solution. In both panels, when present (open circle), 30 /JM ryanodine
was added when the external medium ivas changed to the calciumfree, high potassium solution. Closed circle: control. Data represent
mean of seven preparations with SEM. 'Significantly different from
control (P < 0.05).
Discussion
The results obtained in this study suggest that
ryanodine can diminish the release of calcium from
intracellular stores in vascular smooth muscle without affecting the calcium influx from the extracellular space. The selective inhibition by ryanodine of
only those contractions which involve the release of
calcium from intracellular sites means that this agent
has an advantage over caffeine or local anesthetics
as a tool for the study of smooth muscle contraction.
This selectivity also argues against an effect on
subsequent steps in the smooth muscle contractile
mechanism, such as myosin light chain kinase,
which could be common to all of the agents tested.
Although experimental verification is required, we
are presently assuming from analogies with striated
muscles that the SR is the intracellular calcium store
affected by agents such as ryanodine and caffeine.
This conclusion has been reached by other authors
733
(Casteels and Droogmans, 1981; Itoh et al., 1982;
Bond et al., 1984a, 1984b). Relatively high concentrations of ryanodine are required to affect vascular
smooth muscle. This apparent insensitivity may be
explained by the use- or depolarization-dependence
exhibited by the actions of this agent on both skeletal
and cardiac muscles (Sutko et al., 1985), as well as
by the actual characteristics of the ryanodine-binding site.
Calcium-induced contraction of depolarized muscles seemed to depend on calcium loading of the
cells. However, we must consider an alternative
possibility that this contraction might depend on
superficially bound calcium. Since NE treatment,
used to deplete calcium, can remove superficially
bound calcium (Wheeler and Weiss, 1979; Karaki
and Weiss, 1980a, 1980b), the different amounts of
superficially bound calcium in two groups may explain the difference between the responses to 0.1
mM calcium if calcium at this site contributes to the
calcium-induced contraction. As for this fraction of
calcium, ryanodine does not seem to affect the mobilization of superficially bound calcium, since this
agent did not inhibit the tonic contraction induced
by NE in calcium-free medium. On the contrary,
ryanodine greatly inhibited the contraction induced
by calcium in calcium-loaded muscles. Therefore,
we can conclude that calcium-induced contraction
of depolarized muscle did not depend on superficially bound calcium, but on intracellularly stored
calcium.
It has been suggested that calcium-induced calcium release underlies the caffeine-induced contraction in certain vascular smooth muscles (Itoh et al.,
1981,1982; Saida and Van Breemen, 1984). Caffeine
is thought to increase the affinity of SR for calcium
(Yamamoto and Kasai, 1982; Kirino et al., 1983) and
to induce calcium release at physiological levels of
free calcium in the cytoplasm even in the absence
of calcium influx (Saida, 1982). However, the release
of calcium from intracellular stores by transmembrane calcium influx has not yet been demonstrated
in vascular smooth muscle. Calcium-induced contraction in depolarized muscles has long been believed to be caused directly by transmembrane calcium influx. The present data indicate that this
contraction is dependent on stored calcium rather
than on calcium entry, especially when the external
calcium is low. Since ryanodine does not inhibit
calcium influx, inhibition of this contraction by ryanodine suggests that transmembrane calcium influx
can trigger calcium release from intracellular stores
in vascular smooth muscle as it does in cardiac
muscles (Fabiato, 1983).
The present findings suggest that ryanodine is a
useful tool to assess a contribution of calcium release
from intracellular stores to mechanical activity of
vascular smooth muscle. Our results suggest that
ryanodine inhibits SR calcium release in guinea pig
aortic smooth muscle. Ryanodine can both increase
Circulation Research/Vol. 58, No. 5, May 1986
734
and decrese the calcium permeability of cardiac
and skeletal SR membranes, depending on the ryanodine concentration and experimental conditions
used (Lattanzio and Sutko, unpublished observations; G. Meissner, personal communication). Consequently, the actual effects of ryanodine will have
to be established for different experimental protocols
and types of smooth muscles.
ILS is an Established Investigator of the American Heart Association.
Address for reprints: Dr. Katsuaki Ho, Department of Veterinary
Pharmacology, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-21, ]apan.
Received November 11, 1985; accepted for publication February
14, 1986.
References
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Bond M, Kitazawa T, Somlyo AP, Somlyo AV (1984a) Release
and recycling of calcium by the sarcoplasmic reticulum in
guinea-pig portal vein smooth muscle. ] Physiol (Lond) 355:
677-695
Bond M, Shuman H, Somlyo AP, Somlyo AV (1984b) Total
cytoplasmic calcium in relaxed and maximally contracted rabbit
portal vein smooth muscle. J Physiol (Lond) 357: 185-201
Casteels R, Droogmans G (1981) Exchange characteristics of the
noradrenaline-sensitive calcium store in vascular smooth muscle cells of rabbit ear artery. J Physiol (Lond) 317: 263-279
Deth R, Casteels R (1977) A study of releasable Ca fractions in
smooth muscle cells of the rabbit aorta. ] Gen Physiol 69: 401416
Endo M (1977) Calcium release from the sarcomplasmic reticulum. Physiol Rev 57: 71-108
Fabiato A (1983) Calcium-induced release of calcium from the
cardiac sarcomplasmic reticulum. Am J Physiol 245: C1-C14
Fabiato A (1985) Effects of ryanodine in skinned cardiac cells.
Fed Proc 44: 2970-2976
Godfraind T (1976) Calcium exchange in vascular smooth muscle,
action of noradrenaline and lanthanum. J Physiol (Lond) 260:
21-35
Itoh T, Kuriyama H, Suzuki H (1981) Excitation-contraction coupling in smooth muscle cells of the guinea-pig mesenteric
artery. J Physiol (Lond) 321: 513-535
Itoh T, Kajiwara M, Kitamura K, Kuriyama H (1982) Roles of
stored calcium on the mechanical response evoked in smooth
muscle cells of the porcine coronary artery. J Physiol (Lond)
322: 107-125
Jacobs A, Keatinge WR (1974) Effects of procaine and lignocaine
on electrical and mechanical activity of smooth muscle of sheep
carotid arteries. Br ] Pharmacol 51: 405-411
Jenden DJ, Fairhurst AS (1969) The pharmacology of ryanodine.
Pharmacol Rev 21: 1-25
Johansson B, Somlyo AP (1980) Electrophysiology and excitationcontraction coupling. In Handbook of Physiology, The Cardiovascular System, vol II, Vascular Smooth Muscle, edited by
DF Bohr, AP Somlyo, HV Sparks. Washington, D.C., American
Physiological Society, pp 301-323
Karaki H, Weiss GB (1979) Alterations in high and low affinity
binding of 45Ca in rabbit aortic smooth muscle by norepinephrine and potassium after exposure to lanthanum and low
temperature. J Pharmacol Exp Ther 211: 86-92
Karaki H, Weiss GB (1980a) Effects of stimulatory agents on
mobilization of high and low affinity site 45Ca in rabbit aortic
smooth muscle. J Pharmacol Exp Ther 213: 450-455
Karaki H, Weiss GB (1980b) Dissociation of varied actions of
norepinephrine on 45Ca uptake and release at different sites in
rabbit aortic smooth muscle. J Pharmacol Exp Ther 215: 363368
Kirino Y, Osakabe M, Shimizu H (1983) Ca2+-induced Ca2+
release from fragmented sacroplasmic reticulum: Ca2+-dependent passive Ca2+ efflux. J Biochem 94: 1111 -1118
Leijten PAA, Van Breemen C (1984) The effects of caffeine on
the noradrenaline-sensitive calcium store in rabbit aorta. J
Physiol (Lond) 357: 327-339
Saida K (1982) Intracellular Ca release in skinned smooth muscle.
JGen Physiol 80: 191-202
Saida K, Van Breemen C (1984) Cyclic AMP modulation of
adrenoceptor-mediated arterial smooth muscle contraction. J
Gen Physiol 84: 307-318
Spedding M, Berg C (1985) Antagonism of Ca2+-induced contractions of K+-depolarized smooth muscle by local anaesthetics.
Eur J Pharmacol 108: 143-150
Steinsland OS, Furchgott RF, Kirpekar SM (1973) Biphasic vasoconstriction of the rabbit ear artery. Circ Res 32: 49-58
Sutko JL, Willerson JT (1980) Ryanodine alterations of the contractile state of rat ventricular myocardium. Comparison with
dog, cat, and rabbit ventricular tissues. Circ Res 46: 332-343
Sutko JL, Willerson JT, Templeton GH, Jones LR, Besch HR Jr
(1979) Ryanodine: Its alterations of cat papillary muscle contractile state and responsiveness to inotropic interventions and
a suggested mechanism of action. J Pharmacol Exp Ther 209:
37-47
Sutko JL, Ito K, Kenyon JL (1985) Ryanodine: A modifier of
sarcoplasmic reticulum calcium release in striated muscle. Fed
Proc 44: 2984-2988
Wheeler ES, Weiss GB (1979) Correlation between responses to
norepinephrine and removal of 45Ca from high-affinity binding
sites by extracellular EDTA in rabbit aortic smooth muscle. J
Pharmacol Exp Ther 211: 353-359
Yamamoto N, Kasai M (1982) Kinetics of the actions of caffeine
and procaine on the Ca2+ gated cation channel in sarcoplasmic
reticulum vesicles. J Biochem 92: 477-484
INDEX TERMS: Ryanodine • Vascular smooth muscle. Calcium
release • Contraction • Calcium fluxes
Ryanodine inhibits the release of calcium from intracellular stores in guinea pig aortic
smooth muscle.
K Ito, S Takakura, K Sato and J L Sutko
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Circ Res. 1986;58:730-734
doi: 10.1161/01.RES.58.5.730
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