95
Short Communications
Dependence of Cerebral Arterial Contractions on
Intracellularly Stored Ca+ +
TOMIO SASAKI, M . D . ,
D . M . S c , N E A L F. KASSELL, M . D . ,
AND MARIO ZUCCARELLO,
M.D.
SUMMARY The purpose of the present study was to evaluate the dependence of the arterial contractions
induced by different vasoactive agents upon intracellularly stored calcium in canine versus monkey cerebral
arteries. The potency for inducing contractions in Ca + +-free media was in the order of 9,1 l-epithio-11,12metano-thromboxane A2 (STXAj) > prostaglandin F^ (PGF^) > > serotonin > k + in canine basilar
arteries, and STXA2 >PGF 2a » serotonin = K + in monkey basilar arteries.
Stroke Vol 17, No 1, 1986
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PREVENTION OF THE CEREBRAL VASOSPASM
following subarachnoid hemorrhage (SAH) with calcium channel blockers is of contemporary interest.
Although the pathogenesis of cerebral vasospasm is
still unknown, contraction of arterial smooth muscle
resulting from an increase in transmembrane Ca + +
influx may be the underlying common mechanism.
Furthermore, constrictor responses of cerebral arteries
are more dependent on extracellular Ca + + than those
of other vessels.1^1 It has been reported that calcium
channel blockers increase cerebral blood flow after
ischemic stroke,4-5 and prevent symptomatic vasospasm following SAH. 6 However, the effect of calcium channel blockers on the diameter of spastic cerebral arteries is controversial. Several investigators
have reported that calcium channel blockers showed
little or no vasodilating effect on the spastic cerebral
arteries following SAH.7"9 Presumably, multiple vasoactive agents participate in the genesis of chronic vasospasm, and the dependence of the contractions induced
by different vasoactive agents upon intracellularly
stored Ca + + may not be uniform. The dependence of
cerebral artery contractions, induced by different vasoactive agents, upon intracellularly stored C a + + has
not been extensively investigated.
It also seems to be important to compare the dependence of the contractions upon intracellularly stored
Ca + + among different species, since the role of intracellularly stored Ca + + in the constrictor responses of
cerebral arteries to serotonin has been suggested to be
species-dependent.10- "
The present studies compare the role of intracellularly stored Ca + + in the constrictor responses of cerebral arteries to STXA2, PGF^ and serotonin in canine
versus monkey basilar arteries. The STXA2 described
here is a new stable TXA2 analogue and structurally
more similar to natural TXA2 than carbocyclic TXA 2 . l2
From the Department of Neurological Surgery, University of Virginia, Charlottesville, Virginia 22908.
Address correspondence to: Neal F. Kassell, M D., Department of
Neurological Surgery, University of Virginia, Charlottesville, Virginia
22908.
Received October 18, 1984; revision #1 accepted June 12, 1985.
Physiological action of STXA2 in cerebral vessels has
not been extensively investigated.
Materials and Methods
Adult mongrel dogs and cynomolgus monkeys (macaca fascicularis) of either sex were anesthetized with
sodium pentobarbital (30 mg/kg) and sacrificed by exsanguination from the femoral artery. Under magnification, the basilar arteries were dissected from the
brain and placed immediately in modified Kreb's bicarbonate solution [(mM): NaCl 120: KC1 4.5: MgSo4
1.0; NaHCO3 27.0; KH2PO4 1.0; CaCl2 2.5; and dextrose 10.0] at 37°C, which was gassed with 95% O2
and 5% CO2. The pH of the solution ranged from 7.40
to 7.50. The arteries were cut into 4 mm long ring
segments and were suspended between L-shaped stainless steel holders in organ baths with 10-ml working
volumes. The preparations were allowed to equilibrate
at 37°C for 60 minutes before use. Resting tension was
adjusted to 1.5g. Contractile force was recorded isometrically using a Grass FT .03 force-displacement
transducer. The transducer signal was then amplified
and displayed on a Gould 260 multichannel recorder.
In order to check the contractile activity of each specimen, the contractile response to 40 mM KC1 was first
obtained on each ring segment. Only the specimens
which showed good response to 40 mM KC1 were
used.
It was confirmed in the preliminary experiments that
the exposure of the preparations to Ca ++ -free media
containing 0.5 mM EGTA for 15 minutes inhibited
completely the contractile activity of 80 mM K G .
Therefore, in the present experiments examining the
dependence of the contractions induced by serotonin,
PGF^ or STXA2 upon intracellular Ca + + , the preparations were exposed to Ca + + -free media containing 0.5
mM EGTA for 15 minutes. After 15 minutes exposure
to the Ca ++ -free media containing 0.5 mM EGTA,
either 10" 6 M serotonin, lO' 5 M P G F ^ o r S x 10~ 8 M
STXA2 was added. After the contraction had been stabilized, 2.5 mM Ca + + was then added. Contractile
responses to 10"6 M serotonin, 10" 5 M PGF^ or 3 X
10~8 M STXA2 in Ca ++ -free media containing 0.5 mM
STROKE
96
Monkey Basilar
VOL 17, No
1, JANUARY-FEBRUARY
1986
Artery
(gin)
-
-
-
FIGURE 1. Contractile responses of monkey
basilar arteries to serotonin (10~6 M), PGF2a
(W5 M) and 57X42 (3 X 10~8 M) in the
absence and presence of external Ca++ (2.5
mM). The contraction induced by PGF2a or
STXA2 is dependent on intracellular Ca + + to a
greater extent than that induced by serotonin.
5-HT = serotonin.
^'
i
-
•
^
t
5(1 -
(2.
-
*
•
PGF 2a
(IO-'M)
Ca++
(2.5mM)
TXA2
(3xl0"*M)
5nM)
c a*
)
— Ca*+(-)-
-1
Downloaded from http://stroke.ahajournals.org/ by guest on June 17, 2017
EGTA relative to the responses following the addition
of 2.5 mM Ca ++ were evaluated. With respect to the
concentration of each agonist, the concentration producing approximately the maximum contraction was
used in the present study. The preliminary experiments
revealed that 10"6 M serotonin, 10"5 M PGF^ or 3 x
10"8 M STXA2 produces approximately the maximum
contraction in either canine or monkey cerebral arteries. Statistical analyses of the experimental results
were made using the Student's t test.
Drugs and solutions used in this study were 5-hydroxytryptamine (Sigma Chemical Co.), prostaglandin F2" (Sigma Chemical Co.), 9,11-epithio-l 1,
12-methano-thromboxane A2 (Ono Pharmaceutical
Co., Ltd.) and ethyleneglycol-bis(/3-aminoethylether)-N,N'-tetraacetic acid (Sigma Chemical Co.).
STXA2 was dissolved in '/,5 M phosphate buffer (pH
7.4) to make a stock solution (3 x 10"5 M) and diluted
with distilled water before use.
Results
Serotonin (10~6 M) elicited only slight contractile
responses in monkey basilar arteries exposed for 15
minutes to Ca+ + -free media containing 0.5 mM
EGTA, while the addition of 2.5 mM Ca+ + produced
much stronger contractile responses (fig 1). PGF^
(10"5 M) or STXA2 (3 x 10"8 M) elicited more
marked contractile responses than serotonin (10~6 M)
in Ca++-free media containing 0.5 mM EGTA. The
contractile responses to 10"6 M serotonin, 10~5 M
PGF2a and 3 x 10"8 M STXA2 in Ca+ + , were 0.4 ±
0.3 (mean ± S.E), 15.0 ± 2.7 and 23.2 ± 1.8%,
respectively, in monkey basilar arteries, and 3.3 ±
0.7, 9.4 ± 1.4 and 25.4 ± 1.5%, respectively, in
canine basilar arteries (fig 2). In either monkey or
canine basilar arteries, the mean value of the serotonininduced contractions in Ca++-free media containing
0.5 mM EGTA relative to those induced by the addition of 2.5 mM Ca+ + was significantly less (p < 0.05)
than the value with PGF^ or STXA2. The mean value
of the STXA2-induced contractions in Ca++-free media containing 0.5 mM EGTA relative to those induced
by the addition of 2.5 mM Ca ++ was significantly
larger (P < 0.05) than the value with PGF^. Between
monkey and canine basilar arteries, no significant difference of the value with serotonin, PGF^ or STXA2
was observed.
Discussion
Varying effects of Ca ++ channel blockers on the
constrictor responses of vascular smooth muscles to
different vasoactive agents are considered to relate to
the difference of the role played by intracellularly
stored Ca ++ in the constrictor responses.2
Cerebral artery contractions induced by K + , norepinephrine or serotonin have been well known to be
extently dependent on transmembrane influx of extracellular Ca+ + .3-10- '3 By contrast, constrictor responses
of canine basilar arteries to carbocyclic TXA2 have
recently been reported to be relatively associated with
the release of Ca ++ from intracellular storage sites.14
The present results in canine basilar arteries using
STXA2 appear to be almost consistent with the previous report using carbocyclic TXA2.14 The potency
for inducing contractions of canine basilar arteries in
5-HT(IO
100
9
6
M)
PGF 2 0 (IO
M)
TxA2(3xl0
M]
r
50
0
L
Dog Monkey
<n=!2) (n=3)
Dog Monkey
(n=l2) (n=!2)
Dog Monkey
(n=l2) (n = l2)
FIGURE 2. Values of contractions induced by serotonin,
PGF2a, andSTXA2 in Ca+ + -free media relative to those in the
media contaning 2.5 mM Ca+ + . Contractions induced by the
agonists in the media containing 2.5 mM Ca+ + were taken as
100%. Results are expressed as mean ± SEM. n = number of
arterial specimens examined, a: significantly different from the
value with serotonin, b: significantly different from the value
with PGF2a. 5-HT = serotonin.
PHARMACOLOGICAL ACTION OF THROMBOXANE MSasaki et al
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Ca+ +-free media was in the order of STXA2 >
^
> > serotonin > K + .
On the other hand, it has been reported that contractions of canine basilar arteries induced by serotonin are
almost entirely dependent on transmembrane influx of
extracellular Ca + + , while those of bovine basilar arteries are relatively dependent on intracellularly stored
Ca+ + . Therefore it appears to be important to compare
the potencies of vasoactive agents for inducing contractions in Ca ++ -free media among different species.
Such potencies have not been evaluated in monkey
cerebral arteries, which possess pharmacological characteristics similar to those in human cerebral arteries."
The present results revealed that the ability to release
intracellularly stored calcium for the constrictor responses was in the order of STXA2 > PGF^ > >
serotonin = K + in monkey basilar arteries. No significant difference was observed in the ability of any vasoactive agent to release intracellularly stored calcium
for the constrictor responses between canine and monkey basilar arteries.
The pathogenesis of chronic cerebral vasospasm
following SAH has not been clarified yet. However,
there are several previous studies suggesting that
arachidonic acid metabolites such as PGFjJ 6 - 17 or
TXA218- "• w play important roles in the pathogenesis of
chronic vasospasm. On the other hand, present results
revealed that the ability of STXA2 or PGF^ to release
intracellularly stored Ca + + is significantly greater than
that of serotonin or K + in either monkey or canine
cerebral arteries. The beneficial effects of calcium
channel blockers in the treatment of chronic cerebral
vasospasm may be partial, if vasoactive agents such as
PGF^ or TXA2 are involved in the genesis of the cerebral vasospasm, since their contractile action is relatively dependent on intracellular CA + +
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Dependence of cerebral arterial contractions on intracellularly stored Ca++.
T Sasaki, N F Kassell and M Zuccarello
Stroke. 1986;17:95-97
doi: 10.1161/01.STR.17.1.95
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