970
Effects of Oxygen and Glucose Deprivation on
Vasoactivity in Isolated Bovine Middle
Cerebral Arteries
PHILLIP E. VINALL, AND FREDERICK A.
SIMEONE
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SUMMARY The effects of oxygen and glucose deprivation on vasoactivity were investigated using helical
strips of bovine middle cerebral artery. Hypoxla, created by reducing the PO2 of the bath, or oxidative
inhibition with 2,4 dinitrophenol (DNP) or sodium azide, significantly reduced contractions induced by
serotonin. Normal tonic contractions induced with fresh and aged whole blood, or 5-HT became phasic and
qukkly relaxed to baseline in a hypoxic environment.
Glucose elimination from the Krebs medium, or the inhibition of the glycolytic pathway with iodoacetic
add (IAA), did not significantly reduce serotonin-induced contractions. However, contractions were inhibited more with the combination of oxygen and glucose deprivation, or DNP + IAA, than with oxygen
deprivation alone. Efforts to produce rigor hi this preparation by oxygen/substrate reduction or metabolic
Inhibition were unsuccessful.
Tonic contractions induced by 70 mM potassium became phasic as the Ca + + concentration was reduced.
Contractions resulting from the readdition of C a + + to arteries exposed to calcium-free high potassium
solution were significantly reduced in the presence of oxidative and/or glycolytic inhibitors. The uptake of
45Ca + + , as measured by the lanthanum technique, decreased as the bath PO2 was reduced in both
serotonin stimulated and unstimulated arteries. Glucose deprivation alone did not affect 45Ca + + uptake.
This study suggests that hypoxia has a direct inhibitory affect on cerebral vasoactivity mediated by
reductions in sarcoplasmk Ca + + uptake.
Stroke Vol 17, No 5, 1986
2
34
CORONARY '• AND CEREBRAL ' vasospasm are
an established cause of myocardial and cerebral ischemia. This ischemia is the result of reduced blood flow
secondary to arterial contraction and increased vascular resistance. These constricted arteries are also subjected to this secondary ischemia, which may prolong
the spasm.
Most in vitro smooth muscle studies5"7 have indicated that hypoxia and/or substrate depletion can reduce
vasoactivity. This reduced activity may be the direct
result of reduced influx of calcium into the sarcoplasma.8 Vascular smooth muscle is also capable of
developing rigor tension in response to more dramatic
ischemic conditions.9"" This type of muscle rigor is
defined as an increase in resistance to stretch associated with a loss of ATP." Such irreversible constrictions
induced by stretch injury that do not require oxygen,
and are not reversed by treatment with cyanide or by
removal of calcium, have been demonstrated in the
rabbit cerebral artery.12
The purpose of the following experiments was to
explore the effects of oxygen and glucose reduction on
contractions induced by whole blood and blood products (serotonin) in bovine middle cerebral arteries. Efforts were also made to induce rigor tension in response to metabolic depletion. 45Ca + + uptake was
measured in serotonin stimulated and unstimulated
arteries in relation to glucose and oxygen availability.
Findings are discussed with reference to the possible
existence of rigor in arteries subjected to secondary
ishemia due to vasospasm.
From the Department of Neurosurgery, Pennsylvania Hospital,
Eighth and Spruce Streets, Philadelphia, Pennsylvania 19107 U.S.A.
Address correspondence to: Phillip E. Vinall, Division of Neurosurgical Research, Pennsylvania Hospital, 800 Spruce Street, Philadelphia, Pennsylvania 19107, U.S.A.
Received July 9, 1985; revison # 1 accepted January 13, 1986.
Methods
Tissue Preparation
Bovine middle cerebral arteries (MCA) were obtained from a local slaughterhouse, cut into helical
strips and suspended isotonically from Harvard transducers in modified Krebs solution kept at 37°C. The
mM composition of the Krebs solution was as follows:
NaCl, 118.9; KC1, 4.7; KH 2 PO 4 , 1.2; MgSO 4 , 1.2;
CaCl2 1.2; NaHCO 3 14.9; dextrose, 11.2. The control
gas mixture contained 12% oxygen, 5% carbon dioxide, balance nitrogen. The CO2concentration was always maintained at 5%. Oxygen (95%, 12%, 0%) and
nitrogen (0%, 83%, 95%) concentrations were combined to produce varying gas mixtures. These oxygen
concentrations produced the following bath PO2 (mm
Hg): 95%O 2 = 500^600, 12%O2 = 90-100, 0%O2 =
10—25. The osmolarity of all solutions was 275 and pH
was 7.4. Arteries were allowed to equilibrate for 60
minutes in Krebs solution with 1.25 grams tension
applied. Viability of the vessel was determined by the
rate of adjustment of the strips to resting length upon
the application of tension. Strips that did not stretch at
a rate of 0.5 mm/min were rejected as too damaged.
Contractions were expressed in mm/cm resting length
of the strip (RLo).
Solutions and Drugs
The lanthanum-HEPES solution contained the following (mM): NaCl, 122; KC1, 5.9; MgCl2, 1.2; dextrose, 11.2; LaCL,, 10; HEPES-Tris, 6. The pH of the
HEPES-Tris stock solution was adjusted to 7.4 with
NaOH. Fresh rabbit and human whole blood were extracted and added to the bath before clotting occurred.
Aged whole blood was produced by placing human
and rabbit blood in an incubator at 37°C for seven
days. Cumulative calcium contractions were induced
in arteries washed with calcium-free 70 mM KC1 solu-
METABOLIC INHIBITION OF MCA VASOACTIVITY/Vma// & Simeone
tion. High K + solution was made by increasing the K +
concentration and decreasing the Na + to maintained an
osmolarity of 275 milliosmoles. Metabolic inhibition
in high potassium solution was produced by removal of
glucose and oxygen from the bath medium according
to the method used by Bose & Bose9 to induce rigor in
taenia coli.
PO2 of the bath was measured with an IL pH/gas
analyzer, Model 113. Serotonin hydrochloride (5HT), sodium azide (NaN ? ), 2,4 dinitrophenol (DNP),
and sodium iodoacetic acid (IAA) were obtained from
Sigma Chemical Co. All drugs were dissolved in double deionized water. Concentrations are given as the
salt and expressed as the final molar concentration in
the bath.
Calcium Uptake
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45Ca + + uptake was measured with the lanthanum
technique.13 This method is based on the principle that
10 raM lanthanum displaces extracellular calcium,
blocks both Ca + + uptake and efflux, and does not enter
the cell to alter cellular Ca + + distribution. Cerebral
arteries were placed in Krebs solution for 60 min before the tissue was labelled with 45Ca (0.05uCi/ml).
Radioactive 45Ca + + was obtained as CaCl2 from New
England Nuclear Co. The labelled arteries were allowed to equilibrate for 30 min before 10" 3 M 5-HT
was added to the bath. Arteries were left in contact
with 5-HT or the control medium for one hour before
being washed first with lanthanum-HEPES solution
containing 45Ca + + with the same specific activity as
before, then with a non-radioactive lanthanum-HEPES
solution. The strips were left in the latter solution for
one hour. After blotting and weighing, the arterties
were dried overnight and dissolved in 1 ml Protosol
(New England Nuclear Co.) at 60°C for three hours.
Standard liquid scintillation counting, with appropriate
controls, was performed and the efficiency was determined with internal standards. Calcium content was
expressed as mM/kg wet weight of tissue.
Statistical Treatment of Data
Statistical analysis of calcium uptake values and
dose response contractions were performed with the
Student's t-test using a paired test for correlated variables. Differences were considered significant at the
no,
971
95% confidence level. Mean ± SEM are reported for
all uptake and contractile values.
Results
Bovine MCA were subjected to 95% and 0% oxygen
aeration for 30 min before being contracted with fresh
or aged rabbit, or human whole blood (0.1ml), or 5HT (10~6 M). Normal tonic contractions that can last
up to 6 hours in this preparation became phasic in
the low oxygen environment and relaxed to baseline
(fig. 1). Similar phasic responses also occurred with
aged blood in the hypoxic bath.
Serotonin responses were significantly (p < .01)
depressed in the absence of oxygen (fig. 2), while the
absence of only glucose did not significantly affect
similar contractions. Arteries were washed four times
with glucose-free Krebs solution for 30 min. before 5HT contractions were induced. The combination of 0%
oxygen and glucose-free washings reduced the 5-HTinduced contractions more than oxygen deprivation
alone. Serotonin responses returned to control values
upon the readdition of glucose and oxygen after four
hours of no oxygen or glucose.
A similar depression of 5-HT responses occurred
when the arteries were exposed to the oxidative inhibitors DNP (10' 4 M) orNaN 3 (10~5 M), or the glycolytic
inhibitor IAA (2 x 10~5 M) for 30 min prior to stimulation (fig. 3). NaN3 inhibited the 5-HT responses
throughout the dose response range, while DNP inhibited contractions induced by lfr5 and 10^ M 5-HT
only. Significant (p < .01) reductions in the amount of
contraction induced by 5-HT occurred in the presence
of DNP and NaN3, but not IAA, while the combination
of DNP and IAA totally eliminated all contactile responses. DNP and glucose-free Krebs washings also
completely inhibited all 5-HT responses. Responses to
5-HT returned to control values after washing the vessels with Krebs without metabolic inhibitors.
When MCA were exposed to 70 mM potassium
solution containing 1.2 mM Ca + + , a sustained contraction occurred. As the Ca + + concentration was reduced, the K + response became phasic (fig. 4). If all
calcium was removed from the 70 mM K + solution,
the K + response quickly relaxed almost to baseline
within 20 min. If Ca + + was readded to the above
calcium-free, high K + solution, contractions again oc-
FIGURE 1. Comparison of the effects of 95%
and 0% oxygen on fresh human whole blood
(HWB)-, fresh rabbit whole blood (RWB)- and
serotonin-induced contractions in the MCA.
Note the development of spontaneous activity
in arteries contracted with serotonin in a hypoxic environment.
STROKE
972
VOL 17, No 5, SEPTEMBER-OCTOBER
1986
2.4-
.3 2.0-
• 1 2 % 0 2 - G I U C O M (n«16)
1.6-
DO % O2-Glucose (n=6)
20mki.
1mm
• 12 % ^-Glucose-Free (n=12)
E
E
1.2-
oOSt O9-Glucose-Fret
(n=6)
1.2mM Ca
TOmMK*
1mln.
O820 mm.
1mm
0.4-
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10'
XT
10"
10
SEROTONIN (M)
FIGURE 2. Effects of 0% oxygen and glucose-free Krebs
washings on serotonin cumulative dose responses in MCA.
Each point is the mean ± SEM. Points below (*) are significantly (p < .01) different from their corresponding control
(12% oxygen!glucose) responses. Rio is optimal resting length
of the strip.
curred which were concentration dependent (fig. 5).
Most Ca + + -induced contractions in the presence of 70
mM K + were equally and significantly (p < .01) depressed by the presence of DNP (10~4 M), NaN3 (10" 3
M) or IAA (2 x 10" 3 M). Inhibitors were added 30
min. before C a + + was readded. Like the 5-HT-induced contractions, the combination of DNP and IAA
produced complete inhibition of all C a + + responses.
45Ca + + uptake studies revealed a relationship between the availability of oxygen and the amount of
intracellular calcium (fig. 6). 45Ca + + was measured in
OimMCi
70mM K'
1mm.
20mtn.
1mm
O.OmMCa
70mM K*
i i
1mln.
FIGURE 4. Effects of Ca++ reduction and elimination on tonicity of 70 mM K+ contraction in MCA. Artery was washed with
potassium (K*) solution at arrows.
the presence (stimulated) and absence of 10~3 M 5-HT
by the lanthanum technique. When bath oxygen concentration was decreased from 95% to 12%, the mean
45Ca + + uptake decreased from 0.227 ± .02 to 0.183
± .01 mM/Kg wet wt. in unstimulated arteries (p <
2.0-
I 2.0-1
* CONTROL N=12
•Contro) (n = 24)
• IAA 2xK)~ S M(N=8)
5
• IAA 2 X 1 0 " M ln=7l
Z1.6-
2
o
°DNP 10" 4 M(N=8)
D D N P 1 0 " 4 M ln«7)
•NaN 3 10- 5 M(N=7)
• NaN 3 10~ 5 M (nn5l
1.2-
oONP + IAA(N=8)
oDNP + IAA (n=6)
A.
80.80.410
10
-2
CALCIUM (M)
10
10'
-4
10
SEROTONIN (M)
FIGURE 3. Effects of oxidative (DNP-2,4 dinitrophenol;
NaNySodium azide) and glycolytic (lAA-iodoacetic acid) inhibitors on serotonin cumulative dose responses. Each point is
the mean ± SEM. Points below (*) are significantly (p < .01)
different from their corresponding control (12% oxygenlglucose) responses.
FIGURE 5. Effects of oxidative (DNP-2,4 dinitrophenol;
NaNj-sodium azide) and glycolytic (IAA-iodoacetic acid) inhibition on cumulative Ca++ dose responses in MCA. Ca++ was
added to calcium-free 70 mM K+ solution 30 min after the
addition of the metabolic inhibitors. Each point is the mean ±
SEM. Points below (*) are significantly (p < .01) different from
their corresponding controls (no inhibitors, 12% oxygenlglucose) responses.
METABOLIC INHIBITION OF MCA VASOACTIVITY/V/Vw// & Simeone
QCONTROL
05-HT
10"5M
Dai89±.oi
95X02
GLUCOSE
FIGURE 6.
12X02
GLUCOSE
So.u7±.oi
£0.128 ±.01
12% O j
OXOj
GLUCOSE- GLUCOSE
FREE
D0.130t.01
OX O2
GLUCOSEFREE
Effects of 95% (P02 @ 550 mm Hg), 12% (P02 @
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95 mm Hg), 0% (PO2 @ 18 mm Hg) oxygen and the absence of
glucose on 45Ca++ uptake in stimulated (5-HT) and unstimulated(control)MCA. Absolute values ± SEMof45Ca+ + above
bars. Significant differences in the amount of45Ca++ uptake
between control and 5-HT treated arteries are indicated by(*)p
< .001, (**) p < .01.
.05). In the absence of oxygen (0%), 45Ca + + uptake
decreased to . 120 ± .01 mM/Kg wet wt. This was also
significantly (p < .05) less than the amount of 45Ca + +
uptake seen in the presence of 12% oxygen. The absence of glucose did not appear to significantly affect
45Ca + + uptake in either stimulated or unstimulated
arteries, while the amount of uptake induced by 5-HT
appeared significantly dependent upon the amount of
oxygen being delivered to the artery. Arteries exposed
to 95% O2 had a net 45Ca + + uptake of .79 ± .02
mM/Kg compared to .25 ± .01 mM/Kg exposed to
12%, and .08 ± .01 exposed to 0% oxygen after
contraction with 10~5 M 5-HT. Uptake was significantly (p < .05) increased in stimulated (compared to
unstimulated) arteries exposed to 95% and 12%, but
not 0% oxygen.
Efforts to produce in vitro rigor using the method of
Bose & Bose9 were unsuccessful. Arteries contracted
with glucose-free 70 mM K + solution and subjected to
0% oxygen relaxed, but no secondary contraction followed. Metabolic poisons (DNP + IAA) added to a
high K + medium also did not induce a rigor type response in this preparation. Most vessels contracted
with human whole blood (fresh or aged), or 5-HT
(10~5 M) in glucose-free Krebs solution relaxed when
the all oxygen was replaced with nitrogen, and these
arteries remained relaxed for the duration of the experiment (up to 3 hours). On occasion the introduction of
95% N2 in the above manner produced a contraction
that was quickly followed by relaxation. Arteries left
in an oxygen/glucose-free environment for six hours
showed no signs of rigor-like contraction.
Discussion
This study indicates that hypoxia has a direct inhibitory affect on cerebral vasoactivity that is associated
with a reduction in the uptake of Ca + + into the sarco-
973
plasma. Whole blood- and serotonin-induced contractions were reduced in hypoxic and metabolically inhibited environments. The tonic part of the contraction
appeared more sensitive to hypoxia than initial quick
phasic part. 45Ca + + uptake was significantly reduced
when the bath PO2 was reduced.
Hypoxia can induce a contraction in isolated pulmonary arteries,14'13 aorta,16 and in at least one study,
hypoxic induced contractions in the presence of 5-HT
have been demonstrated in the dog basilar artery.17 On
occasion we also noted small hypoxia-induced contractions in the presence of 5-HT that quickly relaxed
to baseline, but this response was not consistent from
artery to artery. This could be due to our inability to
reduce bath PO2s to low enough levels to consistently
observe this phenomenon. Even in the presence of 0%
oxygen, bath PO2s as high 25 mm Hg were sometimes
detected. In the taenia coli intracellular Ca + + increases
during hypoxia 18 ' l9 due to an increase in extracellular
calcium influx, while a decrease in the sequestration of
Ca + + ions in binding stores occurs in airway smooth
muscle.20 However most studies indicate that hypoxia
induces a depression of vascular activity,21"23 and this
reduced vasoactivity may be associated with reduced
45Ca + + uptake.8
In excitation-contraction coupling in smooth muscle
calcium can be obtained from either extracellular
sources or released from cellular storage sites. However, cerebral arteries appear to be more dependent on
extracellular than on sequestered cellular calcium for
contractile responses than other arteries. 24 ' u The initial
fast part of the contraction appears regulated by a Ca + +
influx process different from that controlling the slow
tonic part of the contraction.26 Contractions activated
by K + and sensitive to D600, and those stimulated by
alpha-adrenoceptor activation and relatively resistant
to D600 are associated with two separate Ca + + entry
pathways, a potential and receptor operated channel,
respectively.27'M In this study hypoxia affected the tonic part of the 5-HT or whole blood contraction more
than the phasic, and also significantly reduced the
Ca + +-induced contraction in 70 mM K + solution. The
reduction of bath Ca + + decreased the steady tonic response normally produced by high K + , more than the
initial fast part of the contraction. The fact that a contraction occurred in the absence of calcium probably
indicates that small amounts of Ca + + may remain in
the extracellular spaces. If arteries are pre-washed with
Ca-free Krebs containing 1.0 mM EGTA, no contraction results when the arteries are stimulated with 70
mM K + calcium-free solution (unpublished observation). Thus, hypoxia may affect the direct influx of
extracellular Ca + + , more than the release of sequestered Ca + + (either intracellular or membrane bound)
associated with the potential and receptor operated calcium channels.
Contractions induced by 5-HT were affected more
by the lack of oxygen or the presence of oxidative
inhibitors, than by the lack of glucose or the presence
of a glycolytic inhibitor. 45Ca + + uptake was also not
affected by the absence of glucose. However, arteries
974
STROKE
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continued to contract even in near anoxic environments when glucose was present. The absence of oxygen and glucose, or the presence of both a glycoltic and
oxidative inhibitor reduced the 5-HT responses more
than the lack of oxygen alone. Other studies have reported total loss of vascular contractility in the absence
of both oxygen and glucose.5'21"23 In this study significant, though reduced, contractions occurred in the absence of both oxygen and glucose. This may indicate a
role for glycolysis in the contractile process in this
artery and/or the presence of large stores of glycogen.
However smooth muscle cells are known to contain
small stores of glycogen.29 A more probable explanation for the relatively large 5-HT response may be that
0% oxygen does not lower bath oxygen below levels
that totally inhibit contraction in this vessel. Even after
three hours of 0% oxygen aeration PO2 levels as high
as 25 mm Hg could still be detected in the bath on
occasion. This may be sufficient oxygen to support the
contractions seen in the absence of oxygen and glucose. These same contractions were totally eliminated
when both the oxidative and glycolytic pathways were
poisoned with the combination of DNP + IAA. The
finding that DNP + glucose-free treatment produced
complete loss of vasoactivity also argues for the existence of small glycogen stores in this artery.
The decrease in Ca + + uptake and contractility due to
hypoxia may be related to the availability of ATP. In
taenia coli hypoxia is associated with decreased ATP
levels. 3031 Similarly, ATP decreases during ischemia
in human cerebral tissue.32 Oxidative phosphorylation
provides most of the energy required to synthesize
ATP directly during contraction of vascular smooth
muscle,33 and it was the oxidative mechanism which
appeared more sensitive to hypoxia in this preparation.
Unlike skeletal muscle, smooth muscle contains meager energy stores of ATP, creatine phosphate and glycogen. 2934 Thus in vivo, cerebral arteries may be dependent upon adequate supplies of oxygen and ATP to
offset pathological states which tend to reduce blood
flow, such as long term vasospasm.
Efforts to demonstrate the existence of rigor in this
preparation were disappointing. Bose & Bose,9 and
later Butler et al," were able to induce rigor in other
smooth muscle in the absence of glucose and oxygen.
Rigor is associated with low ATP levels, increased
resistance to stretch and occurs independent of elevations of cytoplasmic calcium. 1031 An explanation of
long-term vasospasm following subarachnoid hemorrhage in terms of" rigor is an attractive hypothesis. The
secondary ischemia due to reduced blood flow may
reinforce the initial blood induced contraction by reducing the ATP stores required to drive calcium back
into the sarcoplasmic reticulum, mitochrondria and extracellular space, and hence foster relaxation. Again
our inability to demonstrate a rigor type response in
this artery may reflect our inability to reduce bath
oxygen levels, hence ATP levels, to the critical level
required to induce a rigor type contraction.31 Even in
the absence of both glucose and oxygen, significant 5HT contractions still occurred in this preparation indi-
VOL 17, No
5, SEPTEMBER-OCTOBER
1986
cating adequate energy was available for vasoconstriction. Carotid arteries stimulated with high K + solution
containing metabolic inhibitors do not relax when the
vessels are returned to a physiological environment.33
This condition coincides with the almost complete loss
of ATP and phosphorylcreatin. Thus, in the presence
of an extreme hypoxic environment and the significant
loss of smooth muscle ATP, arteries may remain contracted, even in the absence of the original stimulus.
In conclusion, we feel the reduced vasoactivity seen
in response to hypoxia in the cerebral vascular system
is the end result of reduced calcium uptake into the
smooth muscle cell. Cerebral arterial contractions
were depressed by decreasing oxygen availability, and
these reduced responses were reflected by reduced
amounts of 45Ca + + uptake.
Since 45Ca + + uptake seems to be directly related to
oxygen availability, and 10-20% oxygen can produce
physiological bath PO 2 s, we question the use of such
unphysiologic oxygen concentrations as 95% O2 used
in most in vitro smooth muscle experiments, particularly in studies which use thin wall vessels like the
bovine cerebral artery. Such high concentrations of
oxygen may in fact favor unphysiological amounts of
calcium entering the cell, or as documented in porcine
coronary arteries suppress local regulation of arterial
tone.36
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P E Vinall and F A Simeone
Stroke. 1986;17:970-975
doi: 10.1161/01.STR.17.5.970
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Copyright © 1986 American Heart Association, Inc. All rights reserved.
Print ISSN: 0039-2499. Online ISSN: 1524-4628
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