329
Protective Action by Methylprednisolone,
Allopurinol and Indomethacin Against
Stroke-Induced Damage to Adenylate Cyclase
in Gerbil Cerebral Cortex
MICHAEL D . TAYLOR, P H . D . , GENE C. PALMER, P H . D . , AND ALFRED S. CALLAHAN III,
M.D.
Downloaded from http://stroke.ahajournals.org/ by guest on July 31, 2017
SUMMARY Adenylate cyclase activity was investigated in either homogenate or particulate fractions
from the frontal cerebral cortex of the gerbil following five experimental conditions of bilateral ischemia.
After periods of 15 min ischemia, 15 min ischemia plus 15 min of recirculation or 60 min ischemia the
enzyme generally displayed enhanced responses to GTP, norepinephrine (NE), dopamine (DA), NE + GTP
and DA + GTP. Pretreatment of the gerbils with methylprednisolone, allopurinol or indomethacin did not
significantly influence the outcome of these findings. When the animals were subjected to 60 min ischemia
plus 15 min of reflow, enzyme responses to the stimulatory agents including forskolin and NaF were all
reduced. Pretreatment with methylprednisolone, allopurinol or indomethacin prevented the damage to
adenylate cyclase in the 60 min ischemia plus 15 min reflow animals. When animals were made ischemic for
15 min followed by one week of recovery, enzyme sensitivity to GTP, calmodulin-Ca+ + , NE, combinations
thereof and forskolin were reduced in only the particulate fractions. Enzyme damage was reversed following methylprednisolone. Enzyme damage may result from generation of free radicals during reflow and
drugs that either inhibit synthesis pathways generating free radicals, stabilize cell membranes or act as free
radical scavengers may be therapeutically beneficial under specific conditions of stroke.
Stroke Vol 15, No 2, 1984
CYCLIC NUCLEOTIDES, namely adenosine 3 ' , 5'monophosphate (cyclic AMP) and guanosine 3 ' , 5'monophosphate (cyclic GMP) appear to be involved as
a consequence of traumatic insults to the brain. Thus
seizures, cerebral contusion, cerebral concussions,
stab wounds, anoxia and ischemia are all associated
with rapid alterations in brain cyclic nucleotide levels.'"6 With regard to ischemia, bilateral clamping of
the common carotid arteries in gerbils evoked a
marked rise in cerebral levels of cyclic AMP concomitant with a drop in cyclic GMP concentrations. Under
conditions of reflow of blood to the brain cerebral
levels of both cyclic AMP and cyclic GMP were increased.4 The question herein was what perturbations
did bilateral ischemia cause to the enzymes responsible
for formation of the two cyclic nucleotides? Schwartz
et al, 7 demonstrated that adenylate cyclase (EC
4.6.1.1) activity did not change in gerbil cerebral cortex up to an hour after clamping one carotid artery.
However, if recirculation of blood was allowed the
activity of the enzyme declined up to 5 hr post-ischemia. In follow-up investigations we reported that periods of bilateral ischemia (15 min with or without 15
min reflow and 60 min — no reflow) resulted in an
enhanced sensitivity of cerebral adenylate cyclase to
catecholamines and GTP. However, after 60 min of
bilateral ischemia followed by 15 min of recirculation
neurohumoral activation of adenylate cyclase was now
From the Frist-Massey Neurological Institute, 356 24th Avenue N.,
Suite 104, Nashville, Tennessee 37203.
Supported by the Frist-Massey Neurological Institute. A portion of
this work was performed at the Department of Pharmacology, University of South Alabama College of Medicine, Mobile, Alabama where
M.D. Taylor was a doctoral candidate.
Address correspondence to: Dr. Gene C. Palmer, Director of Research, Frist-Massey Neurological Institute, 356 24th Ave. N , Suite
104, Nashville, Tennessee 37203.
Received March 16, 1983; revision 1 accepted August 25, 1983.
depressed. Neither guanylate cyclase (EC 4.6.1.2) nor
cyclic AMP phosphodiesterase (EC 3.1.4.17) activities were altered in these experiments.8
The damage to central enzymes seen after a period
of ischemia followed by recirculation has been termed
"secondary ischemia" and has been attributed to various biochemical processes namely: release of cellular
lysosomes, breakdown of the blood-brain barrier,
Ca + + influx, formation of free radicals, rise in extracellular K + , prostaglandin formation, cellular acidosis
and damage to cell membrane components resulting
from activation of membrane phospholipases.9"13
Therefore, any agent offering protection against one or
more of these processes might afford a degree of therapeutic benefit toward stroke-induced damage. In the
present investigation we report that the greatest damage to the adenylate cyclase system in the gerbil cerebral cortex was seen following reflow of blood subsequent to 60 min of bilateral ischemia. Injections of
methylprednisolone, an agent that stabilizes cell membranes, prevents lysosomal breakdown and acts as a
free radical scavenger prevented the damage to adenylate cyclase. In addition, allopurinol (inhibits xanthine
oxidase) and indomethacin (inhibits cyclo-oxygenase
and prostaglandin synthesis) two agents that inhibit
enzymatic processes associated with free radical formation were likewise effective. Furthermore it is not
known what influence the release of lysosomes, Ca + + ,
free radicals, acidosis, etc. would have on central
acenylate cyclase. In an attempt to circumvent some of
these considerations a particulate form of the enzyme
was evaluated in addition to cortical homogenates.
Methods
Female gerbils (Meriones unguiculatus, 35-50 gm)
were purchased from Tumblebrook Farms, West
Brookfield, Massachusetts. Bilateral ischemia was
performed according to the procedure of Lust and Pas-
330
STROKE
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sonneau.4 The animals were fasted for 24 hours followed by anesthesia (thiopental 35 mg/kg). A midline
cervical incision was made and both common carotid
arteries were isolated and looped with silk thread (60). After a 3 hour recovery period, both carotid arteries
were occluded using aneuerysm clamps. Ischemia was
allowed for either 15 min (reversible) or 60 min (irreversible). 414 In designated experiments the clamps
were removed from the 15 min ischemia animals and
recirculation of blood was allowed for either 15 additional min or 7 days. With the 60 min ischemic period
specified animals received a period of 15 min of reflow.
Reflow was verified by visual inspection of blood
flowing past the point of occlusion. Almost all animals
(95%) survived the period of 15 min ischemia. With
the use of thiopental as an anesthetic 80% survive the
60 min ischemia and associated reflow. At the termination of the ischemia experiments the animals were decapitated and the frontal cortex (rostral to the anterior
commissure) from both sides was rapidly removed and
homogenized in glycyl-glycine buffer (2 mM plus 1
mM MgSO 4 with 1.6 mM EGTA, pH 7.4). In one
group of animals the cortical homogenates were centrifuged at 800 x g for 8 min. The resulting supernatant
was recentrifuged at 27,000 x g for 20 min. This latter
procedure was repeated and the final pellet (calmodulin-free) again was resuspended in glycylglycine buffer
and used as a paniculate form of adenylate cyclase (60
ug enzyme protein per sample).15 The method of
Lowry and coworkers16 was used for determinations of
sample proteins.
Adenylate Cyclase Determination
Our previous method was followed in order to assay
adenylate cyclase. 817 The enzyme preparations (100
ul) were added to a reaction mixture containing the
following at final concentrations and volumes: (a) 40
ul solution containing isobutyl methylxanthine-papaverine (0.2 mM each), phosphoenol pyruvate (1 mM),
pyruvate kinase (8 ug), Hepes buffer (40 mM, pH7.4),
and buffered mercaptoethanol (1 mM); (b) 20 ul of
control solutions, drugs, GTP, or catecholamines. In
addition, in designated paniculate samples 3.42 ug of
calmodulin plus C a + + (10~ 4 M) were added to the
assay mixtures;18 (c) the reactions were initiated by
adding 40 ul of ATP (2 mM) - MgSO4 (4 mM),
vortexing and incubating 8 min at 37° C. Enzyme
reactions were terminated by capping the tubes and
boiling in a water bath (4 min). After cooling and
centrifugation a sample of the supernatant was taken
for assay of cyclic AMP. 19 Adenylate cyclase activity
was expressed as picomoles of cyclic AMP formed per
min per mg protein. All assays for individual samples
were in duplicate.
Drug Injections
Methylprednisolone sodium succinate (Solu-Medrol, a gift from Upjohn Co) was injected (30 mg/kg)
30 min prior to clamping the carotid arteries. In the
VOL 15, No
2, MARCH-APRIL
1984
animals that were alowed to recover for one week,
methylprednisolone was also injected at 8 and 16 hours
post-ischemia. Indomethacin (Sigma Chem. Co.) was
injected 2 mg/kg, 1 hour prior to bilateral ischemia
while allopurinol (Sigma Chem. Co.) was injected 5
mg/kg 30 min before ischemia. Sham operated gerbils
were injected with distilled water containing 0.9%
benzylalcohol used to dissolve methylprednisolone,
30% ethanol used to solubilize indomethacin or 50%
dimethylsulfoxide used to dissolve allopurinol. All
drugs were injected ip. In designated samples these
drugs or equivalent vehicle solutions were added to
adenylate cyclase preparations in vitro. Furthermore
the drugs were injected into selected unoperated gerbils and their lack of influence on the adenylate cyclase
preparations was noted. We also evaluated the effects
of in vivo injections of methylprednisolone on cyclic
AMP dependent phosphodiesterase assayed in vitro
using the cortical homogenates under conditions of
high (0.4 mM) or low (1 uM) substrate (cyclic AMP).
For this experiment previous methodology was followed.8- l7
The Student's paired t test was used to compare data
between sham operated control and ischemic animals.
Data between drug injected or ischemic animals and
sham operated controls was compared using the Student's two-tailed t test. A " P " value of less than 0.05
was considered to be a significant change. Either 4 or 5
animals were used for each experimental condition.
Results
Normal Animals
In homogenates of gerbil frontal cortex both NE and
DA (10- 4 M) or GTP (10~5 M) elevated adenylate
cyclase by 30-40% over respective basal activity. In
the presence of GTP the action of NE and DA was
enhanced in an additive manner to a degree of stimulation of 65-85% over basal enzyme activity. Forskolin
(10~5M) and NaF(5 mM) stimulated adenylate cyclase
to approximate values of 160 and 250% respectively
(fig. 1, tables 1-4).
Adenylate cyclase in paniculate preparations of
frontal cortex was activated by 10" 5 M GTP (37%),
3.4 ug calmodulin- 10~4 M Ca + + (24%), 10" 4 M NE
(20%), NE + calmodulin-Ca ++ in the absence (33%)
or presence of GTP (55%) and 10" 5 M forskolin
(233%) (fig. 2).
a. Fifteen min Ischemia with No Recirculation
Homogenates of frontal cortex displayed an increased adenylate cyclase sensitivity to DA, DA +
GTP, forskolin and NE + GTP following a 15 min
period of bilateral clamping of the carotid arteries.
When methylprednisolone was preinjected 30 min prior to the ischemia the only observation that differed
from sham-operated controls was that the enzyme was
more sensitive to GTP alone (table 1). Neither allopurinol nor indomethacin when preinjected into the animals had any prominent effects on adenylate cyclase
ADENYLATE CYCLASE DURING STROKE/Tnv/or el al
HOMOGENATE
331
TABLE 1 Effect of 15 min Bilateral Ischemia (no reflow) on Adenylate Cyclase in Homogenates of Gerbil Frontal Cortex
Adenylate cyclase (% stimulation
over basal activity)
ISCHEMIA (6Omin+l5mmreflow)
ISCHEMIAtMETHVLPREDNISOLONE)
Ischemia
Additions
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GTP
DA
DA +
GTP
NE+ FORSK- NaF
GTP OLIN
FIGURE 1. Effect of 60 min bilateral ischemia plus 15 min
reflow on adenylate cyclase in homogenates of gerbil frontal
cortex. The values represent the mean ± SE percent enzyme
stimulation over respective basal activities (sham = 43 ±4,
ischemia = 39 ± 3, and ischemia with methylprednisolone =
44 ± 5, expressed as pmol of cyclic AMP formed/minJmg protein). The number of separate experiments are 4. *Denotes a
significant change from the respective sham operated controls.
Concentrations of GTP and forskolin are I0~5 M, NE and DA
are I0~4 M and NaF is 5 mM. Methylprednisolone was injected
at 30 mglkg 30 min before bilateral clamping of the carotid
arteries.
when assessed after 15 min of ischemia (data not
shown).
The observations for the paniculate enzyme preparations were somewhat different to that of the homogenates. In this case sensitivities of adenylate cyclase
were significantly augmented in response to GTP, NE,
DA and forskolin following 15 min ischemia. There
were no differences in enzyme sensitivity between pretreated (methylprednisolone, allopurinol and indomethacin) and untreated ischemic animals (data not
shown).
b. Fifteen Min Ischemia Plus 15 Min Recirculation
In this situation the sensitivity of adenylate cyclase
in the homogenate to GTP, DA, NE, DA + GTP and
NE + GTP was enhanced in the ischemic animals.
The paniculate enzyme was more sensitive to NE and
DA + GTP. Pretreatment of the gerbils with methylprednisolone apparently reduced to sham control values the adenylate cyclase (homogenate) elevation by
NE and NE + GTP (table 2). Pretreatment with either
allopurinol or indomethacin did not produce any
change in the enhanced enzyme activation (both homogenate and paniculate) as a consequence of 15 min
Sham
Control
Methylprednisolone
GTP 1 0 - ' M
36±3
33±3
61±6*t
DA 10~ 4 M
31 ± 2
41±7*
42 ± 5 *
NE 1 0 - " M
37 ± 1
43 ± 7
53 ± 5 *
DA + GTP
65 ± 3
78 ± 2 *
85 ± 5 *
NE + GTP
78±3
98 ± 2 *
110 + 20
Forskolin 10~ 5 M
149 ± 6
133+15
175 + 5
NaF 5 mM
180±3O
190±25
255 ± 1 0
Values are the mean ± SE of 4 experiments
•Significantly different (p < 0.05) than corresponding shamoperated control
tSignificantly different than ischemia control.
Basal enzyme activities (pmol cyclic AMP formed/min/mg protein) are. sham = 43 ± 4; ischemia control = 41 ± 4 ; and
ischemia with methylprednisolone pretreatment = 39 + 7.
ischemia followed by 15 min reflow (data not shown).
c. Sixty Min of Ischemia with No Recirculation
In table 3 are the results of the effects of irreversible
ischemia on the sensitivity of adenylate cyclase in homogenates of gerbil frontal cortex. Enzyme stimulation by GTP, DA + GTP and NE + GTP was elevated
over the sham controls. In ischemic gerbils preinjected
with methylprednisolone activation of adenylate cyclase by GTP, DA, NE, and NE + GTP was augmented when compared to the sham control. Moreover, the
sensitivity of NE differed from the ischemic control
animals. The effects of preinjected allopurinol or indomethacin on the experiment were unremarkable.
Sixty min of bilateral ischemia caused only an elevated sensitivity of the paniculate adenylate cyclase to
TABLE 2 Effect of 15 min Bilateral Ischemia Plus 15 min Reflow
on Adenylate Cyclase in Homogenates of Gerbil Frontal Cortex
Adenylate cyclase (% stimulation
over basal activity)
Ischemia
Methylprednisolone
Sham
Control
GTP 10-5 M
33 ± 2
43 ± 4 *
45 ± 3 *
DA 10- 4 M
27 ± 2
35 ± 3 *
36±9
NE 10-4 M
38 + 2
50 + 2*
39±6
DA + GTP
68 ± 2
79 ± 4 *
83 ± 3 *
NE + GTP
77±3
98 + 7*
82±6
Additions
Forskolin 1 0 " ' M
165±16
152±13
172±6
NaF 5 mM
240+15
300±24
238 ±22
Values are the mean ± SE of 4 experiments.
•Significantly different (p < 0.05) than corresponding shamoperated control.
Basal enzyme activities (pmol cyclic AMP formed/min/mg protein) are: sham = 42 ± 4; ischemia control = 44 ± 3; and
ischemia with methylprednisolone pretreatment = 41 ± 6 .
STROKE
332
VOL
•
PARTICULATE FRACTION
SHAM
220-
•
ISCHEMIA (60mfn+1 week reflow)
200-
[I
ISCHEMIA (METHYLPREDNISOLONE)
Additions
GTP lO- 5 M
DA 10- 4 M
NE 10" 4 M
DA + GTP
NE + GTP
Forskolin 10" 5 M
NaF 5 mM
Sham
35 ± 3
30±3
36+1
62±2
77±4
15O±5
255 ±15
Control
53 ±4*
36±6
31 ±4
83 ±7*
102±8*
163±9
Methylprednisolone
52 + 3*
41 +3*
52±8*t
72±6
100±3*
166±6
275 + 5
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300 + 20
Values are the mean ± SE of 4 experiments.
•Significantly different (p < 0.05) than corresponding shamoperated control.
tSignificantly different than ischemia control.
Basal enzyme activities (pmol cyclic AMP formed/min/mg protein) are: sham = 38 ± 4; ischemia control = 43 ± 4 and ischemia
with methylprednisolone pretreatment = 46 ± 6.
NE. Pretreatment of the gerbils with methylprednisolone, allopurinol or indomethacin failed to influence
the paniculate enzyme under these experimental conditions (data not shown).
d. Sixty Min of Ischemia Plus 15 Min of Recirculation
As noted in our previous work8 this experimental
situation results in the greatest damage to the cerebral
adenylate cyclase system. In cortical homogenates enzyme responses to DA, NE, NE + GTP, NaF and
forskolin were attenuated. Pretreatment with methylprednisolone (30 mg/kg, 30 min prior to bilateral
clamping of the carotids) prevented the damaging effects of 60 min ischemia plus 15 min reflow. In fact the
responses to GTP, DA, DA + GTP and NE + GTP
TABLE 4 Protection by Therapeutic Agents of Damage to Adenylate Cyclase Induced by Secondary Ischemia in Homogenates of
Gerbil Frontal Cortex
60 min ischemia
+ 15 min reflow
Treatments
Conditions
GTP 10- 5 M
DA 10" 4 M
NE 10- 4 M
DA + GTP
NE + GTP
Forskolin 10" 5 M
NaF 5 mM
Sham
30±2
38 ± 3
41 ± 5
71+7
85 + 5
164±2
270±15
None
Allopurinol
Indomethacin
50 + 3*
41±5
40±6
21±2* 51±3*t 68 + 7*t
63 + 3*t
26 ±2* 51±5t
92
+ 2*t
91 ± 10
72 + 3
64 + 2* 92±3t IO2 + 3*t
110±4* 152+15t 153 +lOt
19O±3* 290 ±9t 244+ 5t
Values are the mean ± SE percent stimulation of adenylate
cyclase over respective basal activity of 4 experiments.
*Value is significantly different (p < 0.05) than corresponding
sham-operated control.
tValue is significantly different than ischemia with no treatment.
Allopurinol was injected (5 mg/kg) 30 min before ischemia. Indomethacin was injected (2 mg/kg) 60 min before ischemia.
VER CONT
TABLE 3 Effect of 60 min Bilateral Ischemia (No Reflow) on
Adenylate Cyclase in Homogenates of Gerbil Frontal Cortex
Adenylate cyclase (% stimulation
over basal activity)
Ischemia
15, No 2, MARCH-APRIL
1984
0 iaoin
<
UJ
)t
UJ
wo-
<
• 0-
>-
60-
UJ
20
GTP
Cm-Ca++
NE
NE.Cm-Co-" NE.Cm- FORSKOLIN
FIGURE 2. Effect of 15 min bilateral ischemia plus one week
of reflow on adenylate cyclase in paniculate fractions of gerbil
frontal cortex. The values are the mean ± SE percent enzyme
stimulation over respective basal activities (sham = 56 ± 3,
ischemia = 65 ± 9, and ischemia with methylprednisolone =
55 ± 6, expressed as pmol of cyclic AMP formed/min/mg protein). The number of separate experiments are 5. *Denotes a
significant change from the sham operated controls. Concentrations of agents are: GTP (10'5 M), calmodulin (CM) (3.42
ug), Ca++ (10-" M) andNE (10~4 M). Methylprednisolone
was injected (30 mg/kg) 30 min prior to ischemia and at 8 and
16 hrs post ischemia.
were significantly greater than the sham-operated controls (fig. 1). Furthermore, pretreatment of the gerbils
with allopurinol (5 mg/kg 30 min prior to ischemia) or
indomethacin (2 mg/kg 60 min before ischemia) also
reversed the damage to adenylate cyclase elicited by
the 60 min stroke plus 15 min reflow (table 4). In some
cases pretreatment with allopurinol or indomethacin
elevated the sensitivity of the enzyme above that of the
sham controls. Specific situations when this happened
were: with indomethacin — GTP, DA, NE, DA +
GTP and NE + GTP; and with allopurinol — DA.
Adenylate cyclase in the cortical paniculate fraction
was likewise less responsive to DA, DA + GTP and
forskolin as a consequence of 60 min ischemia plus 15
min reflow. Pretreatment of the animals with methylprednisolone, allopurinol or indomethacin prevented
the deficits in enzyme activity (data not shown).
e. Fifteen Min Ischemia Plus One Week of Recovery
Homogenates of the cortical enzyme from these experimental animals only revealed a tendency (nonsignificant) to be less sensitive to GTP, NE, DA and
forskolin. These deleterious effects were however, not
present when the gerbils were given 3 treatments with
methylprednisolone (30 mg/kg, 30 min prior to ischemia and 8 and 16 hours post ischemia) (data not
shown). On the other hand, the paniculate enzyme
displayed deficits to all parameters of activation,
namely GTP, calmodulin-Ca+ + ,NE, N E + calmod-
ADENYLATE CYCLASE DURING SJKOKEJTaylor et al
ulin-Ca+ + , NE + GTP + calmodulin-Ca+ + , and
forskolin. Treatment with the 3 injections of methylprednisolone prevented the damage to the paniculate
enzyme (fig. 2).
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/ . Lack of Drug Effects in Control Animals
When methylprednisolone, allopurinol or indomethacin were pre-injected into sham-operated gerbils
under similar conditions as the stroke animals, they did
not produce any changes in the sensitivity of either
basal adenylate cyclase (either paniculate or homogenate preparations) or to the degree of enzyme activation by GTP, catecholamines, forskolin or NaF (data
not shown). The drugs at concentrations from 10" 4 to
10 6 M when added in vitro to a control enzyme preparation (homogenates) neither influenced the basal activity of adenylate cyclase nor its activation by NE,
GTP or forskolin. In this case control preparations of
adenylate cyclase contained equal amounts of solvents
used to dissolve the drugs.
In our earlier paper8 the activity of cyclic AMP
phosphodiesterase was not altered by conditions of
bilateral ischemia and reflow. Adrenal corticosteroids,
at high concentrations have been found to inhibit phosphodiesterase.20 In the present experiments (15 and 60
min ischemia with or without 15 min recirculation)
cyclic AMP phosphodiesterase was again measured in
homogenates of cerebral tissue from the sham control,
ischemic or ischemic plus methylprednisolone-treated
animals. When assayed under conditions of high (4
mM) or low (1 uM) cyclic AMP concentrations, the
activity of phosphodiesterase did not vary among the
four experimental conditions (data not shown).
Discussion
The present experiments as well as earlier studies7-8
reveal the presence of a catecholamine, GTP, NaF and
forskolin sensitivity of adenylate cyclase in homogenates of the gerbil frontal cortex. Likewise the action
of catecholamines was additive in the presence of
GTP. The paniculate fraction presumably devoid of
calmodulin-Ca ++ was similarly sensitive to the above
agents. Moreover, addition of calmodulin-Ca+ + back
to the particulate preparation gave progressively greater enzyme responses in the presence of GTP and/or
NE. These latter findings suggest that calmodulinCa + + may play a minor role with regard to the stimulation of adenylate cyclase in the gerbil cortex. Earlier
work had established a link between calmodulin-Ca+ +
and adenylate cyclase activation in specified brain regions from other species. "•21 In our previous study* we
were unable to ascribe a role in the gerbil cerebrum for
calmodulin-Ca+ + activation of cyclic AMP phosphodiesterase as previously described by Uzunov and
Weiss for other species.18 The prominent stimulatory
actions of forskolin on adenylate cyclase in the present
study has been attributed to its effect on the catalytic
site of the enzyme.22
The slight elevation of cerebral adenylate cyclase in
response to catecholamines and/or GTP as observed
under conditions of 15 min ischemia with or without
333
recirculation or 60 min of ischemia (no recirculation) is
in accord with our earlier work.8 In other investigations
using gerbils, mice or rats, ischemic or anoxic insults
to the brain resulted in increased steady-state levels of
cyclic AMP as well as elevated activity of Na + , K + ATPase at various time periods up to an hour. Perhaps
the added sensitivity of the enzyme in our work is
reflected in part to these elevated concentrations of
cyclic AMP. However, ischemic conditions evoke
marked releases of transmitters and adenosine which
most certainly elevate the cyclic nucleotide.23-M In addition, bilateral ischemia does result in varying degrees of seizure activity in gerbils. Seizures readily
elevate cyclic AMP in the brain,5-6 however, it is not
known under acute situations whether or not convulsant activity influences the sensitivity of adenylate cyclase to agonists. In the present study, seizures in gerbils preanesthetized with thiopental were not a major
problem such as we have observed using other modes
of anesthesia (unpublished findings). In most cases the
animals rendered ischemic for 60 min with or without
reflow were extremely lethargic.
Damage to the adenylate cyclase system only occurred when bilateral ischemia (60 min + 15 min
reflow) caused reductions in the ability of DA, NE, NE
+ GTP, NaF and forskolin to activate the enzyme.
Fifteen min of ischemia plus one week of reflow did
reduce mainly the responses of the particulate enzyme
to calmodulin-Ca + + , NE, GTP (and combinations
thereof) and forskolin. This latter instance was the only
situation in which particulate fractions showed a remarkable difference in neurohumoral sensitivity when
compared with the homogenate enzyme. The basis for
these variant findings is currently unknown. Interestingly, basal activity of adenylate cyclase was unaltered
in these reflow experiments. Therefore damage to the
enzyme must be directed toward its sensitivity to agonists acting at receptor (catecholamines), transducer
(GTP) or catalytic (NaF and forskolin) sites. It is presently unknown as to which particular biochemical
event produces this enzyme insensitivity. A likely candidate might be lactate accumulation. Hopefully this
was minimized following fasting of the gerbils. l0 Other
possibilities are considered below. Adenylate cyclase
damage following reflow from ischemia in gerbils was
likewise reported in the initial study by Schwartz et al.7
If methylprednisolone was preinjected before bilateral ischemia the above noted damage to adenylate
cyclase as a consequence of recirculation was prevented. Pretreatment of dogs with methylprednisolone has
likewise been shown to slow the rise in cardiac cyclic
nucleotides, limit the breakdown of lysosomes and
enhance hemodynamic and contractile indices of the
heart following a period of ischemia plus recirculation.31 Corticosteroids have been used with varying
degrees of success to treat brain edema. In addition
these drugs prevent the release of arachidonic acid by
inhibiting the action of phospholipases. Thus during
reflow the metabolic cascade of events resulting in the
breakdown of membrane phospholipids, the synthesis
of vasoactive prostaglandins and the formation of free
334
STROKE
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radicals is inhibited. The stabilization of cell membranes and lysosomes plus the prevention of free radical production along with improved hemodynamic indices are the most likely sites of therapeutic action of
glucocorticoids following an ischemic insult."' 12 - 32 - 34
Pretreatment of gerbils with indomethacin also protected the adenylate cyclase system in the gerbil brain
against the consequences of recirculation following 60
min of bilateral ischemia. Indomethacin has been
shown in ischemic gerbil brain to block the formation
of prostaglandins. Moreover, indomethacin-treated
gerbils were observed to recover more rapidly. 35 - x In
related studies involving experimental ischemia, pretreatment of different species with indomethacin afforded protection to cerebral blood vessels, improved
vascular flow, prevented cerebral edema at low blood
flow states and enhanced recovery.34-37-38 Indomethacin inhibits cyclo-oxygenase and hence the prostaglandin synthesis pathways are blocked. During the subsequent steps of prostaglandin synthesis, especially
following ischemia, free radicals are generated which
have been shown to damage the integrity of phospholipid components in cell membranes. This damage is
especially evident during reflow following ischemia
when the blood source of metabolites is available to the
central tissue due to a breakdown of the blood-brain
barrier. In addition, thromboxane A2 is synthesized
from platelets in the microvasculature thereby promoting platelet aggregation and vasoconstriction of cerebral arteries. For some unexplained reason the proper
balance between prostacyclin synthesis (disperses
platelets and dilates vessels) is upset and the tissue
damage is worsened.10"12
Other agents acting as either free radical scavengers
of inhibitors of other enzymatic pathways that would
form these highly reactive compounds might be expected to provide protection of cell membranes from
the deleterious effects of recirculation following interruption of blood flow to the brain.9 In the present
experiments pretreatment with allopurinol did provide
such protection of adenylate cyclase. Since the enzyme
exists as a complex embedded in the cell membrane it
would be expected to suffer damage as a result of
phospholipase action and free radical disruption of
phospholipids.12-13 The integrity of the phospholipid
components of the cell membrane is necessary for full
expression of receptor mediated adenylate cyclase activity.39 It is presently unknown whether free radicals
actually produce direct damage to brain adenylate cyclase. However, when fat cell membranes were exposed to ionizing radiation at low temperatures subsequent damage to the enzyme was observed. Free
radicals do damage Na + , K + -ATPase under in vitro
conditions,41 however, whether such a mechanism occurs during cerebral ischemia is an equivocal topic.28
Since stroke is the major neurological problem in the
United States, studies directed toward identification of
altered biochemical marker sites within the brain
would provide a rationale to evaluate therapeutic
agents which could lead to either a prevention of stroke
or aid in the recovery of such patients. Future ques-
VOL 15, No
2, MARCH-APRIL
1984
tions to be asked might include: identification of other
molecular markers; evaluation of other potentially
therapeutic agents eg., barbiturates, naloxone, calcium blockers, etc; and a more precise determination
of time and dose schedules for these drugs.
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M D Taylor, G C Palmer and A S Callahan, 3rd
Stroke. 1984;15:329-335
doi: 10.1161/01.STR.15.2.329
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