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receptor regulation
and cerebral blood f low of the newborn
1 Department of Pharmacology
and Therapeutics, Ibuprofen, a cyclooxygenase (COX) inhibitor
nonselective for either COX-1 or COX-2 isoform, upregulates
cerebrovascular prostaglandin E2
(PGE2) and
PGF2
prostaglandin receptors; cyclooxygenase; cerebral
microvessels
TWO ISOFORMS OF cyclooxygenase (COX) have been
identified; COX-1 is constitutively expressed in all tissues (18),
whereas COX-2 can be rapidly induced in various tissues by diverse
stimuli such as mitogenic agents, growth factors, hormones,
inflammatory agents, and muscle stretch/relaxation (18). Several
studies have revealed that brain concentrations of prostaglandins are much higher in the perinatal period than in adult life (19, 24, 25). We
(28) and others (7) recently demonstrated that COX-2 is the main form
of COX expressed in the newborn brain and cerebral vasculature and the
principal catalyst of brain prostaglandin production in the newborn
animal.
Prostaglandins, especially prostaglandin
E2
(PGE2) and
PGF2 The purpose of this study was to establish direct evidence that
COX-2-generated prostaglandins govern
PGE2 and
PGF2 Materials. M&B-28767 was a gift from
Rhone-Poulenc Rorer, L-670596 was from Merck-Frosst, and DUP-697 was
from DuPont-Merck. The following products were purchased: NS-398
(Biomol, Plymouth Meeting, PA);
[3H]PGE2
(191 Ci/mmol),
[3H]PGF2 Animals. Newborn pigs (1 day old) were
obtained from a local breeder (Fermes Ménard) and used according
to a protocol of the Animal Care Committee of St.-Justine Hospital
Research Center. Animals were maintained at the Research Center at
25°C, 50-70% humidity, and a 12:12-h light-dark cycle (lights
on 0700-1900) and fed ad libitum milk and tap water. Brains from
adult pigs (5-7 mo old) were collected from a local abattoir
(Bienvenue-Olympia, St. Valérien, PQ) immediately after death and
transported to the laboratory on dry ice.
Treatments. Newborn pigs were
anesthetized with halothane (2%), and a polyethylene catheter (PE-90)
was placed into the right femoral vein, exteriorized, and attached with
tape on the back of the animal. Animals were randomly assigned to
receive intravenous saline, ibuprofen (40 mg/kg), the COX-1 inhibitor
valerylsalicylate (80 mg/kg) (5), the COX-2 inhibitor DUP-697 (5 mg/kg)
(9), or NS-398 (5 mg/kg) (11) every 8 h for a total of 48 h [time to reach plateau receptor density (23)]; acute single dose
treatments had no effect on receptor density. Doses of ibuprofen and
COX-2 inhibitors were selected based on pilot studies, which revealed that prostaglandin levels of the newborn decreased to those of the
untreated adult; dose of valerylsalicylate decreased prostaglandin levels in tissues expressing COX-1 such as lung vasculature (28). At
the end of this 48 h, animals were either killed (pentobarbital sodium,
120 mg/kg) to obtain brain or subjected to a protocol to determine CBF
autoregulation.
Preparation of brain microvessel
membrane. Brains were homogenized in 5 mM
tris(hydroxymethyl)aminomethane · HCl buffer
(pH 7.4) containing 1.1 mM ASA, 0.5 mM EGTA, 1 mM benzamidine, 0.1 mM
PMSF, and 100 µg/ml soybean trypsin inhibitor (24). The homogenates were filtered through a nylon mesh filter (200 µm) and rinsed with
the above buffer. Microvessels were collected from the nylon mesh,
resuspended in the above buffer, rehomogenized, and filtered as above.
The purified microvessels were homogenized with a hand pestle and then
centrifuged at 1,000 g for 15 min. The
supernatant was recentrifuged at 100,000 g for 45 min, and the pellet was stored at Assay of prostaglandins. Brain tissue
was homogenized and centrifuged at 1,000 g for 10 min at 4°C; the
supernatant was homogenized again and recentrifuged at 50,000 g for 30 min at 4°C. The
extraction and the measurement of prostaglandins in the supernatant was
performed as previously described in detail (23).
[3H]PGE2
and
[3H]PGF2
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
receptors in newborn pigs.
COX-2 was shown to be the predominant form of COX and the main catalyst
of prostaglandin synthesis in the newborn brain. We proceeded to
establish direct evidence that COX-2-generated prostaglandins govern
PGE2 and
PGF2 receptor density and
function in the cerebral vasculature of the newborn. Hence, we
determined PGE2 and
PGF2 receptor density and
functions in brain vasculature by using newborn pigs treated with
saline, ibuprofen, COX-1 inhibitor (valerylsalicylate), or COX-2
inhibitors (DUP-697 and NS-398). Newborn brain
PGE2 and PGF2 concentrations were
significantly reduced by ibuprofen, DUP-697, and NS-398 but not by
valerylsalicylate. In newborn pigs treated with DUP-697, NS-398, and
ibuprofen, PGE2 and
PGF2 receptor densities in
brain microvessels were increased to adult levels; there was also a
significant increase in inositol 1,4,5-trisphosphate (IP3) production and cerebral
vasoconstrictor effects of 17-phenyl trinor
PGE2
(EP1 receptor agonist),
M&B-28767 (EP3 receptor
agonist), PGF2,
and fenprostalene (PGF2
analog). Treatment with ibuprofen or DUP-697 also increased the upper
blood pressure limit of cerebral cortex and periventricular blood flow
autoregulation from 85 to 
125 mmHg (uppermost blood pressure
studied). However, valerylsalicylate treatment did not affect
cerebrovascular PGE2 and
PGF2 receptors,
IP3 production, or vasoconstrictor
effects in newborn animals. These in vivo and in vitro observations
indicate that COX-2 is mainly responsible for the regulation of
PGE2 and PGF2 receptors and their
functions in the newborn cerebral vasculature.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
, play an important role in
the control of the upper range of cerebral blood flow (CBF)
autoregulation in the newborn (8). PGE2 and
PGF2 induce significant
cerebral vasoconstriction in the adult (12, 16). In contrast,
PGF2 exerts minimal constriction and PGE2 produces
dilatation of cerebral vessels in the newborn (16, 21). We recently
demonstrated that these age-dependent differences in the actions of
PGF2 and
PGE2 on cerebral vasculature are
due to a decreased density of
PGF2 and
PGE2 receptors (respectively, FP
and EP) and receptor-coupled second messengers associated with
vasoconstriction in the newborn (24). Further studies revealed that
pretreatment of newborn pigs with the nonselective COX inhibitor
ibuprofen upregulated brain PGE2
and PGF2 receptors to adult
levels (23) and increased the upper limit of CBF autoregulation (8).
However, the relative contributions of COX-1 and COX-2 on the
upregulation of PGE2 and
PGF2 receptors have not been
determined. Also, the relative importance of the COX isozymes on CBF
autoregulation is not known; such information could suggest more
selective approaches to enhance CBF autoregulation in the newborn (30).
receptor density and
function on brain vasculature of the newborn. If such were the case,
selective inhibition of COX-2 in newborns should increase
cerebrovascular PGE2 and
PGF2 receptor densities and
receptor-coupled function, and this could be associated with improved
hypertension-induced autoregulatory control of CBF. To test this
conjecture, newborn pigs were treated with saline, ibuprofen, COX-1
inhibitor (valerylsalicylate) (5), or COX-2 inhibitors (DUP-697 and
NS-398) (9, 11) to determine binding, production of second messengers,
and constrictor responses of brain microvessels to
PGE2 and
PGF2, as well as regional CBF
autoregulation in vivo.
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
(219 Ci/mmol), and enhanced chemiluminescence kit and radioreceptor
assay kits for inositol 1,4,5-trisphosphate (IP3) (Amersham, Mississauga,
ON); ibuprofen, forskolin, soybean trypsin inhibitor (type II-S),
acetylsalicylic acid (ASA), benzamidine, leupeptin, aprotinin,
phenylmethylsulfonyl fluoride (PMSF), dithiothreitol, adenosine
triphosphate, creatine phosphate, creatine phosphokinase, phorbol
12-myristate 13-acetate (PMA), 
-mercaptoethanol, EDTA, ethylene
glycol-bis(-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), penicillin, streptomycin, and 3-isobutyl-1-methylxanthine (Sigma Chemical, St. Louis, MO);
PGE2, 16,16-dimethyl
PGE2, 17-phenyl trinor
PGE2,
PGF2, and valerylsalicylate
(Cayman, Ann Arbor, MI); fenprostalene (Syntex, Mississauga, ON);
radioimmunoassay kits for PGE2 and
PGF2 (Advanced Magnetics,
Boston, MA); and radiolabeled microspheres (DuPont-New England Nuclear,
Boston, MA); all other chemicals were from Fisher Scientific, Montreal, PQ.
80°C until used. The purity of the microvessel
preparation was confirmed by light microscopy and a >15-fold higher
level of 
-glutamyl transpeptidase activity compared with brain
parenchyma (24).
binding assay.
Aliquots of microvessel membranes containing 250 µg protein were
incubated at 37°C for 30 min in 100 µl of 10 mM sodium phosphate buffer (pH 7.4) containing 100 mM NaCl, 2 mM
MgCl2, 1 mM benzamidine, 0.5 mM
EGTA, 0.1 mM PMSF, 1.1 mM ASA, 100 µg/ml soybean trypsin inhibitor,
and varying concentrations of
[3H]PGE2
or
[3H]PGF2
in the absence or the presence of 25 µM unlabeled PGE2 or
PGF2 (24). Receptor densities
(maximal binding; Bmax) and
dissociation constants
(Kd) were
determined from the saturation isotherms (23, 24) using computer
programs (Prism, GraphPad).
were determined after
blockage of the thromboxane receptor by L-670596 (10) to minimize
nonspecific effects via the thromboxane receptors. In some preparations
from saline-treated newborn pigs, the vasomotor effects of COX
inhibitors, ibuprofen (10 µM), DUP-697 (10 µM), and
valerylsalicylate (100 µM) were also tested. Vascular diameter was
recorded with a video camera before and after topical application of
increasing concentration of the agent every 7 min; stable response was
reached within 4 min (23). Digital images were analyzed using a
commercially available software (Sigma Scan; Jandel Scientific, Corte
Madera, CA). Each measurement was repeated three times and had a
variability of <1% (3, 23). To confirm validity of preparations and
assess overall contractility of vessels, the response to PMA was also tested.
Incubation of isolated brain microvessels with COX
inhibitors. Brains from newborn (1 day old) pigs were
collected in Hanks' balanced salt solution (HBSS) containing HEPES (15 mM), penicillin (100 U/ml), and streptomycin (100 µg); brains were
cut into small pieces and centrifuged at 500 g for 10 min at 4°C. The sediment was resuspended in HBSS and 40% Ficoll 400 (1:1, vol/vol) and homogenized with a tissue grinder (Omni, Wheaton, NJ). The homogenates were centrifuged at 20,000 g for 20 min at 4°C; the precipitates were washed three times with HBSS by
centrifugation at 750 g for 15 min at
4°C. The purified microvessels were resuspended in smooth muscle
growth medium and maintained at 37°C in a humidified atmosphere of
21% O2, 5%
CO2, and 74%
N2. The microvessels were treated
with saline, ibuprofen (10 µM), DUP-697 (10 µM), NS-398 (10 µM),
valerylsalicylate (100 µM), ibuprofen (10 µM) plus fenprostalene
(stable PGF2 analog; 10 µM),
or ibuprofen plus 16,16-dimethyl
PGE2 (stable PGE2 analog; 10 µM) and
incubated for 24 h; concentrations of valerylsalicylate and of DUP-697
and NS-398 selectively inhibit COX-1 and COX-2, respectively (5, 9,
11). At the end of the incubation, microvessels were collected and
washed after centrifugation. Microvessel membrane preparations were
used for
[3H]PGE2
and
[3H]PGF2
binding assay as described above.
Protocol for studying CBF
autoregulation. Animals were anesthetized with 2%
halothane for tracheostomy. Catheters were placed into the left
subclavian artery for the withdrawal of blood samples including
reference samples, into the left ventricle via the right subclavian
artery for the injection of radiolabeled microspheres, and into the
femoral artery for continuous blood pressure (BP) recording by means of
a Statham pressure transducer connected to a TA240 Gould multichannel
recorder as described previously (8). A silicone-coated balloon-tipped
catheter was placed in the distal thoracic descending aorta via a
femoral artery; inflation of this balloon produced hypertension in the
aortic arch. Halothane was discontinued after surgery; animals were
maintained on 
-chloralose (bolus intravenous injection of 50 mg/kg
followed by infusion of 10 mg · kg
1 · h1)
and paralyzed with pancuronium (0.1 mg/kg iv). Body temperature was
maintained at 38°C with an overhead radiant lamp, and the animals
were allowed to recover from the surgery for 2 h before the experiments
were started.
Measurement of CBF. CBF was measured
using the radiolabeled microsphere technique at preselected mean BP
(MBP) values at and above the upper limit of the CBF autoregulation
range of the newborn pig (8) by increasing stepwise MBP to 85, 105, and
125 mmHg, and these varied by 
5 mmHg on each animal studied; baseline
MBP was 70 ± 3 mmHg for all animals and was unaffected by the
treatments. This protocol has been described in detail by us (8, 15). For CBF measurements, ~106
microspheres (15 µm diameter) labeled with
141Ce,
113Sn,
95Nb, and
85Sr were injected in a random
sequence into the left ventricle once MBP remained steady for 30 s
after inflation of the balloon. Withdrawal of reference blood samples
from the left subclavian artery catheter was started 10 s before the
injection of each type of microspheres and was continued for 70 s at a
rate of 2 ml/min using a Harvard infusion-withdrawal pump. Immediately
after injections of microspheres, blood samples were withdrawn from the
left subclavian artery to determine blood gases; these remained normally stable. After the experiment, animals were killed with excess
pentobarbital sodium, the location of catheters was verified and the
brains were removed. The brain cortex and periventricular region were
isolated and weighed. Radioactivity in the cortex, periventricular
region, and reference blood samples was counted in a gamma
scintillation counter (Cobra II; Canberra Packard, Meridien, CT).
Regional CBF (milliliters per minute per 100 grams) was calculated with
the formula (counts per minute per 100 grams of tissue × reference blood withdrawal rate)/(counts per minute in the reference
blood) using the computer system online with the counter (PCGERDA,
Charlottesville, VA).
Statistical analysis. Data were
subjected to analysis of variance factoring for treatment and age group
as well as by comparison among means test (Tukey-Kramer method). For
the CBF study, these data were analyzed by regression analysis as
previously described in detail (8, 15); the Pearson's product-moment
coefficient (r) was calculated.
Linear regressions were compared by regression equality test using the
method of least squares. Statistical significance was set at
P < 0.05. Data are expressed as
means ± SE with the exception of CBF as a function of MBP.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Effects of COX-1 or COX-2 inhibition on brain
prostaglandins. Treatment of piglets with the
nonselective COX inhibitor ibuprofen, as well as with the COX-2
inhibitors DUP-697 and NS-398, caused a similar and significant
decrease in brain PGE2 and
PGF2 concentrations
(n = 4 in each treatment group;
P < 0.01 saline compared with
ibuprofen, DUP-697, and NS-398); prostaglandin levels among these
treatments were not significantly different from those in the untreated
adult. The COX-1 inhibitor valerylsalicylate did not affect brain
prostaglandin concentrations in newborn pigs (Fig.
1), whereas in tissues of newborn that
express predominantly COX-1 such as pulmonary vessels (28),
valerylsalicylate decreased PGE2
concentrations from 5,307 ± 175 to 1,591 ± 52 pg/mg protein.
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[3H]PGE2
and
[3H]PGF2
binding in brain microvessels.
Newborn pigs treated with ibuprofen, DUP-697, and NS-398 exhibited
significant increases in PGE2 and
PGF2 receptor densities (Bmax) in brain microvessels;
valerylsalicylate was ineffective (Fig. 2).
The densities of cerebrovascular
PGE2 and
PGF2 receptors in newborn
animals treated with ibuprofen, DUP-697, or NS-398 were not
significantly different from those in the untreated adult (Fig. 2);
Kd values were
not affected by treatments (data not shown).
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, and fenprostalene (FP
receptor agonist) on IP3
production in brain microvessels to levels determined in untreated
adults (Fig. 3). Treatment of newborn pigs
with valerylsalicylate did not modify the effects of these
PGE2 and
PGF2 receptor agonists on
IP3 production.
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receptor
agonists. Cerebral vasoconstrictor responses to PGF2, 17-phenyl trinor
PGE2
(EP1 receptor agonist), and M&B-28767 (EP3 receptor agonist) were significantly
increased in newborn pigs treated with ibuprofen, DUP-697, or NS-398 to
levels observed in the untreated adult. Treatment with
valerylsalicylate did not affect cerebral vasoconstrictor response
(Fig. 4 and Table
1). In separate experiments, COX inhibitors
(ibuprofen, DUP-697, and valerylsalicylate: 10-100 µM) were
found to exert per se no vasomotor effects on brain vessels of
saline-treated newborn pigs; for example, vessel diameter before and
after ibuprofen was 39 ± 5 and 38 ± 6 µm, respectively, which
corresponded to an insignificant 0.8 ± 0.5% decrease in diameter
(n = 4). Also, acute application of COX inhibitors (rather than 48-h treatments as per experimental protocol) did not alter response to prostaglandins and analogs (data
not shown).
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receptor densities of
incubated cerebral microvessels. To ascertain effects of prostaglandins on PGE2 and
PGF2 receptor density on brain vasculature observed in vivo, newborn cerebral microvessels were incubated with COX inhibitors in the presence or absence of stable prostaglandin analogs. Ibuprofen, DUP-697, and NS-398 significantly increased densities (Bmax) of
both PGE2 and
PGF2 receptors (Fig.
5). Ibuprofen-induced increases in
PGE2 and
PGF2 receptors were
specifically prevented by 16,16-dimethyl
PGE2
(PGE2 analog) and fenprostalene
(PGF2 analog), respectively.
Valerylsalicylate marginally affected
PGE2 and
PGF2 receptor densities;
treatment of adult brain vessel preparations with valerylsalicylate
(100 µM) decreased PGE2
concentrations from 71 ± 5 to 29 ± 3 pg/mg protein,
demonstrating the efficacy of the drug.
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have been shown to play an
important role in autoregulatory control of CBF during rises in
perfusion pressure (6, 8), which is effective in the adult (20) but not
in the newborn (8), presumably because of fewer
PGE2 and
PGF2 receptors in the cerebral
microvasculature of the latter (24). The major site for increase in
cerebral vascular resistance during acute hypertension is indeed the
microvasculature (4). We therefore examined whether increased brain
microvascular PGE2 and
PGF2 receptors and
receptor-coupled functions, secondary to inhibition of COX-2 but not of
COX-1 (Figs. 2-5 and Table 1), is associated with enhanced
hypertension-induced autoregulation of CBF in the newborn pig. Basal
cortical and periventricular CBF were 83 ± 6 and 79 ± 5 ml · min1 · 100 g1, respectively, in
saline-treated pigs and did not differ significantly from CBF after COX
inhibitors, consistent with vasomotor effects of COX inhibitors in
vitro (see above). In saline-treated animals, CBF in the cortex and
periventricular region increased linearly with MBP
(r = 0.59-0.79,
P < 0.01) (Fig.
6). Both ibuprofen and DUP-697 treatment
prevented the change in CBF as a function of MBP
(r = 0.05-0.14,
P 0.19). On the other hand, in
animals treated with valerylsalicylate, CBF increased linearly with MBP
(r = 0.61-0.87, P < 0.01). Regression coefficients
for saline-treated animals differed significantly from those of animals
treated with ibuprofen and DUP-697 (P < 0.01, by regression equality test) but did not differ from those of
animals treated with valerylsalicylate.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The concentrations of prostaglandins in blood and brain are higher
during the perinatal period than in normal adults (19, 23, 25). These
higher levels of cerebral prostaglandins in the newborn have been shown
to cause a downregulation of brain PGE2 and
PGF2 receptors and functions
and significantly curtail the upper limit of CBF autoregulation (8,
23). We recently demonstrated that COX-2, rather than COX-1, is the
principal form of COX expressed in the newborn brain and cerebral
vasculature and the main catalyst of high prostaglandin synthesis in
newborn brain (28). However, the relative contribution of COX-1 and COX-2 in the regulation of prostaglandin receptors and their functions in newborn brain blood flow are not known. We hypothesized that COX-2,
via formation of prostaglandins, might be the major regulator of brain
microvascular prostaglandin receptors in the newborn. If so, selective
treatment of newborn pigs with COX-2 inhibitors should inhibit
prostaglandin synthesis and increase brain
PGE2 and
PGF2 receptors and functions,
including possibly an enhancement in hypertension-induced CBF
autoregulation, whereas COX-1 inhibition should be ineffective. Data of
the present study support our working hypothesis.
Ibuprofen treatment significantly decreased brain
PGE2 and
PGF2 levels in the newborn
(Fig. 1) as previously reported (14, 19, 23). The distinct COX-2
inhibitors DUP-697 and NS-398 caused a similar inhibition of
prostaglandin synthesis, whereas the COX-1 inhibitor valerylsalicylate
was ineffective (Fig. 1). These results are consistent with a
predominance of COX-2 expression in the newborn, which is also the main
catalyst of prostaglandin synthesis in newborn brain (28).
The decrease in brain prostaglandin levels in the newborn after
inhibition of both COX-1 and COX-2 by ibuprofen or only of COX-2 by
DUP-697 and NS-398 treatment in vivo was associated with a comparable
increase in cerebral microvessel
PGE2 and
PGF2 receptor densities (Fig.
2) to levels observed in the adult, which contains prostaglandin
concentrations similar to those of treated newborns. Increases in
PGE2 and
PGF2 receptor densities were also observed after incubating isolated brain microvessels with these
agents in vitro (Fig. 5), indicating that the effects of these
inhibitors on prostaglandin receptors were directly produced on
cerebral microvasculature. In addition, because
PGE2 and
PGF2 analogs prevented the
increase in PGE2 and
PGF2 receptors following
inhibition of COX-2, it can also be inferred that these changes in
PGE2 and
PGF2 receptors are specifically
modulated by prostaglandin levels.
We have previously shown that brain microvessels contain
EP1, EP3, and FP
receptors (23, 24); IP3 production
is linked to activation of EP1,
EP3, and FP receptors (1, 13, 26, 29). An
increase in IP3 has been shown to
be associated with vascular contraction (17). Ibuprofen as well as
DUP-697 and NS-398 treatment significantly increased newborn cerebral
microvessel IP3 production and
cerebral vasoconstrictor responses to EP1, EP3, and FP receptor agonists (Fig. 4). These
data provide additional evidence that an inhibition of COX-2 caused an
increase in cerebral microvessel EP1,
EP3, and FP receptor densities and in
receptor-mediated functions. Because two chemically distinct COX-2
inhibitors, DUP-697 and NS-398, caused a comparable increase in
PGE2 and
PGF2 receptor densities and
receptor-coupled IP3 production
and vasoconstrictor response and because the COX-1 inhibitor was
ineffective in the newborn [but effective in newborn lung
vessels, which mainly express COX-1 (28)], it appears that COX-2
rather than COX-1 inhibition was responsible for the upregulation of
brain PGE2 and
PGF2 receptors and their
functions in the neonatal animal.
It has been demonstrated that the narrow range of CBF autoregulation in
the newborn pig was due to a minimal vasoconstrictor activity of
prostaglandins, particularly of
PGE2 and
PGF2, on cerebral vasculature
of the newborn animal (3, 8). In the adult animals, high densities of
PGE2 and
PGF2 in brain vasculature
result in IP3 production and
effective cerebral vasoconstriction (23). An increase in cerebral
vasoconstriction may prevent an increase in CBF when systemic BP rises
and thus CBF remains constant. On the other hand,
PGE2 and
PGF2 produce minimal
IP3 formation in brain
microvessels of the newborn pig due to a relative deficiency of
EP1, EP3, and FP
receptors (23, 24); this causes minimal cerebral vasoconstriction (23),
which is insufficient to counteract the increased perfusion in the
phase of a moderate increase in blood pressure resulting in increased
CBF, and thereby reveals inadequate CBF autoregulatory control (8). In
fact, PGE2 and PGF2 play a major role in
hypertension-induced autoregulatory adjustment of CBF (6, 8). On the
basis of this evidence and because microvessels are the main site for
control of cerebrovascular resistance in response to acute hypertension
(4), we assessed whether upregulation of
PGE2 and
PGF2 receptors secondary to
COX-2 inhibition is associated with improved hypertension-induced control of CBF autoregulation in the newborn. Treatment of newborn pigs
with DUP-697 and NS-398, as seen with the nonselective COX inhibitor
ibuprofen but not with valerylsalicylate (COX-1 inhibition), prevented
the increase in CBF in the cortex and periventricular region in
response to BP increase (Fig. 6). These results indicate that the upper
limit of CBF autoregulation in the newborn could be significantly
raised by selectively inhibiting COX-2 but not by inhibiting COX-1,
consistent with upregulation of
PGE2 and PGF2 receptors and
receptor-coupled IP3 production
and vasoconstrictor responses. However, this association between
PGE2/PGF2
receptor upregulation and enhanced CBF autoregulation can only be
inferred; a direct link would require selective antagonists to these
receptors, which, other than for EP1, are not
currently available.
This enhancement in CBF autoregulation is relevant provided that the effects of PGI2, mostly at the lower end of the autoregulatory range (22), are not compromised by COX inhibition. Indeed, we have recently reported that dilation to PGI2 is unaffected by pretreatment with the nonselective COX inhibitor ibuprofen (2), and furthermore the reduction in prostaglandins after ibuprofen was not associated with impairment of the lower limit of CBF autoregulation in the newborn animal (8).
One could question the origin of prostaglandins required to produce vasomotor actions in vivo once COX-2 is inhibited in the newborn. It must be pointed out that pilot studies were first carefully conducted to establish the dose of COX inhibitors necessary to reduce prostaglandin concentrations in newborn to those comparable to ones in the adult. Hence, COX activity was reduced but not fully inhibited (Fig. 1).
In summary, this study suggests that selective COX-2 inhibition was as
effective as nonselective COX inhibition in reducing prostaglandin
synthesis to increase brain PGE2
and PGF2 receptor densities,
receptor-coupled IP3 production,
cerebral vasoconstrictor responses, and, most importantly, the upper BP
limit of CBF autoregulation in the newborn; in contrast, inhibition of
COX-1 was ineffective. We conclude that COX-2, which is the main source
of prostaglandins in the newborn brain, is responsible for the
regulation of cerebrovascular PGE2
and PGF2 receptors and their
vasomotor/hemodynamic functions in the neonate. These in vivo and in
vitro observations provide additional new information on the
physiological role for the inducible COX-2. Because nonselective COX
inhibitors have been shown and proposed in the prevention of
intraventricular cerebral hemorrhage (30), the present findings may be
of interest in the clinical setting as they suggest a potentially
selective therapeutic target for this purpose, namely COX-2, possibly
without the renal and intestinal complications attributed to COX-1
inhibition (27); future studies will establish efficacy and safety of
COX-2 inhibitors in this regard.
Perspectives
Prostaglandins exert a significant contribution in setting the reduced upper BP limit of CBF autoregulation of the newborn. The present findings reveal that, of the two COX isoforms, COX-2 is the principal isozyme that catalyzes increased prostaglandin synthesis in newborn brain, which in turn leads to downregulation of cerebrovascular PGE2 and PGF2 receptors associated with limited vasoconstrictor response and hypertension-induced CBF autoregulation. Of relevance, a reduced upper BP limit of CBF autoregulation of the newborn compared with that of the adult seems to
contribute in predisposing the immature subject to intraventricular cerebral hemorrhage. Nonselective COX inhibition raises the upper limit
of CBF autoregulation and effectively reduces the incidence of brain
hemorrhage in immature newborns; however, renal and intestinal complications attributed to COX-1 inhibition can be serious drawbacks. The present findings suggest that selective inhibition of COX-2 may be
a potentially preferable therapeutic choice. As COX-2 inhibitors become
clinically available, studies should be considered to evaluate their
efficacy in prevention of intraventricular hemorrhage.
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ACKNOWLEDGEMENTS |
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The authors thank Hendrika Fernandez for her technical assistance and Les Fermes Menard, Quebec, for their generosity in supplying us with newborn pigs.
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FOOTNOTES |
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This work was supported by the Medical Research Council of Canada, The United Cerebral Palsy Foundation, and the Quebec Heart and Stroke Foundation. D.-Y. Li, P. Hardy, and K. Martinez-Bermudez are recipients of fellowships/studentships from the Medical Research Council of Canada. M. Bhattacharya is supported by a Telethon of Stars studentship.
Address for reprint requests: S. Chemtob, Depts. of Pediatrics, Ophthalmology, and Pharmacology, Hôpital Sainte-Justine Research Center, 3175 Côte Sainte-Catherine, Montreal, Quebec, Canada H3T 1C5.
Received 10 January 1997; accepted in final form 19 June 1997.
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REFERENCES |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
1.
Abramovitz, M.,
Y. Boie,
T. Nguyen,
T. H. Rushmore,
M. A. Bayne,
K. M. Metters,
D. M. Slipetz,
and
R. Grygorczyk.
Cloning and expression of a cDNA for the human prostanoid FP receptors.
J. Biol. Chem.
269:
2632-2636,
1994
2.
Abran, D.,
D. R. Varma,
and
S. Chemtob.
Regulation of prostanoid vasomotor effects and receptors in choroidal vessels of newborn pigs.
Am. J. Physiol.
272 (Regulatory Integrative Comp. Physiol. 41):
R995-R1001,
1997
3.
Abran, D.,
D. R. Varma,
D. Y. Li,
and
S. Chemtob.
Reduced responses of retinal vessels of the newborn pig to prostaglandins but not to thromboxane.
Can. J. Physiol. Pharmacol.
72:
168-173,
1994[Medline].
4.
Baumbach, G. L.,
and
D. D. Heistad.
Regional, segmental, and temporal heterogeneity of cerebral vascular autoregulation.
Ann. Biomed. Eng.
13:
303-310,
1985[Medline].
5.
Bhattacharya, D. K.,
M. Lecomte,
J. Dunn,
D. J. Morgans,
and
W. L. Smith.
Selective inhibition of prostaglandin endoperoxide synthase-1 (cyclooxygenase-1) by valerylsalicylic acid.
Arch. Biochem. Biophys.
317:
19-24,
1995[Medline].
6.
Borgdorff, P.,
P. Sipkema,
and
N. Westerhof.
Pump perfusion abolishes autoregulation possibly via prostaglandin release.
Am. J. Physiol.
255 (Heart Circ. Physiol. 24):
H280-H287,
1988
7.
Breder, C. D.,
D. Dewitt,
and
R. P. Kraig.
Characterization of inducible cyclooxygenase in rat brain.
J. Comp. Neurol.
355:
296-315,
1995[Medline].
8.
Chemtob, S.,
K. Beharry,
J. Rex,
D. R. Varma,
and
J. V. Aranda.
Prostanoids determine the range of cerebral blood flow autoregulation of the newborn piglet.
Stroke
21:
777-784,
1990
9.
Copeland, R. A.,
J. M. Willams,
J. Giannaras,
S. Nurnberg,
M. Covington,
D. Pinto,
S. Pick,
and
J. Trzaskos.
Mechanism of selective inhibition of the inducible isoform of prostaglandin G/H synthase.
Proc. Natl. Acad. Sci. USA
91:
11202-11206,
1994
10.
Ford-Hutchinson, A. W.,
Y. Girad,
A. Lord,
T. R. Jones,
M. Cirino,
J. F. Evans,
J. Gillard,
P. Hamel,
C. Leveille,
P. Masson,
and
R. Yong.
The pharmacology of L670,596, a potent and selective thromboxane/prostaglandin endoperoxide receptor antagonist.
Can. J. Physiol. Pharmacol.
67:
989-993,
1989[Medline].
11.
Futaki, N.,
S. Takahashi,
M. Yokoyama,
I. Arai,
S. Higuchi,
and
S. Otomo.
NS-398, a new anti-inflammatory agent, selectively inhibits prostaglandin G/H synthase/cyclooxygenase (COX-2) activity in vitro.
Prostaglandins
47:
55-59,
1994[Medline].
12.
Hadhazy, P.,
B. Malomvolgyi,
and
K. Magyar.
Endogenous prostanoids and arterial contractility.
Prostaglandins Leukot. Essent. Fatty Acids
32:
175-185,
1988[Medline].
13.
Halushka, P. V.,
D. E. Mais,
P. R. Mayeux,
and
T. A. Morinelli.
Thromboxane, prostaglandin and leukotriene receptors.
Annu. Rev. Pharmacol. Toxicol.
10:
213-239,
1989.
14.
Hanamura, T.,
T. Shigeno,
T. Asano,
T. Mira,
and
K. Takakura.
Prostaglandin profiles in relation to local circulatory changes following focal cerebral ischemia in cats.
Stroke
20:
803-808,
1989
15.
Hardy, P.,
A. M. Nuyt,
D. Abran,
J. St.-Louis,
D. R. Varma,
and
S. Chemtob.
Nitric oxide in retinal and choroidal blood flow autoregulation in newborn pigs: interactions with prostaglandins.
Pediatr. Res.
39:
487-493,
1996[Medline].
16.
Hayashi, S.,
M. K. Park,
and
T. J. Kuehl.
Relaxant and contractile responses to prostaglandins in premature, newborn and adult baboon cerebral arteries.
J. Pharmacol. Exp. Ther.
233:
628-635,
1985
17.
Heaslip, R. J.,
and
B. D. Sickels.
Evidence that prostaglandins can contract the rat aorta via a novel protein kinase C-dependent mechanism.
J. Pharmacol. Exp. Ther.
250:
44-51,
1989
18.
Herschman, H. R.
Prostaglandin synthase-2.
Biochim. Biophys. Acta
1299:
125-140,
1996[Medline].
19.
Jones, S. A.,
S. L. Adamson,
I. Bishai,
J. Lees,
D. Engelberts,
and
F. Coceani.
Eicosanoids in third ventricular cerebrospinal fluid of fetal and newborn sheep.
Am. J. Physiol.
264 (Regulatory Integrative Comp. Physiol. 33):
R135-R142,
1993
20.
Lassen, N. A.
Cerebral blood flow and oxygen consumption in man.
Physiol. Rev.
39:
183-238,
1959
21.
Leffler, C. W.,
and
D. W. Busija.
Prostanoids and pial arteriolar diameter in hypotensive newborn pigs.
Am. J. Physiol.
252 (Heart Circ. Physiol. 21):
H687-H691,
1987
22.
Leffler, C. W.,
D. W. Busija,
D. G. Beasley,
and
A. W. Fletcher.
Maintenance of cerebral circulation during hemorrhagic hypotension in newborn pigs: role of prostanoids.
Circ. Res.
59:
562-567,
1986
23.
Li, D. Y.,
D. Abran,
K. G. Peri,
D. R. Varma,
and
S. Chemtob.
Inhibition of prostaglandin synthesis in newborn pigs increases cerebral microvessel prostaglandin F2 and prostaglandin E2 receptors, their second messengers and vasoconstrictor response to adult levels.
J. Pharmacol. Exp. Ther.
278:
370-377,
1996
24.
Li, D. Y.,
D. R. Varma,
and
S. Chemtob.
Ontogenic increase in PGE2 and PGF2 receptor density in brain microvessels of pigs.
Br. J. Pharmacol.
112:
59-64,
1994[Medline].
25.
Mitchell, M. D.,
A. Lucas,
P. C. Etches,
J. D. Brant,
and
A. C. Turnbull.
Plasma prostaglandin levels during early neonatal life following term and preterm delivery.
Prostaglandins
16:
319-326,
1978[Medline].
26.
Namba, T.,
Y. Sugimoto,
M. Negishi,
A. Irie,
F. Ushikubi,
A. Kakizuka,
S. Ito,
A. Ichikawa,
and
S. Naramiya.
Alternative splicing of C-terminal tail of prostaglandin E receptor EP3 determines G-protein specificity.
Nature
365:
166-170,
1993[Medline].
27.
O'Neil, G. P.,
and
A. W. Ford-Hutchinson.
Expression of mRNA for cyclooxygenase-1 and cyclooxygenase-2 in human tissues.
FEBS Lett.
330:
156-160,
1993[Medline].
28.
Peri, K. G.,
P. Hardy,
D. Y. Li,
D. R. Varma,
and
S. Chemtob.
Prostaglandin G/H synthase-2 is a major contributor of brain prostaglandins in the newborn.
J. Biol. Chem.
270:
24615-24620,
1995
29.
Watabe, A.,
Y. Sugimoto,
A. Honda,
A. Irie,
T. Namba,
M. Negishi,
S. Ito,
S. Narumiya,
and
A. Ichikawa.
Cloning and expression of cDNA for a mouse EP1 subtype of prostaglandin E receptor.
J. Biol. Chem.
268:
20175-20178,
1993
30.
Wells, J. T.,
and
L. R. Ment.
Prevention of intraventricular hemorrhage in preterm infants.
Early Hum. Dev.
42:
209-233,
1995[Medline].
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