(Received for publication, June 13, 1995)
From the
In order to understand the molecular basis of the elevated
cerebral prostaglandin levels in the newborn, we compared the
expression of the mRNAs and proteins of prostaglandin G/H synthases
(PGHS), PGHS-1 and PGHS-2, in various regions of the brain and the
microvasculature of newborn (1-2-day-old) and juvenile
(4-7-week-old) pigs and also measured the relative contribution
of PGHS-2 to cerebral prostaglandin synthesis both in vivo and in vitro by using a novel inhibitor of PGHS-2, NS-398.
Ribonuclease protection assays using total RNA isolated from various
regions of the porcine brain revealed that, unlike PGHS-1 mRNA, PGHS-2
mRNA was abundantly expressed in the cortex and the microvasculature of
the newborn compared with those of the juvenile animal. PGHS-2
immunoreactive protein comprised the majority of total PGHS enzyme in
neonatal cerebral microvasculature due to a 2-3-fold lower
expression of immunoreactive PGHS-1 protein. Inhibition of PGHS-2 by
NS-398 decreased the rate of prostaglandin synthesis by purified
cerebral microvessels of the newborn by approximately 65% and of
juvenile pigs by 30%. The decrease in brain tissue prostaglandin
concentrations following intravenous administration of NS-398 was
greater in newborn pigs (90%) than in the juvenile animals
(
30%). Furthermore, NS-398 substantially reduced the net in
vivo cerebrovascular production of prostaglandins in newborn pigs.
Taken together, these results indicate that PGHS-2 is the predominant
form of prostaglandin G/H synthase in the newborn brain and cerebral
microvasculature and the main contributor to the brain prostaglandin
levels in the newborn animal.
Prostaglandins act as modulators in several
neurological(1, 2) and cerebral hemodynamic
functions(3, 4) . During the perinatal period the
concentrations of prostaglandins in blood and brain in the newborn are
higher than those in the normal adult(5, 6) . These
higher levels of cerebral prostanoids in the newborn significantly
affect cerebral blood flow autoregulation as well as cause
down-regulation of prostanoid receptor expression and receptor function
in brain(7, 8) . However, the cause of increased
prostaglandin levels and the relative contributions of the two
prostaglandin G/H synthases (PGHS) ()to prostanoid synthesis
in the neonatal brain are not yet known.
Of the two prostaglandin G/H synthases so far described, PGHS-1 (EC 1.14.99.1) is constitutively expressed in all tissues, albeit to varying degrees (for reviews see (9, 10, 11) ). The other isozyme, PGHS-2, shares significant homology with PGHS-1 in amino acid sequence (11) and exhibits similar enzymatic properties (12, 13, 14) but differs in its pharmacological properties(14, 15, 16) , mRNA size(11) , chromosomal location(17) , and gene organization(18, 19) . Moreover, PGHS-2 can be rapidly induced in various tissues by diverse stimuli such as mitogenic agents, growth factors(20, 21) , hormones(22, 23) , inflammatory agents(24) , synaptic activity(25) , and muscle stretch/relaxation(26) . Elevation of PGHS-2 expression in inflammation (10, 11) and suppression of PGHS-2 gene activity by dexamethasone and other corticosteroids both in tissue cultures (20) and in vivo(27) suggest that PGHS-2 may have a role in inflammatory response. Nonetheless, low but varying levels of PGHS-2 expression have been found in all tissues (28) by using the reverse transcription-polymerase chain reaction technique. Thus, despite its induction in inflammation and mitogenesis, the role of PGHS-2 under normal physiological conditions of increased prostaglandin synthesis in the brain, as seen in the perinatal period(5, 6) , is still a matter of conjecture. This is of particular interest given that expression of the other isozyme, PGHS-1, has been shown to be low in the newborn and increases to reach maximum levels in the adult(29) . We hypothesized that the elevated prostaglandin G/H synthase activity in the newborn brain could be due to increased expression of PGHS-2. For this purpose, we analyzed the expression of porcine PGHS-1 and PGHS-2 mRNAs by ribonuclease protection assays and analyzed the expression of PGHS-1 and PGHS-2 proteins by immunochemical methods in the newborn and the juvenile animals. We also examined the relative contribution of PGHS-2 to the cerebral production of prostaglandins both in vivo and in vitro by using a PGHS-2 inhibitor, NS-398.
The proteins were electrophoretically transferred to nitrocellulose membranes, and the nonspecific binding sites on the membranes were blocked with buffer A (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% (v/v) Tween 20) containing 3% skim milk for 1 h. The membranes were briefly rinsed with buffer B (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl) and incubated for 1 h in buffer A containing 1% skim milk and PGHS-1- or PGHS-2-specific polyclonal rabbit antibodies (1:7500). The membranes were washed six times (5 min each) with buffer B. A second incubation with horseradish peroxidase-conjugated anti-rabbit IgG antibodies (Amersham Corp.) in buffer A containing 1% skim milk for 1 h, followed by several washes of the membranes was conducted as described above. Finally, the immunoreactive bands were visualized by using the enhanced chemiluminescence kit (Amersham Corp.) as instructed by the manufacturer.
In a separate study designed to measure
cerebrovascular production of prostaglandins, newborn pigs were
anesthetized as above, and catheters were placed in the left ventricle
via the right subclavian artery and in the sagittal sinus. After the
animals were allowed to stabilize for 2 h, blood samples were collected
from the femoral artery and sagittal sinus, before and 45 min after
injection of NS-398 (10 mg/kg intravenously) for prostaglandin
determination(41) . Cerebral blood flow was measured using
radiolabeled microspheres as described in detail
previously(4, 41) . The net cerebrovascular production
of prostaglandins was calculated as total cerebral blood flow
difference in prostaglandin concentrations of sagittal sinus and
arterial blood samples and expressed as ng/min/100 g of brain tissue.
Similar studies were not done on juvenile pigs because of surgical
difficulties in catheterization of sagittal sinus.
Figure 1: Distribution of PGHS-1 and PGHS-2 mRNAs in tissues of the newborn and juvenile pig. A, original autoradiograph. B, PhosphorImager-enhanced image of the autoradiogram in A. The antisense cRNA probe for PGHS-1 was 293 nucleotides, and for PGHS-2 it was 209 nucleotides; the protected fragments were 235 and 185 nucleotides for PGHS-1 and PGHS-2, respectively. Lane1, tRNA; lanes2 and 3, brain cortex; lanes4 and 5, cerebellum; lanes6 and 7, medulla oblongata; lanes8 and 9, cerebral periventricular area; lanes10 and 11, hippocampus; lanes12 and 13, thalamus; lanes14 and 15, retina; lanes16 and 17, choroid; lanes18 and 19, lung; lane20, antisense RNA probe. Lanes2, 4, 6, 8, 10, 12, 14, 16, and 18 contain samples from the juvenile tissues, and lanes3, 5, 7, 9, 11, 13, 15, 17, and 19 contain samples from the newborn tissues. Total RNA (50 µg) isolated from various regions of the brain and other tissues was subjected to RNase protection analysis as described under ``Experimental Procedures.'' Autoradiographic exposure was for 7 days.
In contrast to PGHS-1 mRNA, PGHS-2 mRNA in both neural and non-neural tissues was barely detectable; a significant exception was the brain cortex and, to a lesser extent, the choroid of the newborn (Fig. 1, A and B, lanes3 and 17, respectively). Newborn brain cortex contained a 3-fold greater expression of PGHS-2 mRNA compared with juvenile cortex (Fig. 1, A and B, lanes2 and 3). The abundance of PGHS-2 mRNA was variable in different regions of the brain and in other tissues; where PGHS-2 mRNA was detectable, expression of PGHS-2 was higher in the newborn than in juvenile animals.
Figure 2: mRNA levels of PGHS-1 and PGHS-2 in brain cortex and microvasculature from newborn and juvenile pigs. A, PGHS-1; B, PGHS-2. In both panels: lane1, tRNA (control); lane2, juvenile brain cortex; lane3, newborn brain cortex; lane4, juvenile cerebral vasculature; lane5, newborn cerebral vasculature; lane6, antisense RNA probe. The antisense RNA probe for PGHS-1 was 293 nucleotides, and for PGHS-2 it was 209 nucleotides; the protected fragments were 235 and 185 nucleotides for PGHS-1 and PGHS-2, respectively. Total RNA (100 µg) was subjected to RNase protection assay as described under ``Experimental Procedures.'' Autoradiographic exposure was for 6 days. C, newborn/juvenile ratio of mRNA for PGHS-1 and PGHS-2 in cerebral cortex and microvasculature.
Figure 3: Immunoblot of PGHS-1 and PGHS-2 proteins in newborn and juvenile pig cerebral microvasculature. Lane1, juvenile; lane2, newborn; lane3, 50 ng of purified ovine PGHS-1 or PGHS-2 protein (Cayman). Arrows point to PGHS-1 and PGHS-2 polypeptides (70 kDa) that were immunoprecipitated with specific antibodies from the detergent-lysates of cerebral microvasculature (2 mg of protein) isolated from newborn and juvenile pig brains. Following electrophoretic transfer of the proteins to nitrocellulose membranes, PGHS-1 and PGHS-2 polypeptides were detected by Western blotting and visualized by enhanced chemiluminescence (Amersham Corp.).
Figure 4:
Prostanoid synthesis in vitro by
purified cerebral microvasculature from newborn and adult pigs.
Results, calculated as means ± S.E. of quadruplicate
experiments, are expressed as percentages of the basal production of
prostaglandins (in the absence of PGHS inhibitors) in tissues from the
corresponding age group; basal synthesis of PGE,
PGF
, and 6-keto-PGF
was
2-3-fold greater in tissues from newborns than from adults. The
PGHS inhibitors, ibuprofen (1 mM), indomethacin (0.1
mM), and NS-398 (0.1 mM), were preincubated with the
homogenates of cerebral microvessels for 20 min at 25 °C, and the
reaction was initiated by adding 50 µM arachidonic acid.
The reaction rate was linear with time for 10 min. The samples were
boiled for 2 min and clarified by centrifugation. Prostaglandin
concentrations in the supernatants were determined by radioimmunoassay.
*, p < 0.05, newborn versus juvenile. Openbars, juvenile; filledbars,
newborn.
Figure 5: Prostaglandin concentrations in the brain cortex and retina of newborn and juvenile pigs treated with saline or NS-398. Brain cortex and retina from saline- and NS-398-treated animals were homogenized, the prostaglandins were extracted on octadecylsilyl silica columns, and their concentrations were determined by radioimmunoassay as described under ``Experimental Procedures.'' Results are the mean ± S.E. of four experiments. *, p < 0.05;**, p < 0.01, saline-treated versus NS-398-treated. Openbars, saline; filledbars, NS-398.
To assess the effect of NS-398 on cerebrovascular prostaglandins in the newborn animal, their concentrations in blood samples from arterial and sagittal sinus blood were determined before and after the injection of NS-398 to newborn pigs, and in vivo cerebrovascular prostaglandin production was calculated(4) . Net cerebrovascular production of prostaglandins was reduced by >65% in response to NS-398 treatment (Fig. 6); this decrease was unrelated to cerebral blood flow, which actually increased by 33-45% after NS-398 treatment.
Figure 6: In vivo cerebrovascular production of prostaglandins in newborn pigs treated with NS-398. The net cerebrovascular production of prostaglandins in vivo was determined before and 45 min after administration of NS-398 and calculated as the product of total cerebral blood flow times the difference in prostaglandin concentrations of sagittal sinus blood and arterial blood, which was expressed as ng/min/100 g of brain tissue; cerebral blood flow was measured using the microsphere technique and prostaglandins by radioimmunoassay, as described under ``Experimental Procedures.'' Results are mean ± S.E. of four experiments. *, p < 0.05 before (openbars) versus after (filledbars) NS-398 treatment.
Several studies have reported that prostaglandin levels are elevated in the neonatal blood and brain during the perinatal period (5, 6, 8) . However, the reasons for this increase in cerebral prostaglandins are not known. We tested the hypothesis that high prostaglandin levels in brain during neonatal period may be due to increased PGHS-2 activity and provided two main lines of evidence in support of this hypothesis. First, brain cortex and microvasculature of the newborn expressed more PGHS-2 mRNA than the juvenile animal, whereas PGHS-1 mRNA was more abundant in the juvenile than in cerebral cortex and microvessels of the newborn. Moreover, PGHS-2 comprised the majority of immunoreactive PGHS proteins in the newborn brain cortex. Second, NS-398, a relatively specific PGHS-2 inhibitor, produced a much greater decrease in prostaglandin synthesis in the newborn compared with the juvenile pig.
RNase protection assays revealed that PGHS-1 mRNA was ubiquitously expressed but that its abundance differed within various regions of the brains of the newborn and juvenile pigs. The hindbrain and midbrain contained highest expression of PGHS-1. Similar observations have been made by others using different techniques such as Northern analysis and in situ-hybridization(29, 43) . However, the expression of PGHS-1 in brain is considerably lower than that in peripheral tissues such as the choroid and lungs, in accordance with other data(28, 29) . In addition to its diverse tissue expression, PGHS-1 mRNA increased with age in brain (29) and cerebral vasculature (Fig. 2); in other tissues such as fetal cotyledon and amnion, PGHS-1 expression does not increase with gestational age(44, 45) . The ontogenic increase in PGHS-1 mRNA in cerebral vasculature is associated with a corresponding 3-fold increase in immunoreactive PGHS-1 in brain microvessels of the juvenile animal.
In contrast to PGHS-1, PGHS-2 mRNA was not readily detectable in various regions of the porcine brain. However, the highest PGHS-2 expression was observed in brain cortex and microvasculature of newborn animals. PGHS-2 mRNA levels decreased with age, and although immunoreactive PGHS-2 did not exhibit similar ontogenic changes, PGHS-1 protein and mRNA were markedly less in the newborn, thus disclosing the relative abundance of PGHS-2 in newborn brain and microvessels.
Further support for the suggestion that increased PGHS-2 expression accounts for the high prostaglandin G/H synthase activity and, in turn, high prostaglandin levels in newborn comes from studies using PGHS inhibitors. NS-398 is more than 100-fold more potent in inhibiting PGHS-2 than PGHS-1(46) . Furthermore, inhibition of PGHS-1 by NS-398 could be reversed by excess substrate, arachidonic acid, whereas time-dependent inactivation of PGHS-2 by NS-398 renders PGHS-2 refractory to substrate-mediated relief of inhibition(47) . In this context, pretreatment of microvessels with NS-398 may have enabled us to differentiate PGHS-1 and PGHS-2 in their contribution toward prostanoid synthesis by cerebral microvasculature.
NS-398 decreased prostaglandin levels and synthesis by <35% in juvenile tissues and by >60% in those of the newborn. The differential effects of NS-398 in newborn animals compared with the juvenile pigs were consistently observed in prostaglandin synthesis by isolated cerebral vasculature, in prostaglandin levels in brain cortex, and in in vivo cerebrovascular prostaglandin production. Moreover, unlike the brain prostaglandins, NS-398 minimally reduced prostanoids in the newborn retina (Fig. 5) in which PGHS-2 mRNA could not be detected (Fig. 1). Thus, the preponderance of PGHS-2 mRNA and protein in newborn cortex and microvasculature, minimal expression of immunoreactive PGHS-1 in the newborn, and pronounced inhibition of prostanoid synthesis both in vivo and in vitro by NS-398 in newborn tissues compared with those of juvenile animals, taken together, indicate that PGHS-2 is a major contributor to prostaglandin synthesis in newborn brain and cerebral microvasculature.
The factors responsible for the increased expression of PGHS-2 in newborn cerebral cortex and microvasculature are not known but may include estrogens which increase late in gestation and induce a gradual increase in PGHS activity(48, 49) . Indeed, high PGHS-2 expression and concomitant increase in prostanoid synthesis in late gestation and at term was observed in fetal placental tissues(45) . Besides being transcriptionally regulated by various stimuli, PGHS-2 expression is also controlled by factors affecting mRNA stability (50) and its translatability(51) . The mechanisms governing the increased expression of PGHS-2 in neonatal brain remain to be elucidated.
The function of the elevated prostaglandin concentrations in the brain is so far not known. Because prostaglandins have been shown to exhibit neuroprotective properties(52) , It is likely that their increased levels during the perinatal period may provide protection to the fetal brain toward the end of parturition when oxygen tension is markedly reduced (53) and the risk of hypoxic brain injury increases. Being a rapidly inducible enzyme, PGHS-2 would be suited for such a temporary but important role in perinatal life.