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Am J Physiol Regul Integr Comp Physiol 275: R1812-R1821, 1998;
0363-6119/98 $5.00
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Vol. 275, Issue 6, R1812-R1821, December 1998

PGE2, via EP3 receptors, regulates brain nitric oxide synthase in the perinatal period

Isabelle Dumont1,2, Krishna G. Peri1, Pierre Hardy1, Xin Hou1, Ana Katherine Martinez-Bermudez3, Stéphane Molotchnikoff2, Daya R. Varma3, and Sylvain Chemtob1,3

1 Departments of Pediatrics, Ophthalmology, and Pharmacology, Research Center of Hôpital Ste-Justine, Montreal H3T 1C5; 3 Department of Pharmacology and Therapeutics, McGill University, Montreal H3G 1Y6; and 2 Faculty of Biological Sciences, University of Montreal, Montreal, Canada H3C 3J7

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We tested the hypothesis that high prostaglandin levels during the perinatal period might regulate brain nitric oxide synthase (nNOS) expression. nNOS and cyclooxygenase (COX)-2 mRNAs were higher in brain cortex and the periventricular area of newborn rats and pigs compared with adult brain. Nitric oxide synthase activity was also 2.5- to 4-fold higher in newborn than in adult brain. Administration of nonselective COX inhibitor ibuprofen or COX-2 inhibitor nimesulide every 8 h for 24 h to newborn rats and pigs reduced prostaglandin levels and caused comparable reductions in nNOS mRNA, protein, and activity to levels of adults; COX inhibitor-induced changes were prevented by cotreatment with PGE2 analog, 16,16-dimethyl-PGE2, and agonist for the EP3 receptor of PGE2, sulprostone, but not by PGI2 analog carbaprostacyclin, PGD2, EP1 receptor agonist 17-phenyl trinor-PGE2, and EP2 agonist butaprost. Concordant observations were made in vitro and revealed that nNOS expression (detected by NADPH diaphorase reactivity) mostly present in neurons of the deeper cortical layers was reduced by COX inhibitor, and this effect was prevented by EP3 agonist. In conclusion, high levels of PGE2 in neonatal brain contribute to the increased expression of nNOS by acting on EP3 receptors; this positive interaction between PGE2 and nNOS might be required physiologically for normal brain development.

cyclooxygenase-2; neuronal nitric oxide synthase; prostaglandin E2; newborn

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE THREE ISOFORMS of nitric oxide synthase (NOS) include the calcium-independent isoform expressed mainly in macrophages and the calcium-dependent isoforms present in endothelial cells (eNOS) and neuronal tissues (nNOS; see Ref. 22). Nitric oxide (NO), formed via nNOS, acts as a signal for the development and shaping of neuronal cells and their activity (6, 55), as well as the control of blood supply to the brain in response to metabolic demands (30). More than 95% of all NOS activity in the brain is attributed to nNOS (17, 25, 29).

NOS displays a precise pattern of increased activity in the nervous system during the perinatal period in various species, including rodents, pigs, cats, and humans (6, 9, 40, 52, 58). These developmental changes in NOS activity immediately precede a period of maximal synaptogenesis (20, 40, 61) for which it has been implicated (57). Similar functions have been ascribed to cyclooxygenase (COX)-2 (37, 61, 32). The mechanisms regulating this high NOS activity in the perinatal period are not well understood. Although a role for estrogens has been suggested, this contribution is only partial (40). A role for prostaglandins in regulating NOS activity has been proposed under certain conditions. It has been reported that COX inhibitors markedly inhibit NO production in rat alveolar macrophages (2); this has led to the suggestion that the inhibition of prostaglandin synthesis results in an inhibition of NOS gene expression and in turn in a decreased NO synthesis. There is also evidence that constitutive NOS (eNOS and nNOS) can potentially be induced by prostaglandins (51). Furthermore, the suggested role of estrogens in partially governing NOS activity (40) may be partly mediated by prostaglandins, since estradiol can stimulate prostaglandin synthesis (46).

Prostaglandin synthesis is catalyzed by COX-1, which is mostly constitutive, and COX-2, a readily inducible isozyme (59). We have previously shown that COX-2 expression is increased in brain cortex and periventricular white matter of the newborn and is its main source of high prostaglandin levels, such that COX-2 activity accounts for ~80% of total COX activity in perinatal brain, whereas in the adult it is responsible for <10% of total COX activity (37, 49); COX-1 expression is unaltered in brain during this developmental period (37, 49). Because NOS has the potential of being induced by prostaglandins (2, 51), and both prostaglandin levels as well as NOS activity have been found to be high in brain tissue of the perinate (6, 9, 31, 37, 40, 41, 49, 52), we hypothesized that high levels of prostaglandins regulate the expression of nNOS in brain in the newborn. For this purpose, we characterized the ontogeny of nNOS and COX-2 in brain cortex and periventricular region of neonatal rats and pigs and determined the role of COX-2-derived prostaglandins in inducing nNOS expression. Our findings reveal that PGE2 exerts a positive control over nNOS expression in the brain during the perinatal period and that this effect is mediated via the EP3 subtype of PGE2 receptors.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Chemicals. Butaprost was a gift from Miles (West Haven, CT), and M & B-28,767 was from Rhone-Poulenc Rorer. The following products were purchased: polyclonal rabbit antibody specific to nNOS from Calbiochem-Novabiochem (San Diego, CA); beta -actin cDNA from Ambion (Austin, TX); [alpha -32P]CTP (3,000 Ci/mmol), enhanced chemiluminescence kit, and L-[3H]arginine from Amersham (Mississauga, ON, Canada); pepstatin and leupeptin from Boehringer Mannheim (Montreal, PQ, Canada); nimesulide, ibuprofen, aspirin, neutral red, soybean trypsin inhibitor (type II-S), phenylmethylsulfonyl fluoride (PMSF), beta -mercaptoethanol, 1,4-dithiothreitol, HEPES, EDTA, EGTA, L-arginine, NG-nitro-L-arginine (L-NNA), NG-nitro-L-arginine methyl ester (L-NAME), NADPH, FAD, 6R-tetrahydrobiopterin, and beta -NADPH from Sigma-Aldrich (Oakville, ON, Canada); 16,16-dimethyl-PGE2, PGD2, carbaprostacyclin, 17-phenyl trinor-PGE2, and sulprostone from Cayman (Ann Arbor, MI); 7-nitroindazole from Tocris Cookson (St. Louis, MO); ribonuclease A and T7 sequencing kit from Pharmacia Biotech (Montreal, PQ, Canada); pGEM3 plasmid vector and in vitro transcription kit from Promega (Madison, WI); Dowex AG-50W-X8 resin, protein assay, and electrophoretic reagents purchased from Bio-Rad (Mississauga, ON, Canada); guanidinium isothiocyanate, T4 DNA ligase, and restriction enzymes from BRL Life Technologies (Burlington, ON, Canada); radioimmunoassay kits for PGE2, PGD2, and 6-keto-PGF1alpha from Advanced Magnetics (Boston, MA); all others chemicals were from Fisher Scientific (Montreal, PQ, Canada).

Animals. Time-pregnant Sprague-Dawley rats were purchased from Charles River (St. Constant, PQ), maintained on a 12:12-h light-dark (lights on 0700-1900) cycle at 22-25°C and 50-70% humidity, and fed ad libitum rat chow and tap water. Newborn (1-2 days old) Yorkshire pigs were purchased from Fermes Ménard (L'Ange Gardien, PQ). Adult (6-8 mo) pig brains were collected from an abbatoir immediately after death and were brought to the laboratory on ice. Animals were used according to a protocol of the Animal Care Committee of St. Justine Hospital (Montréal, PQ). Pregnant rats were decapitated after anesthesia with pentobarbital sodium (60 mg/kg ip), and 17- and 19-day-old fetuses were immediately delivered thereafter by caesarean section. A group of pregnant rats were allowed to deliver, and pups were studied at 6, 8, 10, 12, and 15 days of age. Frontoparietal brain cortex and periventricular area from rat pups were collected after decapitation. Tissues from adult animals were obtained from males and nonpregnant females; results were independent of gender. Newborn pigs were anesthetized with halothane, and polyethylene catheters were placed in the jugular vein for intravenous injections. Piglets were killed with pentobarbital sodium (120 mg/kg) for the removal of brain.

Treatments. Rat pups (9 days old) were randomly assigned to intraperitoneal treatment every 8 h for 24 h with saline, nonselective COX inhibitor ibuprofen (40 mg/kg), COX-2 inhibitor nimesulide (5 mg/kg; see Ref. 21), or a combination of nimesulide with one of the following: 16,16-dimethyl-PGE2 (stable PGE2 analog, 10 µg/kg), carbaprostacyclin (PGI2 analog, 1 µg/kg), PGD2 (10 µg/kg), 17-phenyl trinor-PGE2 (agonist of the EP1 receptor of PGE2, 20 µg/kg), butaprost (agonist of the EP2 receptor of PGE2, 100 µg/kg), sulprostone (EP3 agonist, 10 µg/kg; see Ref. 13); animal age selected was based on pilot studies that revealed near-peak expression of nNOS (see confirmation in Fig. 1). Newborn pigs were treated with the same agents as described for rats. The 24-h treatment duration was based on pilot experiments which revealed that acute (<= 2 h) administration of COX inhibitors was ineffective in altering nNOS expression. Doses of ibuprofen and nimesulide used in this study have previously been shown to reduce prostaglandin levels to those found in the adult pig (23, 39); and doses of prostaglandins and analogs administered have been demonstrated to alter prostaglandin levels and/or cause effects in vivo (3, 34, 47). In addition, conversely, we tested the role of NO on COX-2 expression; accordingly, animals were treated every 8 h for 24 h with L-NAME (1 mg/kg; see Ref. 27).


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  		Fig. 1.   Expression of neuronal nitric oxide synthase (nNOS; C and D) and cyclooxygenase (COX)-2 (A and B) mRNA in rat brain cortex (A and C) and periventricular region (B and D) at different developmental ages. Total RNA (60 µg) isolated from fetal, neonatal, and adult rat brains was subjected to ribonuclease protection assay. F and P in front of the ages refer to fetal and postnatal, respectively. Values are means ± SE of 3 experiments for each age. * P < 0.05 compared with all other values; dagger  P < 0.05 compared with values at P8-P15 (by analysis of variance and Tukey-Kramer method).

In separate experiments, slices (2-3 mm) of newborn pig brain cortex were incubated for 12-18 h in culture medium without or with (10 µM) ibuprofen, nimesulide, or a combination of nimesulide (10 µM) and a PGE2 analog (1 µM): PGD2, carbaprostacyclin, 16,16-dimethyl-PGE2, 17-phenyl trinor-PGE2, butaprost, sulprostone, or M & B-28,767 (selective EP3 agonist; see Ref. 13).

Preparation of cRNA probes for rat and pig nNOS, pig destrin and rat COX-2, and ribonuclease protection assays. The partial cDNAs encoding rat and pig nNOS and pig destrin mRNAs (45) were synthesized by RT-PCR, respectively, from rat and porcine cerebellar total RNA. Reverse transcription of total RNA followed by amplification of the cDNA using the gene specific primer sets were conducted as previously described (49, 50). The primer pairs for porcine destrin were as follows: 5'-ATG ATG C<UNL>AA GCT T</UNL>TG AAA CC-3' and 5'-GG<UNL>A AGC TT</UNL>T CGA TCT GTG G-3'. The primer pairs for porcine nNOS were as follows: 5'-GGG <UNL>GGA TCC</UNL> ARG ART AYG ARG ART GGA ART GG-3' (QEYEEWKW) and 5'-GGG <UNL>GAA TTC</UNL> GAT RTC RAA YTG CGY TGY TGC CA-3' (WQQRQFDIQ), based on the consensus amino acid sequences (in parentheses) of human and rat nNOS sequences. The amplified products (0.4 kb) were digested with appropriate restriction enzyme (underlined sequences in the primers denote the restriction sites) and cloned into pGEM4 vector.

Rat nNOS cDNA was amplified as above with the following primer pairs: 5'-GCA CAT TTG CAT GGG CTC GA-3' and 5'-CCT CTG CAG CGG TAT TAT TC-3'. The amplified product (1.0 kb) was digested with BamH I and the 0.214-kb fragment was cloned into pGEM4. The nucleotide sequences of rat and pig nNOS partial cDNAs were determined by sequencing multiple clones using T7 sequencing kit. The partial cDNAs of pig (49) and rat (50) COX-2 were previously described.

32P-labeled cRNA probes for nNOS and COX-2 were prepared using an in vitro transcription kit (Promega). Total RNAs from brain cortex were obtained as described previously (11). Aliquots of the total RNAs were subjected to ribonuclease protection assays according to a published protocol (4) with minor modifications. Briefly, 60 µg of total RNA were mixed with 105 counts/min of nNOS or COX-2 probes in 20 µl of hybridization buffer (80% deionized formamide, 40 mM PIPES, pH 6.8, 1 mM EDTA, and 0.4 M NaCl), denatured at 90°C for 5 min, and incubated overnight at 50°C. The RNA hybrids were digested with ribonuclease A (10 µg/ml) and ribonuclease T1 (200 U/ml) in 200 µl of digestion buffer (10 mM Tris · HCl, pH 7.5, 5 mM EDTA, and 0.3 M NaCl) for 30 min at 25°C. Proteinase K treatment followed by precipitation of protected fragments was conducted exactly as described (4). The protected RNA fragments were resolved on urea-8% polyacrylamide gels, and the bands were visualized by phosphorimaging (Molecular Dymamics) and quantified densitometrically.

Western blotting. Western blotting for nNOS was performed using a method previously described (1, 49, 50). Brain cortex was homogenized in a buffer (20 mM Tris · HCl, pH 8.0, 1 mM EDTA, 137 mM NaCl, 1% Nonidet P-40, 10 µg/ml each of leupeptin, pepstatin, and soybean trypsin inhibitor, and 0.2 mM PMSF) with an Omni tissue grinder for 15-20 s; whole tissue lysates were used, since nNOS is contained in both membrane and cytosolic fractions (8, 28). Protein was determined by dye-binding assays using bovine serum albumin as the standard. After the addition of beta -mercaptoethanol to aliquots of supernatants (100 µg protein) to a final concentration of 10%, the samples were denatured by boiling for 5 min and resolved by electrophoresis on an 8% SDS-polyacrylamide gel. The transfer of tissue lysate proteins to membranes and immunoblotting using antibodies (1:2,000) against nNOS were conducted exactly as previously described (1, 49). Immunoreactive bands were visualized by chemiluminescence (Amersham) as recommended by the supplier.

NOS activity. NOS activity was determined by the conversion of L-arginine to L-citrulline (53) as previously described (27). Briefly brain tissue was homogenized in 5 ml ice-cold homogenization buffer (50 mM Tris · HCl, pH 7.5, 1 mM EDTA, 1 mM 1,4-dithiothreitol, 5 mM glucose, 1 mM PMSF, and 20 µg/ml each of aprotinin, leupeptin, and soybean trypsin inhibitor). The homogenate was centrifuged at 12,000 g for 15 min, and the protein content of the supernatant was determined by the dye-binding method. An aliquot of the supernatant (100-200 µg protein) was incubated in the absence or the presence of the NOS inhibitor L-NNA (1 mM) or the selective nNOS inhibitor 7-nitroindazole (100 µM; see Ref. 44) in incubation buffer (in mM: 50 HEPES, pH 7.5, 1 1,4-dithiothreitol, 1 EDTA, and 1.25 CaCl2) with 0.1 mM L-arginine [containing 1 µCi L-[3H]arginine (1 µCi/mmol specific activity)], 1 mM NADPH, 15 µM 6R-tetrahydrobiopterin, 1 µM FAD, and 1 µM calmodulin for 10 min at 37°C. The reaction was terminated by addition of 1 ml ice-cold 100 mM HEPES buffer (pH 5.5) containing 10 mM EGTA and 500 mg Dowex AG-50W-X8 cation exchange resin and immediately centrifuged at 10,000 g for 20 min. Radioactivity in the L-[3H]citrulline-containing supernatant was counted. Total NOS and nNOS activities were measured, respectively, from the L-NNA- and 7-nitroindazole-sensitive production of L-[3H]citrulline from L-[3H]arginine. Inducible NOS (iNOS) activity was determined by adding EGTA (10 mM) to the incubation buffer. Constitutive NOS activity, mostly all nNOS in brain tissue (25, 29), was obtained by subtracting iNOS activity from total L-NNA-sensitive NOS activity. 7-Nitroindazole-sensitive NOS activity was measured as total NOS activity minus that after addition of 7-nitroindazole.

Prostaglandin assays. 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. Extraction and measurement of PGE2, PGD2, and 6-keto-PGF1alpha (stable PGI2 metabolite) in the supernatant were performed as previously described in detail (39, 49).

NADPH diaphorase histochemistry. NADPH diaphorase reactivity, which is currently a well-established marker for NOS (5, 15), was performed using a method previously described (36). Brain slices were fixed by immersion in 4% paraformaldehyde in 0.1% phosphate-buffered saline (pH 7.4) overnight at 4°C and then placed in 30% sucrose buffer for 2 days. Tissue sections 40 µm thick were made with a cryotome. The free-floating sections were incubated in 0.1 M phosphate buffer (pH 7.4) containing 0.1% beta -NADPH at 37°C for 60 min. After the reaction, the sections were rinsed in phosphate buffer and mounted on slides. The slides were air-dried and treated in chloroform for 30 min to remove background staining and counterstained with neutral red (0.13 g/100 ml).

Statistics. Data were analyzed by analysis of variance, comparison among means test (Tukey-Kramer method), and Student's t-test. Statistical significance was set at P < 0.05. Data are presented as means ± SE.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Ontogeny of nNOS and COX-2 mRNA in rat and pig brain. nNOS and COX-2 mRNAs were expressed in rat fetal brain cortex and periventricular area on days 17 and 19 of gestation and exhibited an age-related increase that peaked on postnatal day 10, after which they declined (Fig. 1). In pig, expression of nNOS and COX-2 mRNA was approximately threefold greater in newborn than in adult brain cortex (Fig. 2, A and B) and periventricular area (data not shown).


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  		Fig. 2.   COX-2 (A) and nNOS (B) mRNA, nNOS immunoreactive protein (C and D), and nitric oxide synthase (NOS) activity (E) in newborn and adult pig brain cortex. A and B, total RNA (60 µg) isolated from pig brain of newborn (1-2 days old) and adult was subjected to ribonuclease protection assay. C: Western blot lanes were each loaded with 100 µg protein (Bio-Rad). Arrow points to the nNOS 155-kDa protein, the only band detected in the range of interest, 100-165 kDa. Immunoreactive band densitometry was quantified by chemiluminescence (D). Constitutive brain NOS activity was measured from the NG-nitro-L-arginine-sensitive production of L-[3H]citrulline from L-[3H]arginine after subtracting inducible NOS activity from total NOS activity as described in MATERIALS AND METHODS (E); similar NOS activity was measured by determining 7-nitroindazole-sensitive NOS activity, indicating that the latter reflects mostly nNOS activity. Values are means ± SE; n = 3-4 experiments for each value; * P < 0.05 compared with value for the adult.

nNOS protein immunoreactivity and NOS activity in newborn and adult pig brain. As was the case with nNOS mRNA, nNOS immunoreactive protein and constitutive NOS activity in newborn pig brain cortex were 2.5- and 4-fold greater than that in the adult (Fig. 2, C-E). Similar age-related differences were observed in the periventricular region. L-NNA- and 7-nitroindazole-sensitive NOS activities yielded equivalent results, which reflect the predominant nNOS activity in brain (25, 29); for instance, L-NNA and 7-nitroindazole-sensitive NOS activity in newborn brain cortex were, respectively, 62 ± 12 and 55 ± 10 pmol · min-1 · mg protein-1. iNOS activity was approximately one-tenth that of nNOS and did not significantly differ between newborn and adult.

Modulation of nNOS mRNA expression by prostaglandins in the newborn rat. Treatment of 9-day-old rat pups for 24 h with COX-2 inhibitor nimesulide or nonselective COX inhibitor ibuprofen reduced brain PGE2 concentrations (pg/mg protein) from 201 ± 54 to 87 ± 14 and 72 ± 15, respectively; comparable reductions were observed for PGD2 and 6-keto-PGF1alpha . Prostaglandin levels in the adult (e.g., PGE2: 79 ± 10 pg/mg protein) were similar to those in treated newborns. Treatment with COX inhibitors for 24 h (but not acute, <= 2 h) caused a significant reduction in the expression of nNOS mRNA in brain cortex and periventricular region of the newborn to levels found in the adult (Fig. 3, A-D). Effects of COX inhibitors on nNOS mRNA were prevented by cotreatment with 16,16-dimethyl-PGE2 (stable PGE2 analog) but unaltered by the other major prostaglandins, namely PGI2 (utilizing carbaprostacyclin) and PGD2, at similar doses; prostaglandin and analog doses used have previously been shown to change prostaglandin levels and/or cause effects in vivo (3, 34, 47). Treatments did not affect mRNA expression of beta -actin.


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  		Fig. 3.   Modulation of nNOS mRNA expression by prostaglandins in newborn rat brain cortex (A and B) and periventricular area (C and D). Rat pups (9 days old) were treated ip every 8 h for 24 h with saline, ibuprofen (40 mg/kg), nimesulide (5 mg/kg), or a combination of nimesulide (5 mg/kg) and carbaprostacyclin (PGI2 analog, 1 µg/kg), PGD2 (10 µg/kg), or 16,16-dimethyl (DM)-PGE2 (10 µg/kg). Adult rats were untreated. Total RNA (60 µg) was subjected to ribonuclease protection assay. nNOS cRNA probe and protected RNA fragment contained, respectively, 255 and 235 nucleotides (nt), and the cRNA probe and protected RNA fragment for beta -actin contained 200 and 127 nt, respectively. Autoradiographic exposure was overnight and was visualized by phosphorimaging. Values in histograms are means ± SE of 3-4 experiments each. * P < 0.01 compared with other values.

To assess if conversely high NO synthesis in the newborn affected COX-2 expression, a separate group of rat pups was treated with L-NAME (1 mg/kg; see Ref. 27). L-NAME did not affect COX-2 mRNA expression or tissue PGE2 levels (data not shown) as we previously reported for the latter (26). Studies were therefore pursued to ascertain the role of COX-2 products on nNOS mRNA, protein, and activity in the perinate.

Modulation of nNOS mRNA and protein expression by prostaglandins in the newborn pig. Experiments conducted in the rat pup utilizing prostaglandin synthesis inhibitors and prostaglandin analogs were repeated in the newborn pig for confirmation. In addition, because PGE2 seemed to be the major prostaglandin involved in the regulation of nNOS expression (Fig. 3), the PGE2 receptor(s) (EP1, EP2, EP3, and/or EP4; see Ref. 13) mediating these actions was investigated using receptor agonists. Treatment of newborn pigs with nimesulide reduced brain PGE2 concentrations (pg/mg protein) from 297 ± 43 to 68 ± 17, levels similar to those of the adult (80 ± 15). Nimesulide caused a significant decrease in the expression of nNOS mRNA and immunoreactive protein (Fig. 4, A and B). This effect was prevented by 16,16-dimethyl-PGE2 and sulprostone (agonist of EP3 receptors of PGE2) but not by 17-phenyl trinor-PGE2 (EP1 agonist) and butaprost (EP2 agonist); the role for EP4 was not investigated because this receptor is not detected in newborn brain tissue (38).


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  		Fig. 4.   Modulation of nNOS mRNA (A) and protein (B) expression by prostaglandins in newborn pig brain. Newborn pigs (1-2 days old) were treated iv every 8 h for 24 h with saline, nimesulide (5 mg/kg), or a combination of nimesulide (5 mg/kg) and 16,16-dimethyl PGE2 (10 µg/kg), 17-phenyl trinor-PGE2 (EP1 agonist, 20 µg/kg), butaprost (EP2 agonist, 100 µg/kg), or sulprostone (EP3 agonist, 10 µg/kg). A: total RNA (60 µg) was subjected to ribonuclease protection assay as described in MATERIALS AND METHODS. nNOS cRNA probe and protected RNA fragment contained, respectively, 446 and 401 nucleotides (nt), and the cRNA probe and protected RNA fragment for destrin contained 237 and 165 nt, respectively. Autoradiographic exposure was overnight and visualized by phosphorimaging. B: for Western blot, each lane was loaded with 100 µg protein (Bio-Rad). Arrow points to 155-kDa nNOS immunoreactive band revealed by chemiluminescence; a single band was detected in the range of interest, 100-165 kDa. Figure is a typical representation of 4 experiments.

Effects of COX inhibitors and prostaglandins on NOS activity in newborn brain. We examined if PGE2-elicited changes in nNOS mRNA and protein are reflected in NOS activity. NOS activity in newborn rat and pig brain cortex and periventricular region was similarly decreased to adult levels by COX inhibitors ibuprofen and nimesulide (shown in Fig. 5, A and B). This reduction was prevented by cotreatment with 16,16-dimethyl-PGE2 and EP3 agonist sulprostone but not with the same dose (10 µg/kg every 8 h 3 times) of PGD2.


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  		Fig. 5.   Effects of COX inhibitors and prostaglandins on NOS activity in newborn rat (A) and pig (B) brain. Newborn rats (9 days old) and pigs (1-2 days old) were treated for 24 h with saline, ibuprofen, or nimesulide with and without 16,16-dimethyl-PGE2, sulprostone, or PGD2, as described in Figs. 3 and 4 and in MATERIALS AND METHODS. Adult animals were untreated. 7-Nitroindazole-sensitive NOS activity was determined as described in Fig. 2 and in MATERIALS AND METHODS. Values are means ± SE of 3-4 experiments each. * P < 0.01 compared with all other values (by analysis of variance and Tukey-Kramer method).

In vitro modulation of newborn brain nNOS activity by COX inhibitors, prostaglandins, and PGE2 receptor agonists. To examine whether effects of PGE2 on NOS expression in vivo and dependent on EP3 receptors (Figs. 3-5) are mediated directly on brain, experiments were conducted in vitro. Incubation of slices of newborn pig brain cortex with ibuprofen or nimesulide for 18 h caused a significant and equivalent reduction in 7-nitroindazole-sensitive NOS activity (Fig. 6A). 16,16-Dimethyl-PGE2, sulprostone, and the selective EP3 receptor agonist M & B-28,767 prevented COX inhibitor-induced decrease in NOS activity. In contrast, PGD2, carbaprostacyclin, 17-phenyl trinor-PGE2, or butaprost (at high concentrations of 1 µM) did not alter effects of nimesulide. Prevention of nimesulide-induced decrease in NOS activity by EP3 agonists in vitro was also reflected on nNOS mRNA, and this effect was EP3 agonist (sulprostone) concentration dependent (Fig. 6B).


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  		Fig. 6.   A: in vitro modulation of nNOS activity in newborn pig brain by COX inhibitors in the absence or presence of PGE2 receptor agonists. Brain cortex slices from 1- to 2-day-old newborn pigs were incubated for 18 h in culture medium without or with 10 µM ibuprofen, nimesulide, or a combination of nimesulide and 1 µM PGD2, carbaprostacyclin (PGI2 analog), 16,16-dimethyl-PGE2, 17-phenyl trinor-PGE2 (EP1 agonist), butaprost (EP2 agonist), sulprostone, or M & B-28,767 (EP3 agonists). 7-Nitroindazole-sensitive NOS activity was measured as described in Fig. 2 and in MATERIALS AND METHODS. Values are means ± SE; n = 3 experiments for each value. * P < 0.01 compared with all other values (by analysis of variance and Tukey-Kramer method). B: dose response of EP3 receptor agonist sulprostone on nNOS mRNA expression. Newborn (1-2 days old) pig brain cortex slices were incubated for 18 h in culture medium without or with nimesulide (10 µM) or a combination of nimesulide (10 µM) and sulprostone (0.1, 1, and 10 µM). Total RNA (60 µg) was subjected to ribonuclease protection assay as described in MATERIALS AND METHODS. Values are means ± SE; n = 3 experiments for each value. * P < 0.05 compared with all other values (by analysis of variance and Tukey-Kramer method).

Modulation of nNOS-containing brain cells by prostaglandins. To localize the neural elements producing NO that are modulated by prostaglandins, slices of newborn pig brain incubated for 12 h in culture medium without or with nimesulide or a combination of nimesulide with sulprostone were stained for NADPH diaphorase activity. This approach was based on numerous reports demonstrating that NOS fully accounts for NADPH diaphorase activity as identified histochemically (5, 15). The major modulation of NADPH diaphorase staining by prostaglandins was located in neurons of the deepest layers of the brain cortex and in the immediate underlying white matter, consistent with prominent location of NOS in forebrain (5, 36), whereas NADPH diaphorase reactivity of cerebral microvessels due to eNOS expression (5, 42) was unaffected by prostaglandin modulation (Fig. 7). Incubation of slices of newborn pig brain with COX-2 inhibitor nimesulide for 12 h caused a threefold reduction in NADPH diaphorase positive cells (Fig. 7). This effect was prevented by the EP3 receptor agonist sulprostone. Hematoxylin and eosin staining (10) of brain slices revealed no adverse effect of prostaglandin modulation on cell number (data not shown) as previously reported (7, 48); hence, the effect of prostaglandins was on the expression of nNOS.


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  		Fig. 7.   Modulation of NADPH diaphorase staining by prostaglandins. Brain cortex slices from newborn pig incubated for 12 h in culture medium without (a) or with (b) nimesulide (10 µM) or a combination of nimesulide (10 µM) and sulprostone (c; EP3 agonist, 1 µM) were stained for NADPH diaphorase as described in MATERIALS AND METHODS; counterstaining by neutral red is shown in d. Calculated distribution of NADPH diaphorase-positive individual cells per field is presented in e. Small arrows point to individual NADPH diaphorase-positive cells (which are neurons), and large arrows point to blood vessels. Scale bar represents 200 µm. Values in histogram (e) are means ± SE of 3 experiments each. * P < 0.01 compared with other values (by analysis of variance and Tukey-Kramer method).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

NO has been attributed with important roles in neural development and synaptogenesis in early life (33, 57, 60), and its formation is relatively high during the perinatal period in nearly all species (6, 9, 27, 31, 40, 41, 49, 52, 58). Similar functions and ontogenic changes have been observed for COX-2-derived prostaglandins (32, 37, 49, 61). Based on evidence that prostaglandins can regulate NOS expression in vitro (2, 51), we set out to investigate whether these eicosanoids contribute to the perinatal regulation of nNOS expression and activity. Our findings reveal that the high levels of PGE2 in the brain of the neonate, arising from COX-2, contribute to the increased expression of nNOS in brain cortex and periventricular region by acting on EP3 receptors, as demonstrated on two distinct species, namely rat and pig, and shown both in vivo and in vitro.

Evidence that nNOS is regulated by prostaglandins, specifically PGE2, in the perinatal period is based on numerous observations. 1) The main COX isoform responsible for catalysis of prostaglandin generation in brain of the young neonate, namely COX-2 (37, 49), exhibits a profile of ontogenic changes similar to that of nNOS mRNA in the rat (Fig. 1). Although it was not practical to follow the ontogeny of NOS from fetal to adult life in pigs, we demonstrated nonetheless an equivalent abundance of nNOS in brain of the newborn pig relative to that of the adult, consistent with changes observed for COX-2 (Fig. 2). 2) More convincingly, a reduction in prostaglandin synthesis [sustained (24 h), but not acute] in newborn brain to levels in the adult, using either nonselective or COX-2-selective inhibitors, resulted in decreased expression of nNOS mRNA, immunoreactive protein, and activity in brain cortex and periventricular region of rats and pigs. 3) Among the major prostaglandins, PGE2 was the one that prevented COX inhibitor-induced reduction in nNOS expression and activity. This effect was observed in vivo (Figs. 3 and 5) and in vitro after incubation of newborn brain slices with COX inhibitors and PGE2 analogs (Fig. 6); these data also suggest that the in vivo modulation of nNOS by PGE2 is the result of a direct action of this prostaglandin, which is most specifically observed on brain neuronal cells (Fig. 6B). Inefficacy of PGD2 and carbaprostacyclin in vivo cannot be explained by insufficient dosing, which was comparable for all prostaglandins and analogs and previously shown to alter prostaglandin levels and/or cause effects in vivo (3, 34, 47). Moreover, inability of high concentrations (1 µM) of PGD2 and carbaprostacyclin to reverse effects of COX inhibitors in vitro additionally supports their inefficacy (Fig. 6).

The effect of PGE2 on nNOS expression in newborn brain seems to depend on EP3 receptor activation. Correspondingly, actions of PGE2 in preventing COX inhibitor-induced reduction in nNOS expression and activity were reproduced specifically by EP3 receptor agonists sulprostone and M & B-28,767 (13) and demonstrated in vivo and in vitro (Figs. 3, 4, and 6). It is relevant to point out that EP3 was previously shown to be the only PGE2 receptor detected in newborn cerebrum (38). The mechanism through which EP3 activation induces nNOS expression is not known; stimulation of c-fos (55), for which binding sites on the nNOS gene promoter are found in the 5' regulatory sequence (24), is a possibility. Altogether, our observations reveal that PGE2, via EP3 receptors, is the principal prostaglandin involved in the regulation of brain nNOS in the newborn. Along these lines, one may suggest that the previously reported partial role of estrogens in regulating NOS activity in brain (40) may in part be attributed to prostaglandins (46).

At the cellular level, modulation of NADPH diaphorase staining by prostaglandins was located in the deepest layers of the cortex and in the immediately adjacent white matter in the area corresponding to the subplate neurons, which are the first to be generated in the developing mammalian cortex (12) and are critical in synaptogenesis and maturation of neuronal phenotype (14). Of interest, the developmental and regional profile of COX-2 (16, 32, 49), nNOS (6, 16), along with that of N-methyl-D-aspartate receptor expression (54, 56) which per se is coupled to NO production (43), coincides with the critical period for activity-dependent synaptic remodeling (19, 35). In addition, an intimate relationship between neural cells and blood vessels (18), which are also known to contain abundant NOS and COX-2 in the perinatal period (1, 49, and present paper; Fig. 7), may also suggest a significant contribution for products of these enzymes in coupling developing synaptic activity with local cerebral microcirculation (18). Because NO and COX-2 products seem to play important roles in neural development and synaptogenesis (32, 33, 37, 49, 57, 60, 61), our observations provide a link for these distinct factors in shaping neural development by disclosing a major role for PGE2 in modulating nNOS expression through action on EP3 receptors.

In conclusion, our data reveal a mechanism for increased activity of nNOS in brain during the perinatal period, contributed by actions of EP3 receptors of PGE2 which per se is generated by equivalently high catalytic activity of COX-2 (37, 49, and present study). Of interest, a similar role for prostaglandins has recently been suggested in the regulation of NOS in the eye (1). At present, the mechanisms responsible for increased COX-2 expression in the perinate remain unknown; these are currently under investigation. We speculate that the positive interaction between COX-2 and nNOS in brain disclosed in this study might be required physiologically for normal neural development (19, 35, 57, 60, 61).

Perspectives

NO and prostaglandin formation is abundant in the brain of the perinate relative to that of the older subject, at a time critical for synaptogenesis and cortical development. Both of these factors (NO and prostaglandins) have independently been attributed significant roles in these crucial functions of neural development as well as in the control of coupled cerebral microcirculation. The present study clearly demonstrates an interaction between prostaglandins and NO. Specifically, PGE2, via its EP3 receptors, was found to regulate nNOS mRNA and protein expression as well as its activity in brain cortex, mostly in its deepest layers and adjacent white matter, predominantly in neurons; of relevance, EP3 is the main PGE2 receptor found in the perinatal brain parenchyma. Based on our findings, we propose a concerted interaction between NO and PGE2 at a crucial period of ontogeny, which may be implicated in synaptogenesis and cortical brain development.

    ACKNOWLEDGEMENTS

We thank Hendrika Fernandez for technical support and Dr. P. Russo (pathologist) for assisting in interpretion of histological data. We are also grateful to Les Fermes Ménard, L'Ange Gardien, Québec, for generosity in supplying piglets.

    FOOTNOTES

This study was supported by grants from the Medical Research Council of Canada, the Heart and Stroke Foundation of Québec, the Hospital for Sick Children Foundation, the March of Dimes Birth Defects Foundation, the United Cerebral Palsy Foundation, the Fonds pour la Formation de Chercheurs et l'Aide à la Recherche, and the Fonds de la Recherche en Santé du Québec.

I. Dumont is a recipient of a studentship from the Ministry of Indian and Northern Affairs, Canada, and A. K. Martinez-Bermudez was awarded a studentship from the Medical Research Council of Canada. P. Hardy is recipient of a fellowship award from the Medical Research Council of Canada. S. Chemtob is a recipient of a scholarship from the Fonds de la Recherche en Santé du Québec.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: S. Chemtob, Research Center of Hôpital Ste Justine, 3175 Côte Ste. Catherine, Montréal, Québec, Canada H3T 1C5.

Received 29 January 1998; accepted in final form 5 August 1998.

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Abstract
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Results
Discussion
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