Research ArticleArticle

Developmental Regulation of Endothelial Nitric Oxide Synthase in Cerebral Vessels of Newborn Pig by Prostaglandin E2

Isabelle Dumont, Xin Hou, Pierre Hardy, Krishna G. Peri, Martin Beauchamp, Taline Najarian, Stéphane Molotchnikoff, Daya R. Varma and Sylvain Chemtob
Journal of Pharmacology and Experimental Therapeutics November 1999, 291 (2) 627-633;

Abstract

We investigated whether prostaglandins regulate endothelial nitric oxide synthase (eNOS) in the pig cerebral vasculature during the neonatal period. Prostaglandins, eNOS mRNA, eNOS protein, and NO production were higher in cerebral microvessels of newborn (1 day old) than in those of adult (6- to 8-month-old) pigs. The treatment of isolated cerebral microvessels of newborn animals with ibuprofen for 24 h reduced eNOS mRNA and nitrite production to levels in the adult; this effect of ibuprofen was prevented by concurrent treatment with prostaglandin (PG)E2 analog 16,16-dimethyl-PGE2, nonselective PGE2 receptor analog 11-deoxy PGE1, and prostaglandin EP3receptor agonists sulprostone and M&B 28,767 but was not modified by PGI2 analog carbaprostacyclin, PGD2, and EP1 receptor agonist 17-phenyl trinor PGE2. Correspondingly, 16,16-dimethyl-PGE2 and M&B 28,767 increased eNOS mRNA expression of adult microvessels to values in the newborn. Data similar to those with isolated cerebral vessels were obtained through histochemical analysis (NADPH-diaphorase positivity) of brain from newborn animals treated in vivo with ibuprofen in combination or not with sulprostone. Furthermore, substance P-induced NO-mediated cerebral vasorelaxation was decreased to adult values through the treatment of newborn pigs with ibuprofen; this effect was prevented by concomitant treatment with sulprostone. It is concluded that PGE2 regulates eNOS in newborn pig cerebral microvessels via EP3 receptors; this may be physiologically required during normal neurovascular development.

Endothelial nitric oxide synthase (eNOS) plays an important role in the control of cerebral blood flow, metabolism, and, in turn, neuronal activity (Prado et al., 1992; Lo et al., 1996; Ohashi et al., 1998). Mice with a disrupted eNOS gene are hypertensive and exhibit a reduction in cerebral blood flow (Huang et al., 1995), which could favor cerebral ischemia (Prado et al., 1992; Lo et al., 1996). NOS activity is developmentally regulated in cerebral vasculature (Northington et al., 1996, 1997). Increased cerebrovascular levels of NOS activity during the perinatal period could have functional significance during brain development (Bredt and Snyder, 1994; Northington et al., 1996, 1997;Estrada and DeFelipe, 1998). However, the mechanisms regulating the ontogeny of cerebrovascular NOS are not known. A role for prostaglandins in regulating inducible NOS expression in macrophages has been proposed (Gaillard et al., 1992; Raddassi et al., 1993;Kuchiiwa et al., 1994; Aeberhard et al., 1995), but this type of regulation has never been reported for constitutive NOS, specifically eNOS, and especially not during development.

There is some evidence that the ontogeny of cerebral prostaglandin synthesis parallels that of NOS (Leffler et al., 1993; Peri et al., 1995; Busija, 1997). Based on evidence presented and because brain microvessels (<150 μm) are major regulators of cerebral circulation (Baumbach and Heistad, 1985), we hypothesize that high levels of prostaglandins regulate eNOS expression and activity in cerebral microvessels. For this purpose, we characterized the expression of eNOS in cerebral microvessels of newborn and adult pigs and determined whether increased prostaglandin levels in the former modulate eNOS expression, activity, and function. Our findings reveal that prostaglandin (PG)E2 positively regulates eNOS expression in the cerebral microvessels of the newborn and that this effect is mediated via the EP3 subtype of PGE2 receptors.

Materials and Methods

Chemicals.

M&B 28,767 was a gift from Rhone-Poulenc Rorer (Dagenham Essex, UK). Carbaprostacyclin, PGD2, 16,16-dimethyl-PGE2, 11-deoxy-PGE1, 17-phenyl trinor PGE2, sulprostone, and U46619 were obtained from Cayman (Ann Arbor, MI). Polyclonal rabbit antibody specific to eNOS was obtained from Calbiochem-Novabiochem (San Diego, CA). [α-32P]CTP (3000 Ci/mmol) and enhanced chemiluminescence kit were purchased from Amersham (Mississauga, Ontario, Canada). Pepstatin and leupeptin were purchased from Boehringer Mannheim (Montreal, Quebec, Canada). Ribonuclease A and T7 sequencing kit were obtained from Pharmacia Biotech (Montreal, Quebec, Canada). pGEM4 plasmid vector and in vitro transcription kit were purchased from Promega (Madison, WI). Protein assay and electrophoretic reagents were purchased from Bio-Rad (Mississauga, Ontario, Canada). Guanidinium isothiocyanate, T4 DNA ligase, and restriction enzymes were purchased from BRL Life Technologies (Burlington, Ontario, Canada). The radioimmunoassay for PGE2 was obtained from Advanced Magnetics (Boston, MA). All other chemicals were obtained from Fisher Scientific (Montreal, Quebec, Canada) and Sigma-Aldrich (Oakville, Ontario, Canada).

Animals.

Newborn (1-day-old) Yorkshire pigs were used according to a protocol of the Hôpital Sainte-Justine Animal Care Committee. Animals were anesthetized with 2% halothane and then sacrificed with 120 mg/kg intracardiac pentobarbital, and the brains were removed. Brains from adult (6- to 8-month-old) pigs were collected from an abbatoir immediately after sacrifice and transported on ice to the laboratory.

Preparation of Microvessels.

Cerebral microvessels were prepared as described previously (Li et al., 1994). To compare eNOS mRNA and immunoreactivity, as well as nitrite production in newborn and adult microvessels, brains were gently homogenized with a Wheaton pestle in 5 mM Tris-HCl buffer (pH 7.4) containing 1.1 mM acetylsalicylic acid (ASA), 0.5 mM EGTA, 1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and 5 μM soybean trypsin inhibitor; for all other experiments that involved the effects of modulation of prostaglandin levels on eNOS expression, ASA was omitted from the buffer for the microvessel preparations. ASA was initially added to avoid potential effects of prostaglandins on eNOS expression; subsequent experiments revealed that this action required several hours (see Results), whereas the full microvessel preparation only took 75 to 90 min. After homogenization of brain, the homogenates were mixed with equal volume of Ficoll 400 and centrifuged at 20,000g for 20 min. The pellets containing the microvessels were washed three times in the above buffer and then filtered through a 150-μm filter. The filtrates containing the microvessels were centrifuged at 1000g for 15 min and stored at −80°C until assayed. The morphology and purity of microvessels were monitored by light microscopy and enrichment of γ-glutamyl transpeptidase activity (Sasz, 1969; Golstein et al., 1975). The activity of γ-glutamyl transpeptidase was greater in the microvessels (5.6–5.9 mU/mg protein) than in brain parenchyma (0.30–0.35 mU/mg protein).

Incubation of Microvessels.

Cerebral microvessels of newborn pigs were incubated for 24 h in Dulbecco’s modified Eagle’s medium in the absence or presence of 10 μM ibuprofen or 10 μM ibuprofen plus 1 μM concentration of one of the following agents: carbaprostacyclin (stable PGI2 analog), PGD2, 16,16-dimethyl-PGE2 (stable PGE2 analog), 11-deoxy-PGE1, 17-phenyl trinor PGE2, sulprostone, or M&B 28,767. Microvessels of adult pigs were similarly treated with 16,16-dimethyl-PGE2 and M&B 28,767. Concentrations of agents used have been previously reported to be effective (Kennedy and Doktorcik, 1988; Ando et al., 1995; Li et al., 1995). After incubation, eNOS mRNA and nitrite production were determined.

In Vivo Experiments.

Newborn pigs were anesthetized with 2% halothane, and polyethylene catheters were placed in the jugular vein for i.v. injection. Animals were randomly assigned to receive every 8 h for 24 h i.v., saline, ibuprofen (40 mg/kg), or ibuprofen plus EP3 agonist sulprostone (10 μg/kg; Coleman et al., 1994). Animals were sacrificed at the end of the 24-h period, and the brain was immediately removed for NADPH-diaphorase staining and vasomotor experiments as described below; experiments were conducted on frontoparietal cortex because of its neuronal organization and segregated vasculature.

eNOS and Destrin Ribonuclease Protection Assays.

The primer pair for porcine eNOS synthesized by RT-PCR was 5′-GCTTTTCCCTGCAGGAGCGAC-3′ and 5′-GCCAGTCTCTGCAGACTCTGG-3′ (Zhang et al., 1996). The primer pair for (loading control) destrin was 5′-ATGATGCAAGCTTTGAAACC-3′ and 5′-GGAAGCTTTCGATCTGTGG-3′. 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. The nucleotide sequences of eNOS and destrin partial cDNAs were determined using a T7 sequencing kit. The 32P-labeled cRNA probes for eNOS and destrin were prepared with the use of an in vitro transcription kit (Promega).

Total RNAs from brain microvessels was aliquoted and subjected to ribonuclease protection assays according to a published protocol (Bordonaro et al., 1994) with minor modifications. Briefly, 20 μg of total RNA was mixed with 105 cpm of eNOS and destrin probes in 20 μl of hybridization buffer [80% deionized formamide, 40 mM piperazine-N,N′-bis(2-ethanesulfonic acid), 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 (Bordonaro et al., 1994). The protected RNA fragments were resolved on urea-6% polyacrylamide gels, and the bands were visualized by phosphorimaging (Molecular Dynamics, Sunnyvale, CA) and quantified by densitometry.

Western Blotting.

Western blotting for eNOS was performed using a method described previously (Abran et al., 1997). Cerebral microvessels were homogenized in a buffer containing 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. After centrifugation at 13,000g for 10 min to remove fibrous material, the supernatants were saved and protein was determined (Bio-Rad DC assay). After the addition of β-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 8% SDS-polyacrylamide gels. The transfer of tissue lysate proteins to membranes and immunoblotting using specific antibodies against eNOS (1:1000 titer) was conducted exactly as described previously (Abran et al., 1997). Immunoreactive bands were visualized by chemiluminescence (Amersham) as recommended by the supplier.

Nitrite Production.

NO formation was estimated by determination of its stable metabolite, nitrite (Verdon et al., 1995), as reported previously (Abran et al., 1997). NOS-dependent generation of NO was estimated as the difference in nitrite production in the absence or presence of 1 mMNω-nitro-l-arginine.

NADPH-Diaphorase Histochemistry.

NADPH-diaphorase reactivity was performed using a method described previously (Kuchiiwa et al., 1994). Tissue from frontoparietal cortex was fixed by immersion in 4% paraformaldehyde in 0.1% PBS (pH 7.4) overnight at 4οC and then placed in 30% sucrose buffer for 2 days. Tissues were sectioned (40 μm) using a microtome. The free-floating sections were incubated in 0.1 M phosphate buffer (pH 7.4) containing 0.1% β-NADPH at 37οC for 60 min. After the reaction, the sections were rinsed in phosphate buffer and mounted on slides. The slides were air dried, treated in chloroform for 30 min to remove background staining, and counterstained with neutral red. Densitometry of tonality was analyzed after normalization adjustment of background tone; tonality was determined on similar number of pixels delineating microvessels (which differed by ≤1% between photographs) using software program Photoshop 5 (Adobe).

Vasomotor Response of Brain Microvessels.

Newborn pigs treated with ibuprofen in combination or not with sulprostone were prepared as described above; adult animals were not treated (tissues obtained from abbatoir). Brains from these animals were sectioned (1 mm thick) to study vascular responses using videoimaging techniques as reported previously (Li et al., 1997). Cumulative concentration-relaxant response curves to NO-dependent substance P (Jansen et al., 1991; Rosenblum et al., 1993) were determined on tissues precontracted with U46619 to approximately 75% of maximum contraction; relaxant response was calculated as percent decrease in the induced-tone.

Prostaglandin Synthesis.

PGE2 synthesis in cerebral microvessels was measured as described previously (Peri et al., 1995). Cerebral microvessels from newborn and adult pigs were homogenized in ice-cold buffer containing 50 mM Tris-HCl buffer (pH 7.4), 1 mM EDTA, 0.2 mM PMSF, and 10 μg/ml each of leupeptin, aprotinin, and soybean trypsin inhibitor. An aliquot of the homogenate (400–600 μg of protein/ml) was preincubated for 20 min at 25°C, and the reaction was initiated by the addition of 50 μM arachidonic acid for 10 min at 25°C; the reaction rate was linear over this time period.

Statistical Analysis.

Data were analyzed by ANOVA, comparison among mean values test (Tukey-Kramer method), and Student’st test. Statistical significance was set atp < .05. Data are presented as mean ± S.E.

Results

Prostaglandin Level, eNOS Expression, and Nitrite Generation in Newborn and Adult Brain Microvessels.

PGE2 levels were approximately 6-fold higher in newborn than in adult cerebral microvessels (Fig. 1A). This was associated with 3- to 5-fold greater eNOS mRNA, immunoreactive protein, and nitrite production in newborn compared with adult (Fig. 1, B–F).

Figure 1
Figure 1

Rate of PGE2 synthesis (A), eNOS mRNA (B and C), eNOS protein (D and E), and nitrite production (F) in cerebral microvessels from 1-day-old (NB) and adult (A) pigs. B and C, 20 μg of total RNA was subjected to RNase protection assay. The unprotected and protected fragments are 414 and 356 nucleotides for eNOS and 237 and 165 nucleotides for destrin, respectively. D and E, 100 μg of protein was loaded for Western analysis; the arrow points to the eNOS 140-kDa protein, the only band detected in the range (120–180 kDa) of interest. The production of nitrite measured was that sensitive toNω-nitro-l-arginine (F). Values are mean ± S.E. of three or four experiments, each performed in duplicate. *Different (p < .01) from the value for the adult.

Modulation of eNOS mRNA and Nitrite Production on Isolated Microvessels by Prostaglandins.

Incubation of cerebral microvessels from newborn pigs with ibuprofen for 24 h (but not acute, ≤2 h) caused a significant reduction in the expression of eNOS mRNA and nitrite production (Fig. 2, A–C). Effects of ibuprofen were prevented by concurrent treatment with 16,16-dimethyl-PGE2 but not with other major prostaglandins, carbaprostacyclin, and PGD2.

Figure 2
Figure 2

Modulation of eNOS mRNA expression (A and B) and nitrite production (C) by prostaglandins in isolated newborn cerebral microvessels. Cerebral microvessels of newborn pigs were incubated for 24 h with saline, ibuprofen (10 μM), or a combination of ibuprofen (10 μM) and 1 μM concentration each of carbaprostacyclin (carba-PGI2), PGD2, and 16,16-dimethyl-PGE2 (16,16 PGE2). Cerebral microvessels of adults were treated with saline. RNase protection assays were performed as described in the legend to Fig. 1. Autoradiographic exposure was overnight and visualized by phosphorimaging. Values are mean ± S.E. of three or four experiments, each performed in duplicate. ∗, Different (p < .01) from values without an asterisk.

Effects of PGE2 Receptor Agonists on eNOS mRNA on Isolated Microvessels.

Incubation of newborn pig cerebral microvessels with 11-deoxy-PGE1(EP2/EP3/EP4 agonist), EP1/EP3 agonist sulprostone, or selective EP3 receptor agonist M&B 28,767 prevented ibuprofen-induced decrease in eNOS mRNA (Fig. 3, A and B); the effects of M&B 28,767 on eNOS mRNA expression were concentration dependent (Fig. 3C). In contrast, 17-phenyl trinor PGE2(EP1 agonist), even at relatively high concentrations (1 μM), did not modify the effect of ibuprofen on eNOS mRNA. Furthermore, treatment of adult cerebral microvessels with 16,16-dimethyl-PGE2 and M&B 28,767 increased eNOS mRNA values to those in saline-treated newborns (Fig. 3, A and B).

Figure 3
Figure 3

Modulation of eNOS mRNA expression in pig cerebral microvessels by PGE2 analogs. Isolated newborn cerebral microvessels were incubated 24 h with saline, ibuprofen (10 μM), or a combination of ibuprofen with 1 μM concentration of one of the following: 16,16-dimethyl-PGE2 (16,16 PGE2), 11-deoxy-PGE1, 17-phenyl trinor PGE2 (17-ph-PGE2), sulprostone (Sulp), or M&B 28,767. Isolated cerebral microvessels of adult pig were treated with saline, 16,16 PGE2, or M&B 28,767. Values are mean ± S.E. of three or four experiments. ∗, Different (p < .01) from values without an asterisk.

In Vivo Modulation of Brain Tissue NADPH Diaphorase Staining by Prostaglandins.

To determine whether findings ex vivo also applied in vivo, newborn pigs were treated with ibuprofen with or without sulprostone; ibuprofen reduced PGE2 levels in brain cortex from 201 ± 54 to 87 ± 14 pg/mg protein, comparable to levels in adult brain. In ibuprofen-treated animals, there was a decrease in overall NADPH-diaphorase staining of brain microvessels (as well as neurons), which was prevented by sulprostone (Fig.4); these changes can be further appreciated by tonality densitometry of microvessels (Fig. 4). Hematoxylin and eosin staining revealed no adverse effects of prostaglandin modulation on cell number (data not shown) as reported previously (Patel et al., 1993).

Figure 4
Figure 4

In vivo modulation of NADPH-diaphorase staining by prostaglandins. Newborn animals were treated every 8 h for 24 h with saline, ibuprofen (40 mg/kg), or a combination of ibuprofen plus sulprostone (Ibu and Sulp, respectively; 10 μg/kg). At the end of treatment period, animals were sacrificed, and the brain was stained for NADPH-diaphorase. Tonality densitometry was analyzed as described in the text (n = 3 for each treatment); note that higher arbitrary tonality units correspond to reduced densitometry, and vice versa for lower units. Large arrows point to individual NADPH-diaphorase-positive vessels, and small arrows point to neurons. 1 cm represents 200 μm. ∗, Different (p < .05) from values without an asterisk.

Effects of Prostaglandin Modulation on NO-Dependent Vasorelaxant Response of Brain Vasculature.

To assess whether modulation of eNOS on vessels by prostaglandins was reflected functionally, newborn pigs were treated with ibuprofen with or without sulprostone, and the vasomotor effects of NO-dependent substance P (Jansen et al., 1991;Rosenblum et al., 1993) were tested. The treatment of newborn pigs with ibuprofen decreased vasorelaxant response to substance P (Fig.5); this effect was prevented by concomitant treatment with sulprostone. Correspondingly, in adult animals, substance P caused less vasorelaxation than that seen in saline-treated newborns and approached dilatation of ibuprofen (alone)-treated newborns. Vasorelaxation to substance P was inhibited by the NOS blocker l-nitro-monomethyl arginine(l-NMMA; Fig. 5).

Figure 5
Figure 5

Vasorelaxant effects of substance P on cerebral microvessels. Newborn animals were treated as described in Fig. 4; adults were untreated. Some tissue preparations from saline-treated newborn and from adult animals were exposed to L-NMMA (1 mM). Ibu, Sulp, and NB refer to ibuprofen, sulprostone, and newborn, respectively. ●, NB-Saline; ♦, NB-Ibu; ▪, NB-Ibu + Sulp; ▴, NB-L-NMMA; ○, Adult; ▵, Adult-L-NMMA. Values are mean ± S.E. of four experiments each. ∗, different (p < .01) from adult and ibuprofen (alone)-treated newborns and froml-NMMA-treated preparations (two-way ANOVA factoring for substance P concentration and group).

Discussion

NO has been attributed important roles in the regulation of cerebral hemodynamics (Prado et al., 1992; Lo et al., 1996), particularly in the newborn when the metabolic demand is high (Northington et al., 1996, 1997). Similar functions and ontogenic changes have been observed for prostaglandins (Leffler et al., 1993;Peri et al., 1995; Busija, 1997). Thus far, the factors that regulate cerebrovascular eNOS expression in the perinate were not known. Based on evidence that prostaglandins can regulate NOS activity in macrophages (mostly iNOS), we investigated whether these prostanoids contribute to the perinatal regulation of eNOS expression and activity in cerebral vasculature. Our findings reveal that through actions on EP3 receptors, high levels of PGE2 in cerebrovascular tissues of the perinate regulate eNOS expression and NO generation in brain microvessels, and this in turn affects vasomotor responses.

The ability of ibuprofen (which reduced prostaglandin levels) to decrease eNOS expression and NO formation in the newborn cerebral microvessels suggests that prostaglandins regulate eNOS; because this effect of ibuprofen could be prevented by PGE2and not by other major prostanoids (Fig. 2), it seems that PGE2 is the major prostaglandin playing a critical role in eNOS regulation (Fig. 2). This regulation of eNOS expression in the newborn cerebral microvessels by PGE2 seems to be mediated via EP3 receptors because EP3receptor agonists sulprostone and M&B 28,767 prevented the effect of ibuprofen as did PGE2 (Figs. 2 and 3). As a corollary to observations made in the newborn, PGE2 and M&B 28,767 increased eNOS expression in adult microvessels to values in the newborn (Fig. 3). Moreover, the regulation of eNOS expression by EP3 stimulation both ex vivo and in vivo was also manifested functionally by studying the vasorelaxant response to NO-dependent substance P (present study;Jansen et al., 1991; Rosenblum et al., 1993); substance P-induced vasorelaxation was diminished by treatment (24 h) of newborn pigs with ibuprofen to values seen in the adult (Fig. 5) and could be prevented by concomitant treatment with ibuprofen plus sulprostone. It is of interest to point out in this regard that EP3receptors were among the two PGE2 receptors detected in porcine cerebral microvessels, with the other one being EP1 (Li et al., 1994); EP2and EP4 are not detectable in porcine brain microvessels (Li et al., 1994). Altogether, findings suggest that via EP3 PGE2 positively regulates NOS expression as well as NOS-dependent functions in cerebral vessels.

NADPH-diaphorase staining of cerebral vessels in the cerebral cortex was modulated by prostaglandins (Fig. 4). Interestingly, a majority of the NADPH-diaphorase positive cerebral vessels were found near neurons, which also exhibited strong NADPH staining and were also affected by prostaglandins. The spatial relationship between cerebral vessels and neurons, together with the fact that prostaglandins modulate NADPH-diaphorase positivity, could suggest that via EP3 PGE2 may be involved in the concerted regulation of cerebral blood flow and neuronal development; this inference has been proposed for nNOS (Dumont et al., 1998).

The mechanism by which PGE2 induces eNOS expression is not clear; however, certain inferences can be drawn. EP3 stimulation may lead to the activation of protein kinases (Burkey and Regan, 1995); eNOS promoter contains a site for activator protein-1 that could be stimulated by phosphorylation (Barchowsky et al., 1995). Alternatively, we recently reported the activation directly of functional perinuclear PGE2 receptors that can induce gene transcription (Bhattacharya et al., 1998). In support of this suggestion, inhibition of prostaglandin transporter using bromcresol green (Kanai et al., 1995) prevented PGE2-induced up-regulation of eNOS expression (data not shown).

In conclusion, our results reveal an important mechanism for the developmental regulation of eNOS by PGE2 through its action on EP3 receptors in cerebral microvessels. Because parturition is associated with significant increases in PGE2 levels in brain (Jones et al., 1993), we speculate that PGE2 is developmentally required in the perinate to maintain via increased NO formation adequate brain circulation, particularly at the end of labor, when fetal blood oxygen tension decreases (Dildy et al., 1994).

Acknowledgments

We thank Hendrika Fernandez for technical support and Les Fermes Ménard Inc. (L’Ange Gardien, Québec, Canada) for their generous supply of piglets.

Footnotes

  • Send reprint requests to: Sylvain Chemtob, M.D., Ph.D., Departments of Pediatrics, Ophthalmology and Pharmacology, Research Center of Höpital Sainte-Justine, 3175 Cöte Ste. Catherine, Montreal, Quebec, Canada H3C 1C5.

  • ↵1 This study was supported by grants from the Medical Research Council of Canada, the Quebec Heart and Stroke Foundation, 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. P.H. and S.C. are recipients of Medical Research Council of Canada fellowship and scientist awards, respectively.

  • Abbreviations:
    eNOS
    endothelial nitric oxide synthase
    ASA
    acetylsalicylic acid
    PMSF
    phenylmethylsulfonyl fluoride
    L-NMMA
    l-nitro-monomethyl arginine
    PG
    prostaglandin
    • Received May 17, 1999.
    • Accepted July 15, 1999.

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Developmental Regulation of Endothelial Nitric Oxide Synthase in Cerebral Vessels of Newborn Pig by Prostaglandin E2

Isabelle Dumont, Xin Hou, Pierre Hardy, Krishna G. Peri, Martin Beauchamp, Taline Najarian, Stéphane Molotchnikoff, Daya R. Varma and Sylvain Chemtob
Journal of Pharmacology and Experimental Therapeutics November 1, 1999, 291 (2) 627-633;
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