Krishna G. Peri

Localization of Functional Prostaglandin E2 Receptors EP3 and EP4 in the Nuclear Envelope

The effects of prostaglandin E2are thought to be mediated via G protein-coupled plasma membrane receptors, termed EP. However recent data implied that prostanoids may also act intracellularly. We investigated if the ubiquitous EP3 and the EP4 receptors are localized in nuclear membranes. Radioligand binding studies on isolated nuclear membrane fractions of neonatal porcine brain and adult rat liver revealed the presence of EP3 and EP4. A perinuclear localization of EP3α and EP4receptors was visualized by indirect immunocytofluorescence and confocal microscopy in porcine cerebral microvascular endothelial cells and in transfected HEK 293 cells that stably overexpress these receptors. Immunoelectron microscopy clearly revealed EP3α and EP4 receptors localization in the nuclear envelope of endothelial cells; this is the first demonstration of the nuclear localization of these receptors. Data also reveal that nuclear EP receptors are functional as they affect transcription of genes such as inducible nitric-oxide synthase and intranuclear calcium transients; this appears to involve pertussis toxin-sensitive G proteins. These results define a possible molecular mechanism of action of nuclear EP3 receptors.

Augmented Vasoconstriction and Thromboxane Formation by 15-F2t-Isoprostane (8-Iso-Prostaglandin F2α) in Immature Pig Periventricular Brain Microvessels

BACKGROUND AND PURPOSE Oxidant stress, especially in the premature, plays a major role in the pathogenesis of hypoxic-ischemic encephalopathies mostly manifested in the periventricular region. We studied the vasomotor mode of actions of the peroxidation product 15-F(2t)-isoprostane (15-F(2t)-IsoP) (8-iso-prostaglandin F(2alpha)) on periventricular region during development. METHODS Effects of 15-F(2t)-IsoP on periventricular microvessels of fetal, newborn, and juvenile pigs were studied by video imaging and digital analysis techniques. Thromboxane formation and intracellular Ca(2+) were measured by radioimmunoassay and by using the fluorescent indicator fura 2-AM. RESULTS 15-F(2t)-IsoP-mediated constriction of periventricular microvessels decreased as a function of age such that in the fetus it was approximately 2.5-fold greater than in juvenile pigs. 15-F(2t)-IsoP evoked more thromboxane formation in the fetus than in the newborn, which was greater than that in the juvenile periventricular region; this was associated with immunoreactive thromboxane A(2) (TXA(2)) synthase expression in the fetus that was greater than that in newborn pigs, which was greater than that in juvenile pigs. 15-F(2t)-IsoP-induced vasoconstriction was markedly inhibited by TXA(2) synthase and receptor blockers (CGS12970 and L670596). Vasoconstrictor effects of the TXA(2) mimetic U46619 on fetal, neonatal, and juvenile periventricular microvessels did not differ. 15-F(2t)-IsoP increased TXA(2) synthesis by activating Ca(2+) influx through non-voltage-gated channels in endothelial cells (SK&F96365 sensitive) and N-type voltage-gated channels (omega-conotoxin sensitive) in astrocytes; smooth muscle cells were not responsive to 15-F(2t)-IsoP but generated Ca(2+) transients to U46619 via L-type voltage-sensitive channels. CONCLUSIONS 15-F(2t)-IsoP causes periventricular brain region vasoconstriction in the fetus that is greater than that in the newborn, which in turn is greater than that in the juvenile due to greater TXA(2) formation generated through distinct stimulatory pathways, including from endothelial and astroglial cells. The resulting hemodynamic compromise may contribute to the increased vulnerability of the periventricular brain areas to oxidant stress-induced injury in immature subjects.

Nitric Oxide Signaling via Nuclearized Endothelial Nitric-oxide Synthase Modulates Expression of the Immediate Early Genes iNOS and mPGES-1

Stimulation of freshly isolated rat hepatocytes with lysophosphatidic acid (LPA) resulted in LPA1 receptor-mediated and nitricoxide-dependent up-regulation of the immediate early genes iNOS (inducible nitric-oxide synthase (NOS)) and mPGES-1 (microsomal prostaglandin E synthase-1). Because LPA is a ligand for both cell surface and intracellular receptor sites and a potent endothelial NOS (eNOS) activator, we hypothesized that NO derived from activated nuclearized eNOS might participate in gene regulation. Herein we show, by confocal microscopy performed on porcine cerebral endothelial cells expressing native LPA1-receptor and eNOS and on HTC4 rat hepatoma cells co-transfected with recombinant human LPA1-receptor and fused eNOS-GFP cDNA, a dynamic eNOS translocation from peripheral to nuclear regions upon stimulation with LPA. Nuclear localization of eNOS and its downstream effector, soluble guanylate cyclase, were demonstrated in situ in rat liver specimens by immunogold labeling using specific antibodies. Stimulation of this nuclear fraction with LPA and the NO donor sodium nitroprusside resulted, respectively, in increased production of nitrite (and eNOS phosphorylation) and cGMP; these separate responses were also correspondingly blocked by NOS inhibitor l-NAME and soluble guanylate cyclase inhibitor ODQ. In addition, sodium nitroprusside evoked a sequential increase in nuclear Ca2+ transients, activation of p42 MAPK, NF-κB binding to DNA consensus sequence, and dependent iNOS RNA. This study describes a hitherto unrecognized molecular mechanism by which nuclear eNOS through ensuing NO modulates nuclear calcium homeostasis involved in gene transcription-associated events. Moreover, our findings strongly support the concept of the nucleus as an autonomous signaling compartment.

Key role for cyclooxygenase-2 in PGE2 and PGF2α receptor regulation and cerebral blood f low of the newborn

Ibuprofen, a cyclooxygenase (COX) inhibitor nonselective for either COX-1 or COX-2 isoform, upregulates cerebrovascular prostaglandin E2(PGE2) and PGF2α 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 PGF2α receptor density and function in the cerebral vasculature of the newborn. Hence, we determined PGE2 and PGF2α 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 PGF2α 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 PGF2α 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), PGF2α, and fenprostalene (PGF2αanalog). 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 PGF2α 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 PGF2α receptors and their functions in the newborn cerebral vasculature.

Prolonged Hypercapnia-Evoked Cerebral Hyperemia via K+ Channel– and Prostaglandin E2–Dependent Endothelial Nitric Oxide Synthase Induction

Abstract— Mechanisms for secondary sustained increase in cerebral blood flow (CBF) during prolonged hypercapnia are unknown. We show that induction of endothelial NO synthase (eNOS) by an increase in prostaglandins (PGs) contributes to the secondary CBF increase during hypercapnic acidosis. Ventilation of pigs with 6% CO2 (Paco2≈65 mm Hg; pH ≈7.2) caused a ≈2.5-fold increase in CBF at 30 minutes, which declined to basal values at 3 hours and gradually rose again at 6 and 8 hours; the latter increase was associated with PG elevation, nitrite formation, eNOS mRNA expression, and in situ NO synthase (NOS) reactivity (NADPH-diaphorase staining). Subjecting free-floating brain sections to acidotic conditions increased eNOS expression, the time course of which was similar to that of CBF increase. Treatment of pigs with the cyclooxygenase inhibitor diclofenac or the NOS inhibitor N&ohgr;-nitro-l-arginine blunted the initial rise and prevented the secondary CBF increase during hypercapnic acidosis; neuronal NOS blockers 1-(2-trifluoromethylphenyl) imidazole and 3-bromo-7-nitroindazole were ineffective. Diclofenac abolished the hypercapnia-induced rise in cerebrovascular nitrite production, eNOS mRNA expression, and NADPH-diaphorase reactivity. Acidosis (pH ≈7.15, Pco2≈40 mm Hg; 6 hours) produced similar increases in prostaglandin E2 (PGE2) and eNOS mRNA levels in isolated brain microvessels and in NADPH-diaphorase reactivity of brain microvasculature; these changes were prevented by diclofenac, by the receptor-operated Ca2+ channel blocker SK&F96365, and by the KATP channel blocker glybenclamide. Acidosis increased Ca2+ transients in brain endothelial cells, which were blocked by glybenclamide and SK&F96365 but not by diclofenac. Increased PG-related eNOS mRNA and NO-dependent vasorelaxation to substance P was detected as well in rat brain exposed to 6 hours of hypercapnia. PGE2 was the only major prostanoid that modulated brain eNOS expression during acidosis. Thus, in prolonged hypercapnic acidosis, the secondary CBF rise is closely associated with induction of eNOS expression; this seems to be mediated by PGE2 generated by a KATP and Ca2+ channel–dependent process.

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

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.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 EP3agonist. 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.

The role of prostaglandin receptors in regulating cerebral blood flow in the perinatal period

Prostaglandins exert significant effects on the range of cerebral blood flow autoregulation. However, the newborn exhibits a narrow cerebral blood flow autoregulatory range compared to the adult, and this apparently contributes to the susceptibility of the newborn to major perinatal complications such its intraventricular cerebral haemorrhage. Reduced vasoconstriction in response to prostaglandins due to the fewer prostaglandin receptors, especially for PGE2 (EP) and PGF2α (FP), seems to contribute in part to the narrower range of cerebral blood flow autoregulation in the newborn. Evidence suggests that high levels of prostaglandins in the perinatal period are responsible for the down‐regulation of neurovascular EP and FP receptors. We review the pharmacology of prostaglandin receptors, in particular PGE2 and PGF2α receptors, their ontopeny on the neural vasculature. the perinatal regulation of their expression, and how these changes relate to the control of neural blood flow autoregulation.

Developmentally increased cerebrovascular NO in newborn pigs curtails cerebral blood flow autoregulation

We tested the hypothesis that a reduced ability of the newborn (1–2 d old) to autoregulate cerebral blood flow (CBF) during acute hypertension is contributed by an increased synthesis of nitric oxide (NO) from endothelial (e) and neuronal NO synthase (nNOS). As previously reported, CBF (measured by radiolabeled microsphere technique) in newborn pigs remained constant only between 50 and 90 mm Hg of mean arterial blood pressure. Treatment of newborn pigs with Nω-monomethyl-L-arginine or specific nNOS inhibitors 7-nitroindazole monosodium, 3-bromo-7-nitroindazole, and 1-(2-trifluoromethylphenyl) imidazole extended the upper limit of CBF autoregulation as seen in saline-treated (control) juvenile (4–6-wk-old) animals. Cerebrovascular production of nitrite (stable NO oxidation product) in vivo was markedly increased during hypertension (mean arterial blood pressure >90 mm Hg) in newborn but not in the juvenile pigs. Inhibition of NOS with Nω-monomethyl-L-arginine, 7-nitroindazole monosodium, 3-bromo-7-nitroindazole, or 1-(2-trifluoromethylphenyl) imidazole prevented the hypertension-induced increase in nitrite levels. In addition, eNOS and nNOS protein expression and activity were 2- to 3-fold higher (p< 0.05) in the cerebral microvasculature of newborn than in the tissues of juvenile pigs. It is concluded that during acute hypertension, excess production of NO associated with increased activity of NOS curtails the upper limit of CBF autoregulation in the newborn subject; in addition, nNOS seems to serve a significant role in this important physiologic function.

Stimulation of prostaglandin G/H synthase-2 expression by arachidonic acid monoxygenase product, 14,15-epoxyeicosatrienoic acid

The relationship between arachidonic acid (AA) mobilization and transcription of immediate‐early genes, particularly of prostaglandin G/H synthase‐2 (PGHS‐2), in intestinal crypt epithelial cells was analyzed. PGHS‐2 mRNA and protein synthesis were stimulated by its own substrate, AA; actinomycin D, a transcription inhibitor, prevented the AA‐induced increase in PGHS‐2 mRNA. Eicosatetraynoic acid, an inhibitor of AA utilization, significantly reduced PGHS‐2 mRNA synthesis elicited by AA. Inhibitors of cytochrome P450 monoxygenases, ketoconazole and miconazole, also prevented PGHS‐2 mRNA synthesis in a dose‐dependent manner. Phenyl chalcone oxide, an epoxide hydrolase inhibitor, potentiated AA‐induced PGHS‐2 mRNA synthesis. Of the four regioisomers of arachidonic acid epoxides, only 14,15‐epoxyeicosatrienoic acid elicited the expression of PGHS‐2 in intestinal crypt epithelial cells. This is the first direct evidence of stimulation of an immediate‐early gene product, specifically PGHS‐2, by an AA epoxygenase metabolite, 14,15‐epoxyeicosatrienoic acid, as well as of a heterologous regulation of PGHS‐2 synthesis by these monoxygenase products.

Association of -158(C |[rarr]| T) (XmnI) DNA Polymorphism inG|[gamma]|-Globin Promoter with Delayed Switchover from Fetal to Adult Hemoglobin Synthesis

In this study, the effect of -158(C → T) (XmnI) polymorphism on the synthesis of fetal Hb and its Gγ component during the switchover from fetal to adult Hb was examined using cord blood samples from normal Caucasian term infants. The presence of -158(C→T) mutation was determined by amplification of Gγ- and Aγ-globin gene promoter fragments from the DNA isolated from cord blood samples, followed by XmnI restriction enzyme digestion. The syntheses of fetal and adult Hb in cord blood were measured by [3H]leucine incorporation in globin synthesis, separation of the globin polypeptides by HPLC, and scintillation counting of the fractions. The presence of -158(C→ T) substitution in the Gγ-globin promoter region was positively correlated with elevated synthesis of fetal Hb and itsGγ-globin component in term newborn infants and is associated with delayed switchover from fetal to adult Hb. In addition, analysis of cord blood samples from 100 normal Caucasian French Canadian term infants revealed that the frequency of -158(C → T) substitution inGγ-promoter was 0.32.