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(Stroke. 2000;31:516.)
© 2000 American Heart Association, Inc.

Original Contributions

Augmented Vasoconstriction and Thromboxane Formation by 15-F_2t-Isoprostane (8-Iso-Prostaglandin F₂) in Immature Pig Periventricular Brain Microvessels

Xin Hou, MD, PhD; Fernand Gobeil, Jr, PhD; Krishna Peri, PhD; Giovanna Speranza, BSc; Anne Marilise Marrache, BSc; Pierre Lachapelle, PhD; Jackson Roberts, II, MD; Daya R. Varma, MD, PhD Sylvain Chemtob, MD, PhD

From the Centre de Recherche de l’Hôpital Sainte-Justine, Department of Pediatrics and Pharmacology, Université de Montréal, Montréal, Québec, Canada (X.H., F.G., K.P., G.S., A.M.M., S.C.); Departments of Pharmacology and Therapeutics (G.S., A.M.M., D.R.V., S.C.) and Ophthalmology (P.L.), McGill University, Montréal, Québec, Canada; and Departments of Pharmacology and Medicine, Vanderbilt University, Nashville, Tenn (J.R.).

Correspondence to Sylvain Chemtob, MD, PhD, Research Center, Hôpital Sainte-Justine, Department of Pediatrics and Pharmacology, 3175 Côte Sainte-Catherine, Montréal, Québec, Canada, H3T 1C5. E-mail chemtobs{at}ere.umontreal.ca

	Abstract

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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 F₂) 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 Ca²⁺ 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 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 A₂ (TXA₂) 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 TXA₂ synthase and receptor blockers (CGS12970 and L670596). Vasoconstrictor effects of the TXA₂ mimetic U46619 on fetal, neonatal, and juvenile periventricular microvessels did not differ. 15-F_2t-IsoP increased TXA₂ synthesis by activating Ca²⁺ influx through non–voltage-gated channels in endothelial cells (SK&F96365 sensitive) and N-type voltage-gated channels (-conotoxin sensitive) in astrocytes; smooth muscle cells were not responsive to 15-F_2t-IsoP but generated Ca²⁺ 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 TXA₂ 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.

Key Words: fetus • ischemia • newborn • peroxidation • prostaglandins

	Introduction

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Oxidant stress plays a major role in the pathogenesis of various disorders such as hypoxic-ischemic encephalopathies,^{1 2 3} including periventricular leukomalacia in premature subjects.^{2 4 5 6} Free radicals can alter brain hemodynamics by causing vasoconstriction^{7 8} and increase thromboxane A₂ (TXA₂).^{9 10 11 12 13} Although TXA₂ has been implicated in peroxidation-induced vasoconstriction,^{9 10 11 12} the mechanisms of TXA₂ production by brain vasculature during oxidant stresses remain unknown.

A series of prostaglandin F₂–like compounds produced nonenzymatically by free radical–induced peroxidation of arachidonic acid independent of cyclooxygenase have been shown to be produced in vivo and their formation to increase markedly during oxidant injury.^{13 14 15 16 17 18 19} 15-F_2t-Isoprostane (15-F_2t-IsoP) (8-iso-prostaglandin F₂)²⁰ is one of the abundantly generated isoprostanes in vivo; it is a potent renal, coronary, pulmonary, retinal, and cerebral vasoconstrictor.^{7 18 21 22 23 24} Although the vasomotor effects of 15-F_2t-IsoP are largely inhibited by TXA₂ receptor blockers,^{7 22 23 24} 15-F_2t-IsoP does not seem to bind directly with the TXA₂ receptor.^{25 26 27} We recently reported that 15-F_2t-IsoP causes marked constriction of retinal vessels of piglets by stimulating TXA₂ synthesis,²¹ but such cyclooxygenase-dependent action of this isoprostane has not been uniformly observed in other vascular beds.²⁸ Moreover, the direct effects of 15-F_2t-IsoP on brain intraparenchymal vasculature, which is intimately involved in the genesis of encephalopathies, are not known.

Because oxidant stress–induced encephalopathies are mostly localized in the periventricular region in immature compared with older subjects,² we postulated that the constrictor effects of the product of peroxidation 15-F_2t-IsoP on microvessels of the periventricular brain region are more pronounced in immature than in older subjects. In this process we evaluated the effects of 15-F_2t-IsoP on these microvessels as well as the role and cellular sources of TXA₂ in these vascular responses. It was found that 15-F_2t-IsoP caused periventricular microvessel constriction in fetal more than in newborn more than in juvenile animals by stimulating increased formation of TXA₂ through distinct pathways in endothelial and astroglial cells from brains of younger subjects.

	Materials and Methods

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Tissue Preparation
Animals were used according to a protocol of the Animal Care Committee of the Hôpital Sainte-Justine in accordance with the principles of the Guide for the Care and Use of Experimental Animals of the Canadian Council on Animal Care. Fetal pigs (78 to 90 days’ gestation [term, 114 days]) were obtained from an abattoir (St-Hélène, Québec, Canada) immediately after exsanguination of sows. Newborn (1 to 3 days old) and juvenile pigs (4 to 5 weeks old) were obtained from Fermes Ménard Inc (L’Ange-Gardien, Québec, Canada). Animals were anesthetized with halothane (2.5% to 5%), and india ink (1.5 mL/kg) was injected intracardially to facilitate visualization of the cerebral microvessels; animals were killed with pentobarbital (120 mg/kg), and brains were removed and placed immediately in ice-cold Krebs’ buffer (pH 7.4) of the following composition (mmol/L): NaCl 120, KCl 4.5, CaCl₂ 2.5, MgSO₄ 1.0, NaHCO₃ 27, KH₂PO₄ 1.0, and glucose 10; 1.5 U/mL heparin was added to the buffer. For biochemical measurements, tissues were frozen in liquid N₂ and stored at -80°C.

Vasomotor Response of Brain Periventricular Microvessels
Slices of brain (1 mm thick) exposing the periventricular brain region were prepared as previously described^{29 30} to study relatively undisturbed penetrating microvessels (30 to 50 µm), reported to contribute significantly in the control of cerebral vascular resistance.³¹ The brain slices were pinned securely to a wax base of a 20-mL bath containing Krebs’ buffer (pH 7.4) equilibrated with 21% O₂, 5% CO₂, and 74% N₂ and maintained at 37°C. The preparations were washed 2 to 3 times with fresh buffer and allowed to equilibrate for 45 minutes before the start of the experiment.

Cerebral microvessels were visualized and recorded with a video camera (model CCD72, MTI) mounted on a dissecting microscope (model M-400, Nikon) as previously reported.^{29 30} Vascular diameter was measured with a digital image analyzer (Sigma Scan software, Jandel Scientific) and repeated 3 times with a variability of <1%; pilot experiments indicated that the inert india ink did not modify vascular responses to constrictors (eg, U46619 and phenylephrine) and relaxants (eg, carbaprostacyclin and sodium nitroprusside). Vascular diameter was recorded before and after topical application of increasing concentration of test agents (15-F_2t-IsoP, thromboxane mimetic U46619, and prostaglandin F₂ [PGF₂]) in the presence and absence of 30 minutes’ pretreatment with the following agents at known effective concentrations^{9 21 32} : nonselective cyclooxygenase inhibitor ibuprofen (10 µmol/L); nonselective phospholipase A₂ blocker oleoyloxyethyl phosphocholine³³ (OPPC) (50 µmol/L); TXA₂ synthase inhibitor CGS12970 (1 µmol/L); TXA₂ receptor antagonist L670596 (0.1 µmol/L); endothelin ET_A receptor blocker BQ-123 (1 µmol/L); non–voltage-dependent Ca²⁺ entry and receptor-mediated Ca²⁺ channel blocker SK&F96365³⁴ (20 µmol/L); L-type voltage-gated Ca²⁺ channel blocker nifedipine (5 µmol/L); and N-type voltage-gated Ca²⁺ channel blocker -conotoxin³⁵ (10 µmol/L). Focus was placed on receptor-operated and N- as well as L-type voltage-gated Ca²⁺ channels since endothelial cells are not excitable and are mostly devoid of voltage-gated Ca²⁺ channels,³⁶ whereas astrocytes contain voltage-gated Ca²⁺ channels primarily of the N- and L-types.^{37 38}

Microvascular Endothelial and Smooth Muscle Cell Culture
Microvessels from fetal brain were prepared as previously described.^{29 30 39} Microvessels were suspended in selective endothelial or smooth muscle growth media (Clonetics). Confluent individual endothelial and smooth muscle cells were trypsinized, centrifuged, reseeded in culture flasks, and subcultured; cell viability was verified by trypan blue exclusion and was >90%. Endothelial cells were identified by their cobblestone morphology at confluence, positive reactivity to factor VIII antibody, and negative reactivity to smooth muscle–specific actin and glial fibrillary acidic protein (GFAP) antibodies (Dako). Smooth muscle cells were recognized by their spindle-shaped appearance, positive reactivity to smooth muscle–specific actin antibody, and negative reactivity to factor VIII and GFAP antibodies. Confluent cultures of endothelial and smooth muscle cells of passages 5 to 15 were used for experiments.

Astroglial Cell Culture
Astrocytes were cultured from fetal brain.⁴⁰ Brains were collected in Ham’s F-12 medium containing penicillin (50 U/mL) and streptomycin (50 mg/mL). Brain homogenate was sequentially filtered through 230- and 150-µm nylon mesh, and the filtrate was centrifuged at 1000g for 7 minutes and resuspended in DMEM with 10% fetal calf serum and incubated in air and 5% CO₂ at 37°C. Mixed glial cultures were grown for 9 to 11 days, and loosely attached macrophages were removed with a rotary shaker at 225 rpm for 3 hours. Media were changed, and culture was equilibrated for 6 hours and shaken thereafter for 18 hours to remove oligodendrocyte progenitors. Cultures were trypsinized and reseeded. Purity of astrocytes was assessed by immunoreactivity to GFAP, which was >95%.

Thromboxane Assay
The effects of 15-F_2t-IsoP on thromboxane formation were studied in fetus, newborn, and juvenile pig brain slices stimulated (15 minutes) with 15-F_2t-IsoP (1 µmol/L); the reaction was stopped with liquid N₂. Thromboxane B₂ (stable TXA₂ metabolite) was determined on the homogenized tissue by radioimmunoassay, as previously described.^{9 21} TXB₂ concentration was also measured in the culture media of astroglial, endothelial, and smooth muscle cells stimulated for 15 minutes with 15-F_2t-IsoP (1 µmol/L) in the absence or presence of ibuprofen (10 µmol/L), CGS12970 (1 µmol/L), SK&F96365 (20 µmol/L), nifedipine (5 µmol/L), -conotoxin (10 µmol/L), or EGTA (100 µmol/L).

Ca²⁺ Signals
Intracellular Ca²⁺ ([Ca²⁺]_i) signals were measured with the fluorescent indicator fura 2-AM, as we reported.²¹ For this purpose, confluent endothelial, smooth muscle, and astroglial cells of fetal pigs were trypsinized in a solution containing 0.05% trypsin and 0.02% EDTA for 2 minutes, then 5 mL of HBSS was added. Cells were centrifuged at 250g for 10 minutes and resuspended in a buffer containing (in mmol/L) HEPES 20, D-glucose 10, KCl 4.6, NaCl 118, and CaCl₂ 0.5, as well as 1% fetal bovine serum. Cell viability was determined by trypan blue exclusion and was >90%. Fura 2-AM (2 µmol/L) and 0.2% Pluronic F-127 were added to cell suspensions, which were incubated at 37°C for 30 minutes. The loaded cells were then washed twice and resuspended in HBSS with Ca²⁺ (2.5 mmol/L) and 1% fetal bovine serum with or without pretreatment for 15 minutes with SK&F96365 (20 µmol/L), nifedipine (5 µmol/L), -conotoxin (10 µmol/L), or EGTA (100 µmol/L) followed by stimulation with 15-F_2t-IsoP (1 µmol/L) or U46619 (1 µmol/L). The [Ca²⁺]_i was determined in 2 mL of fura 2–loaded cell suspension (2x10⁶ cells per milliliter) continuously stirred and measured with a spectrofluorometer (model LS 50, Perkin-Elmer) by using excitation wavelengths of 340 and 380 nm and emission at 510 nm. Calibration of the fluorescent signal was determined on 2 mL of cell suspension by sequential addition of 10 mmol/L ionomycin to obtain the maximal fluorescence ratio (R_max) and to 5 mmol/L EGTA plus 0.2% Triton X-100 to obtain the minimal fluorescence ratio (R_min). Autofluorescence was determined by measuring fluorescence from nonloaded cells and subtracting it from the fluorescence produced by fura 2-AM–loaded cells to calculate the fluorescence ratio R corresponding to the values produced at 340 and 380 nm. The [Ca²⁺]_i was calculated from the equation of Grynkiewicz et al⁴¹ : [Ca²⁺]_i=K_d[(R-R_min)/(R_max-R)](S_f2/S_b2), where K_d (224 nmol/L) is the effective dissociation constant of fura 2-AM/Ca²⁺ complex and S_f2/S_b2 is the ratio of fluorescence intensity at 380-nm wavelength in the presence of EGTA to that in the presence of Triton X-100.

Immunoblotting of Thromboxane Synthase
TXA₂ synthase immunoreactivity on brain was determined as we previously described for other membrane-bound enzymes.⁴² Briefly, homogenized tissues of periventricular regions of all age groups studied were preabsorbed with 50 mL of immunoprecipitin for 30 minutes and then centrifuged at 12 000g for 10 minutes to remove immunoprecipitin. The supernatant was incubated with porcine TXA₂ synthase–specific polyclonal antibodies (Cayman Chemicals) for 1.5 hours, and immune complexes were collected by incubation with 50 mL immunoprecipitin for 30 minutes followed by centrifugation. Immune precipitates were denatured in SDS buffer and centrifuged at 12 000g for 15 minutes to remove the immunoprecipitin; samples were loaded on SDS-polyacrylamide gels. Proteins were electrophoretically transferred to nitrocellulose membranes and incubated with TXA₂ synthase–specific antibodies. After they were washed, membranes were incubated with horseradish peroxidase–conjugated anti-rabbit IgG antibody followed by several washes. Immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Canada), as recommended by the supplier, and analyzed by densitometry.

Chemicals
L670596 and CGS12970 were generous gifts from Merck-Frosst (Pointe-Claire, Québec, Canada) and Ciba-Geigy (Summit, NJ), respectively. The following products were purchased: 15-F_2t-IsoP (>99% pure), U46619, and TXA₂ synthase polyclonal antibodies (Cayman Chemicals); ATP, EDTA, EGTA, OPPC, ibuprofen, ionomycin, nifedipine, Triton X-100, -conotoxin, and Tris-HCl (Sigma Chemical); SK&F96365 (BioMol); fura 2-AM (Calbiochem); TXB₂ radioimmunoassay kits (Amersham); endothelial cell, smooth muscle cell, and astrocyte growth medium (Clonetics); factor VIII antibody, smooth muscle–specific actin antibody, and GFAP antibody (Dako); and all other chemicals (Fisher Scientific).

Statistical Analysis
All results are expressed as mean±SEM. Results were analyzed with Student’s t test and 2-way ANOVA, factoring for concentrations and age or treatments. Post-ANOVA comparisons among means were performed with the Turkey-Kramer method. Statistical significance was set at P0.05.

	Results

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Effects of 15-F_2t-IsoP on Brain Periventricular Microvessels
15-F_2t-IsoP caused concentration-dependent constriction of periventricular microvessels from all animals (Figure 1). The EC₅₀ values of 15-F_2t-IsoP on fetal, newborn, and juvenile pig microvessels were comparable: 38.7±4.8, 24.8±3.6, and 17.6±3.1 nmol/L, respectively. On the other hand, maximal constriction to 15-F_2t-IsoP in fetus (30.8±1.7%) was greater than in newborn (21.3±2.3%), which was greater than in juvenile pig microvessels (12.2±2.1%). In contrast, PGF₂ was more effective on juvenile than on fetal and neonatal pig microvessels (P<0.05).

View larger version (14K): [in this window] [in a new window]   Figure 1. Concentration-vasoconstrictor response to 15-F2t-IsoP, PGF2, and U46619 on brain periventricular microvessels of fetal, neonatal, and juvenile pigs. Constriction is the percent reduction in vascular diameter from basal values, which were 36.5±3.2, 34.5±2.6, and 35.1±2.8 µm, respectively, for fetus, newborn, and juvenile, as previously reported.27 28 Effects of agents were studied in situ on brain slices, as described in Methods. Data are mean±SEM of 5 to 6 separate experiments. *P<0.05 compared with fetus and newborn; P<0.05 compared with fetus and juvenile (2-way ANOVA and comparison among means test).

Relationship Between Vasoconstrictor Effects of 15-F_2t-IsoP and TXA₂
The vasoconstrictor effects of 15-F_2t-IsoP on microvessels from all 3 groups of animals were almost fully inhibited by ibuprofen, phospholipase A₂ inhibitor OPPC, TXA₂ synthase inhibitor CGS12970, and TXA₂ receptor antagonist L670596 (Figure 2); the endothelin ET_A receptor blocker BQ-123 was ineffective. PGF₂-induced constriction was unaffected by all the above inhibitors. Correspondingly, 15-F_2t-IsoP increased TXB₂ generation in periventricular tissue of fetal, newborn, and juvenile pigs; basal and 15-F_2t-IsoP–induced TXA₂ production exhibited an age-dependent profile that was greater in fetal than in newborn than in juvenile microvessels (Figure 3A). A similar developmental pattern of expression of immunoreactive TXA₂ synthase was observed (Figure 3B and 3C). On the other hand, vasoconstriction to the TXA₂ mimetic U46619 did not differ between the 3 age groups (Figure 1C). Hence, developmental changes in 15-F_2t-IsoP–induced constriction seem largely dependent on ontogenic differences in TXA₂ formation, which is highest in the fetus.

View larger version (23K): [in this window] [in a new window]   Figure 2. Contribution of cyclooxygenase products and more specifically thromboxane in 15-F2t-IsoP–induced periventricular microvascular constriction. Tissues were pretreated 30 minutes with saline (control), ibuprofen (10 µmol/L), phospholipase A2 inhibitor OPPC (50 µmol/L), thromboxane synthase inhibitor CGS12970 (1 µmol/L), the thromboxane receptor antagonist L670596 (0.1 µmol/L), or the endothelin ETA receptor blocker BQ-123 (1 µmol/L). Experimental preparations are similar to those for Figure 1. Data are mean±SEM of 5 to 6 separate experiments. *P<0.05 compared with saline-treated preparations (2-way ANOVA).

View larger version (29K): [in this window] [in a new window]   Figure 3. Effects of 15-F2t-IsoP on thromboxane formation (A) and representative immunoblot and relative densitometry of thromboxane synthase in periventricular brain region of fetal, newborn, and juvenile pigs (B, C). A, Data are mean±SEM of 5 to 6 separate experiments. *P<0.01 compared with basal values; P<0.05 compared with corresponding values for newborn and juvenile; P<0.05 compared with corresponding values for fetus and juvenile (2-way ANOVA and comparison among means test). B, Representative immunoblot of 3 experiments. C, Compiled densitometry of the immunoblots relative to that of the fetus set at 100%. P<0.05 compared with corresponding values for fetus and juvenile.

Effects of 15-F_2t-IsoP on Thromboxane Formation by Cultured Cerebral Endothelial, Smooth Muscle, and Astroglial Cells
To determine the potential source of increased thromboxane in response to 15-F_2t-IsoP in immature animals (a principal interest of this study), thromboxane formation was measured on primary cultures of fetal cerebrovascular cells, namely, endothelial and smooth muscle cells, as well as astroglial cells, which are not only perivascular but also are the most abundant cell type in brain; experiments were conducted only on cells from fetal animals since fetus exhibited responses to 15-F_2t-IsoP and immunoreactive expression of TXA₂ synthase that were similar to those of newborn (Figures 1 to 3). 15-F_2t-IsoP stimulated formation of TXB₂ by endothelial and astroglial cells (Figure 4); effects of 15-F_2t-IsoP on TXA₂ generation by smooth muscle cells were negligible (<2 pg/10⁶ cells per 15 minutes). The stimulatory effects of 15-F_2t-IsoP on TXB₂ formation in endothelial and astroglial cells were diminished by inhibitors of TXA₂ synthesis (ibuprofen and CGS12970).

View larger version (24K): [in this window] [in a new window]   Figure 4. Effects of 15-F2t-IsoP on TXB2 production by cultured endothelial (A) and astroglial cells (A) from fetal pig brain. Cells were pretreated 20 minutes with saline or one of the following: ibuprofen (10 µmol/L), CGS12970 (1 µmol/L), nifedipine (5 µmol/L), SK&F96365 (20 µmol/L), -conotoxin (10 µmol/L), and EGTA (100 µmol/L). Data are mean±SEM of 5 to 6 separate experiments. *P<0.01 compared with all other values without asterisk.

Because prostanoid formation is Ca²⁺ dependent through phospholipase A₂ requirements, we attempted to identify the type of channel involved in 15-F_2t-IsoP–induced TXA₂ generation. We focused on receptor-operated and N- as well as L-type voltage-gated Ca²⁺ channels since endothelial cells are not excitable and are mostly devoid of voltage-gated Ca²⁺ channels,³⁶ whereas astrocytes contain voltage-gated channels, primarily of the N- and L-types.^{37 38 43} The putative receptor-operated Ca²⁺ channel blocker SK&F96365³⁴ selectively reduced TXB₂ formation in endothelial cells, and the selective N-voltage–gated Ca²⁺ channel blocker -conotoxin³⁵ caused a similar effect only in astrocytes (Figure 4); Ca²⁺ chelator EGTA was effective in both cells. Therefore, TXB₂ formation induced by 15-F_2t-IsoP is dependent on extracellular Ca²⁺, which seems to influx through activation of distinct Ca²⁺ channels in endothelial and astroglial cells.

Effects of 15-F_2t-IsoP on Ca²⁺ Transients
The effects of 15-F_2t-IsoP on Ca²⁺ transients (using fura 2-AM) corroborated data on TXB₂ formation. 15-F_2t-IsoP induced an increase in Ca²⁺ signals in endothelial cells, which was significantly reduced by SK&F96365 and EGTA but not by nifedipine or -conotoxin (Figure 5A and 5B). This effect of 15-F_2t-IsoP on astrocytes was not significantly affected by nifedipine and SK&F96365 but was markedly inhibited by -conotoxin and EGTA (Figure 5C and 5D). In contrast, 15-F_2t-IsoP did not affect Ca²⁺ transients in smooth muscle cells (Figure 5E and 5F). On the other hand, TXA₂ mimetic U46619 induced Ca²⁺ transients in smooth muscle cells, which was inhibited by nifedipine but not by SK&F96365 (Figure 5E and 5F).

View larger version (33K): [in this window] [in a new window]   Figure 5. Intracellular calcium transients [Ca2+]i in fetal pig brain endothelial (A, B) and astroglial cells (C, D) in response to 15-F2t-IsoP, and in smooth muscle cells (E, F) in response to U46619. Cells were pretreated 20 minutes with saline (control), nifedipine (5 µmol/L), SK&F96365 (20 µmol/L), -conotoxin (10 µmol/L), and EGTA (100 µmol/L). Intracellular calcium transients were measured with fura 2-AM, as described in Methods. Arrows in B, D, F point to moment of addition of 15-F2t-IsoP (B, D) and U46619 (F). Values are mean±SEM of 4 to 5 separate experiments. *P<0.05 compared with all other values without asterisk.

Effects of Ca²⁺ Channel Blockers on Vasoconstriction of Fetal Periventricular Microvessels in Response to 15-F_2t-IsoP and U46619
The relative role of Ca²⁺ channels involved in 15-F_2t-IsoP–induced TXB₂ formation was assessed on periventricular vasoconstriction. Vasoconstriction to 15-F_2t-IsoP was decreased by SK&F96365 and reduced further by -conotoxin and nifedipine (Figure 6A), whereas the vasoconstrictor response to U46619 was inhibited by nifedipine but not by -conotoxin or SK&F96365 (Figure 6B).

View larger version (18K): [in this window] [in a new window]   Figure 6. Vasoconstrictor response of periventricular microvessels of fetal pigs to 15-F2t-IsoP (A) and U46619 (B) in the presence of saline, SK&F96365 (20 µmol/L), nifedipine (5 µmol/L) or -conotoxin (10 µmol/L). Effects of agents were studied in situ on brain slices, as described in Methods. Data are mean±SEM of 4 to 5 separate experiments. *P<0.05 compared with 15-F2t-IsoP+saline as well as with 15-F2t-IsoP+nifedipine or -conotoxin; P<0.01 compared with 15-F2t-IsoP+saline; P<0.01 compared with all other values (2-way ANOVA and comparison among means tests).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
The present study reveals that 15-F_2t-IsoP causes greater constriction of periventricular microvessels of the fetus than of newborn and juvenile animals due to greater thromboxane generation through a previously undescribed mechanism of action of 15-F_2t-IsoP, which seems to involve activation of distinct calcium channels and an interaction between vascular and perivascular cells. Data indicate that 15-F_2t-IsoP elicits vasoconstriction by increasing the release of thromboxane from brain astroglial and endothelial but not vascular smooth muscle cells; the increased thromboxane synthesis seems to result from increased entry of extracellular calcium into cells, possibly through N-type voltage-dependent calcium channels in astrocytes and non–voltage-dependent calcium channels in endothelial cells.

The effects of 15-F_2t-IsoP have been found to be markedly inhibited by thromboxane receptor blockers^{7 22 23 24} ; however, ligand binding studies suggest that 15-F_2t-IsoP does not directly interact with the thromboxane receptor^{25 26 27} but possibly with distinct binding sites.²⁵ In the present study 15-F_2t-IsoP–induced constriction of periventricular microvessels was almost completely suppressed by inhibition of thromboxane synthesis as well as by thromboxane receptor blockade (Figure 2). Inhibition of phospholipase A₂ (with OPPC) and of cyclooxygenase (with ibuprofen) caused comparable suppression of 15-F_2t-IsoP–induced constriction. In accordance, 15-F_2t-IsoP markedly increased synthesis of thromboxane in periventricular brain as well as in astrocytes and cerebrovascular endothelial cells (Figures 3 and 4). Hence, it can be inferred that in periventricular brain region, 15-F_2t-IsoP seems to act primarily by activating thromboxane formation; this observation is consistent with a recent report on another neural tissue, the retina.²¹

An important observation in this study is the greater constriction evoked by 15-F_2t-IsoP in fetus than in newborn, which in turn was also greater than that in juvenile pigs, whereas constriction to U46619 did not differ during development. For all ages, thromboxane synthase inhibitors nearly totally suppressed 15-F_2t-IsoP–induced constriction (Figure 2); this would suggest that greater constriction in younger subjects is mainly due to increased release of thromboxane, as confirmed, and was associated with more abundant immunoreactive thromboxane synthase protein (Figure 3). The reason for increased expression of TXA₂ synthase in the periventricular brain region of immature subjects is not clear; however, its role in the migration of astrocytes from the germinal matrix in the periventricular region to others in the developing brain has been proposed.⁴⁴

Of the vascular and perivascular cells studied, thromboxane generation in response to 15-F_2t-IsoP arose largely from endothelial and astroglial cells (Figure 4); 15-F_2t-IsoP was ineffective on smooth muscle cells. Because astrocytes, which release vasoactive substances,^{45 46} are the most abundant cell type in brain parenchyma, it is reasonable to suggest that astrocytes are the main source of thromboxane formation and contribute most to 15-F_2t-IsoP–mediated constriction; this inference is supported by inhibition of constriction by -conotoxin (Figure 6), which inhibits thromboxane generation only in astrocytes (Figure 4). Thus, 15-F_2t-IsoP–induced constriction is mediated by thromboxane released mainly from astrocytes as well as from vascular endothelial cells, but not from smooth muscle.

This 15-F_2t-IsoP–evoked thromboxane formation was dependent on extracellular calcium since EGTA virtually abolished it (Figure 4). Astrocytes contain various calcium channels.^{37 38 43} The increase in thromboxane formation and calcium signals in astrocytes was inhibited by the selective N-type voltage-gated calcium channel blocker -conotoxin³⁵ but not by L-type voltage-gated channel blocker nifedipine and putative inhibitor of non–voltage-gated calcium channels SK&F96365.³⁴ This finding would suggest that in astrocytes, 15-F_2t-IsoP stimulates thromboxane formation by enhancing entry of calcium mainly via N-type voltage-gated calcium channels. In contrast in endothelial cells, SK&F96365 was found to inhibit 15-F_2t-IsoP–induced thromboxane formation and the increase in intracellular calcium (Figures 4 and 5). SK&F96365 has been reported to inhibit receptor-mediated calcium entry at 30 µmol/L concentration, whereas at concentrations >100 µmol/L, SK&F-96365 also blocks voltage-gated calcium channels.³⁴ In this study SK&F96365 was used at 20 µmol/L. Hence, influx of calcium in cerebrovascular endothelial cells in response to 15-F_2t-IsoP is possibly via receptor-operated channels; similar observations have been made in retinal endothelial cells.²¹ Collectively, data suggest that 15-F_2t-IsoP increases influx of calcium through distinct channels in astrocytes and endothelial cells, and this in turn leads to activation of phospholipase A₂ and metabolism of arachidonic acid into thromboxane. The involvement of separate pathways (channel activation) resulting in thromboxane formation would be consistent with existence of distinct receptor sites for 15-F_2t-IsoP, which remain to be characterized.

Taken together, these data suggest that 15-F_2t-IsoP exerts little direct effects on cerebrovascular smooth muscle; rather, its vasoconstrictor effects are mediated indirectly by release of thromboxane from other vascular or perivascular cells, including astroglial and endothelial cells. This inference is based on a number of observations: (1) 15-F_2t-IsoP stimulated thromboxane production and calcium signals from astroglial and endothelial cells but was ineffective on smooth muscle (Figures 4 and 5); (2) periventricular vasoconstriction to 15-F_2t-IsoP is thromboxane dependent (Figure 2); (3) thromboxane mimetic U46619 evoked a nifedipine-sensitive calcium transient in smooth muscle and a vasoconstriction (Figures 5E, 5F, and 6); and (4) 15-F_2t-IsoP–induced vasoconstriction was nearly totally abolished by nifedipine and partly by -conotoxin and SK&F96365 (Figure 6), according to the relative abundance of cells containing corresponding channels (astroglial and endothelial cells).

In conclusion, the present study shows that 15-F_2t-IsoP causes more pronounced vasoconstriction in the periventricular brain region of the fetus than in that of older subjects because of greater thromboxane formation, dependent on a newly described complex mechanism involving interaction between astrocytes and endothelial and smooth muscle cells; a model depicting this interrelationship is shown in Figure 7. We speculate that 15-F_2t-IsoP may be a contributory factor in the hemodynamic compromise and periventricular brain injury in the premature neonate during oxidant stress; cyclooxygenase inhibitors, thromboxane synthase, and/or receptor blockers may attenuate the deleterious effects of oxidant stress.^{47 48}

View larger version (59K): [in this window] [in a new window]   Figure 7. Model based on data presented, depicting mechanism of action of 15-F2t-IsoP on periventricular brain microvessels of immature subjects involving activation of specific channels in endothelial and astroglial cells resulting in thromboxane formation and their interaction with smooth muscle cells to elicit 15-F2t-IsoP–induced vasoconstriction, found to be greater in fetal and newborn than juvenile animals (Figure 1). F indicates fetus; NB, newborn; A, adult; R, receptive site for 15-F2t-IsoP (unknown); ROC, receptor-operated Ca2+ channel; PLA2, phospholipase A2; COX, cyclooxygenase; AA, arachidonic acid; N-VOC, N-type voltage-gated Ca2+ channels; TP, thromboxane receptor; and L-VOC, L-type voltage-gated Ca2+ channels.

	Acknowledgments

 
This work was supported by grants from the Medical Research Council of Canada, the Hospital for Sick Children Foundation, the March of Dimes Birth Defects Foundation, the Heart and Stroke Foundation of Québec, and the Fonds de la Recherche en Santé du Québec. Drs Gobeil and Chemtob are recipients of fellowship and scientist awards, respectively, from the Medical Research Council of Canada. We wish to thank Hensy Fernandez for her technical assistance.

Received August 27, 1999; revision received October 28, 1999; accepted November 1, 1999.

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Editorial Comment

Earl F. Ellis, PhD, Guest Editor

Department of Pharmacology/Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
This study by Hou et al provides further evidence for the possible role of oxidant stress-induced formation of 15-F_2t-isoprostane (8-isoprostaglandin F₂) as a contributor to local reductions in brain blood flow associated with prematurity. The authors use a diversity of approaches to substantiate that 8-isoprostanglandin F₂ causes an increase in intracellular free calcium in endothelial and astroglial cells, which in turn activates phospholipase A₂, thereby liberating arachidonic acid substrate for subsequent formation of vasoconstrictor thromboxane A₂. This action is antagonized by thromboxane A₂ receptor blockers. The authors’ findings are relevant not only to changes in cerebral blood flow associated with prematurity but also are globally relevant to changes in flow associated with other pathological events known to produce free radicals and oxidant stress. It has been shown extensively in the literature that oxidant stress will increase nonenzymatic formation of 8-isoprostaglandin F₂. In this regard, an area of additional possible applicability of the current results is the area of traumatic brain injury. Experimental traumatic brain injury is known to be associated with increased oxygen radical formation and a reduction in cerebral blood flow in fluid percussion brain-injured rats. While this reduction in posttraumatic cerebral blood flow can be prevented by free radical scavengers such as superoxide dismutase, the particular pharmacological species that induce vasoconstriction and subsequent decreases in cerebral blood flow are uncertain. The current study provides evidence suggesting that posttraumatic synthesis of 8-isoprostaglandin F₂ may be a candidate for causing the posttraumatic decrease in CBF. In this regard, it is also of relevance that Hoffman et al^R1 have shown in a preliminary communication that traumatically injured astrocytes produce increased amounts of 8-isoprostaglandin F₂ and that the antioxidant deferoxamine can reduce the injury-induced proliferation of 8-isoprostaglandin F₂. Thus the current study, along with the work of others, implies that brain cells such as astrocytes and endothelial cells may be the source and site of action of 8-isoprostaglandin F₂ and its capacity to stimulate thromboxane formation with subsequent action on adjacent vascular smooth muscle cells. Hou et al are to be commended for their creative and multidisciplinary approach to addressing the possible role of 8-isoprostaglandin F₂ in the modulation of cerebral blood flow.

Received August 27, 1999; revision received October 28, 1999; accepted November 1, 1999.

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1. Hoffman SW, Raigalinski BA, Willoughby KA, Ellis EF. Astrocyte injury increases free radical–dependent formation of vasoconstrictive isoprotanes. J Neurotrauma.. 1997;14:784. Abstract.

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