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Am J Physiol Regul Integr Comp Physiol 274: R1406-R1416, 1998;
0363-6119/98 $5.00
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Vol. 274, Issue 5, R1406-R1416, May 1998

A novel mechanism for vasoconstrictor action of 8-isoprostaglandin F2alpha on retinal vessels

Isabelle Lahaie1, Pierre Hardy1, Xin Hou1, Haroutioun Hasséssian2, Pierre Asselin1, Pierre Lachapelle3, Guillermina Almazan4, Daya R. Varma4, Jason D. Morrow5, L. Jackson Roberts II5, and Sylvain Chemtob1,4

1 Departments of Pediatrics, Ophthalmology, and Pharmacology, Research Center of Hôpital Sainte Justine, University of Montréal, 2 Department of Ophthalmology, Guy-Bernier Research Center of Hôpital Maisonneuve-Rosemont, University of Montréal, Montreal, Quebec H3T 1C5; 3 Department of Ophthalmology, Montreal Children's Hospital Research Center, McGill University, 4 Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec H3A 2B2, Canada; and 5 Departments of Pharmacology and Medicine, Vanderbilt University, Nashville, Tennessee 37232

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Using a video-imaging technique, we characterized the effects of 8-isoprostaglandin F2alpha (8-iso-PGF2alpha ) on retinal vasculature from piglets. 8-Iso-PGF2alpha potently contracted (EC50 = 5.9 ± 0.5 nM) retinal vessels. These effects were completely antagonized by the cyclooxygenase inhibitor indomethacin, the thromboxane synthase blocker CGS-12970, the thromboxane receptor antagonist L-670596, and the putative inhibitor of the non-voltage-dependent receptor-operated Ca2+ pathway SKF-96365; constrictor effects of 8-iso-PGF2alpha were also partly attenuated by the ETA-receptor blocker BQ-123 and an inhibitor of endothelin-converting enzyme, phosphoramidon, but was negligibly affected by the L-type voltage-gated Ca2+ channel blocker nifedipine. Correspondingly, 8-iso-PGF2alpha elicited endothelin release from retinal preparations, which was markedly reduced by SKF-96365. 8-Iso-PGF2alpha also increased thromboxane production in the retina and cultured endothelial cells, but not on retinovascular smooth muscle cells; these effects of 8-iso-PGF2alpha were blocked by indomethacin, CGS-12970, SKF-96365, and EGTA, but not by nifedipine. 8-Iso-PGF2alpha also increased Ca2+ transients in retinal endothelial cells, which were inhibited by SKF-96365 and EGTA, but not by nifedipine, whereas in smooth muscle cells U-46619, but not 8-iso-PGF2alpha , stimulated a rise in Ca2+ transients. Finally, H2O2 + FeCl2 (in vitro) and anoxia followed by reoxygenation (in vivo) stimulated formation of 8-iso-PGF2alpha in the retina. In conclusion, 8-iso-PGF2alpha -induced retinal vasoconstriction is mediated by cyclooxygenase-generated formation of thromboxane and, to a lesser extent, by endothelin after Ca2+ entry into cells, possibly through receptor-operated channels. Retinal vasoconstriction to 8-isoprostanes might play a role in the genesis of ischemic retinopathies.

peroxidation; calcium influx; cyclooxygenase; thromboxane; endothelin

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

OXIDANT STRESS leads to the formation of reactive oxygen species, which have been implicated in numerous diseases (49). One of the main targets of oxygen free radicals is unsaturated fatty acids from cellular membranes, leading to peroxidation and cellular injury (49). In the retina, peroxidation appears to play an important role in the genesis of various disorders such as ischemic retinopathies, most notably retinopathy of prematurity and diabetes (42, 52, 55). Oxidant stress can alter retinal hemodynamics (9) by causing marked vasoconstriction, which is sustained in the newborn (1, 3, 9). We and other investigators have shown that reactive oxygen species stimulate the cyclooxygenase pathway to produce thromboxane, which appears to be involved in this constriction (3, 9, 29, 51). However, the cascade of events leading to this production of thromboxane is not known.

A series of prostaglandin-like compounds, termed isoprostanes, have recently been shown to be produced during oxidant stresses in vivo and in vitro in animals and humans (40, 41). Isoprostanes are produced independently of the cyclooxygenase pathway, and their formation results from oxidation of the ubiquitous arachidonic acid by free radicals (40, 41). In contrast to prostaglandins formed by cyclooxygenase, the isoprostanes are formed in situ on esterified phospholipids and are released in free form presumably by phospholipases (36). Isoprostanes are stable products, and their formation increases markedly in animal models subjected to free radical injury (32, 36, 38, 46).

8-Isoprostaglandin F2alpha (8-iso-PGF2alpha ), which is an abundantly produced isoprostane in vivo, is a highly potent renal vasoconstrictor with an EC50 in the low nanomolar range (38, 50). It has also been shown that 8-iso-PGF2alpha constricts bronchioles, as well as coronary, pulmonary, and cerebral vessels, which can be inhibited by thromboxane receptor antagonists (7, 21, 26, 27). However, binding studies suggest that 8-iso-PGF2alpha does not directly interact with the thromboxane receptor (13, 43, 56). 8-Iso-PGF2alpha has also been shown to stimulate endothelin-1 release from aortic endothelial cells, but the mechanism of endothelin release is not known (14). Altogether, the mechanism of isoprostane action remains unclear, such that the potency and efficacy of 8-iso-PGF2alpha in tissues studied so far vary markedly (7, 21, 26, 27, 38, 50) and cannot be extrapolated to other tissues such as the retina in the present case.

Unlike other tissues, including the brain, the retina contains a specific profile of membrane phospholipids (6). The retina and its vasculature predominantly generate prostaglandin I2 (PGI2) (45, 47), rather than prostaglandin E2 (PGE2), which is generated by most other neural and nonneural tissues (23, 30). Although in general the responses of the retinal vasculature resemble those of other surface neural vessels, namely, pial ones, the retinal vasculature exhibits distinct vasomotor responses to a variety of agents, including prostanoids and peroxides (1, 3, 4, 9, 31). Hence, because of the susceptibility of the retina to peroxidation, we proceeded to investigate the effects of 8-iso-PGF2alpha on the retinal vasculature of piglets and propose a new mechanism of action for 8-iso-PGF2alpha .

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

Tissue preparation. Animals were used according to a protocol of the Animal Care Committee of 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. Piglets (1-3 days old) were obtained from Fermes Ménard (L'Ange-Gardien, PQ, Canada). Animals anesthetized with halothane (2.5%) and injected with pentobarbital sodium (90 mg/kg) were subjected to thoracotomy. Blood was removed from the circulation by perfusion with systemically heparinized (1 U/ml) saline (~200 ml; corresponding to ~2 blood volumes) through a beating left ventricle through which a 16-gauge needle was inserted; blood was concomitantly disposed from the circulation via a right atriotomy. At the end of the procedure, additional pentobarbital (30 mg/kg) was injected into the heart to ensure that the animals were killed. The eyes were removed and placed immediately in ice-cold Krebs buffer (pH 7.4) of the following composition (mM): 120 NaCl, 4.5 KCl, 2.5 CaCl2, 1.0 MgSO4, 27 NaHCO3, 1.0 KH2PO4, and 10 glucose; 1.5 U/ml heparin was added to the buffer. Previous histological studies confirmed the absence of platelets and other blood elements in the ocular vasculature (9). For biochemical measurements, tissues were frozen in liquid N2 and stored at -80°C.

Vasomotor response to 8-iso-PGF2alpha and other agents. Eyecup preparations were used to study the response in situ of the relatively undisturbed retinal vasculature, as previously described (1, 3, 9, 18). Briefly, a circular incision was made 3-4 mm posterior to the ora serrata to remove the anterior segment and vitreous body with minimal handling of the retina. The remaining eyecup was fixed with pins to a wax base in a 20-ml tissue bath containing Krebs buffer (pH 7.35-7.45) equilibrated with 21% O2 and 5% CO2 and maintained at 37°C (3). The preparations were allowed to stabilize for 30-45 min, during which they were rinsed two or three times with fresh buffer.

Cumulative concentration-response curves to 8-iso-PGF2alpha , the thromboxane A2 (TxA2) mimetic U-46619, and endothelin-1 were constructed separately on nonperfused primary arterioles (100-200 µm diameter) of fresh tissue. The outer vessel diameter was recorded with a video camera mounted on a dissecting microscope (model M-400, Zeiss), and responses were quantified by a digital image analyzer (Sigma Scan software, Jandel Scientific, Corte Madera, CA). Vascular diameter was recorded before and 10 min after topical application of each concentration of agent, at which time a stable response was achieved. Each measurement was repeated three times, and variability was <1%. The vasomotor effects of 8-iso-PGF2alpha were also studied 20 min after pretreatment with the following agents at concentrations shown to inhibit desired targets: indomethacin (10 µM), a cyclooxygenase inhibitor (29); oleoyloxyethyl phosphocholine (OPPC, 50 µM), a phospholipase A2 blocker (17); L-670596 (100 nM), a thromboxane receptor antagonist (12); CGS-12970 (1 µM), a thromboxane synthase inhibitor (5); phosphoramidon (10 µM), an endothelin-converting enzyme inhibitor (53); BQ-123 (1 µM), a selective ETA-receptor antagonist (24); BQ-788 (25 µM), an ETB-selective antagonist (25); nifedipine (1-5 µM), an L-type voltage-gated Ca2+ channel blocker (15); SKF-96365 (20 µM), a Ca2+ entry blocker (and putative inhibitor of non-voltage-dependent receptor-mediated Ca2+ entry) (33, 34); or econazole (10 µM), a Ca2+ influx blocker (17). The responses are expressed as percent change in the outer diameter of the vessel from baseline.

Retinal microvascular endothelial and smooth muscle cell cultures. For primary cultures of retinovascular endothelial and smooth muscle cells, retinas were collected in Hanks' balanced salt solution (HBSS) buffer (pH 7.4) of the following composition (mM): 2.8 KCl, 0.2 KH2PO4, 68 NaCl, 0.16 Na2HPO4, 2.8 glucose, 100 HEPES, and 0.01 phenol red. Retinal microvessels were prepared as previously described (2). Briefly, retinas were gently homogenized with a Wheaton pestle in 5 mM Tris · HCl buffer (pH 7.4) containing (mM) 1.1 acetylsalicylic acid, 0.5 EGTA, 1 benzamidine, and 0.1 phenylmethylsulfonyl fluoride; 100 µg/ml soybean trypsin inhibitor was added to the buffer. The homogenate was mixed with Ficoll 400 (40%) at a 1:1 (vol/vol) ratio and centrifuged at 20,000 g for 20 min at 4°C. The pellet, which contains the microvessels, was washed in HBSS three times. Purity of the microvessel preparation was confirmed by high-power microscopy and by gamma -glutamyl transpeptidase activity, which was higher in vasculature (5.6-6.1 mU/mg protein) than in neural parenchyma (0.3-0.35 mU/mg protein) (2).

Microvessels were resuspended in endothelial or smooth muscle growth medium (Clonetics) containing gentamicin (5 µg/ml), kanamycin (20 µg/ml), and nystatin (10 U/ml) and placed in a humidified atmosphere with 95% O2-5% CO2 at 37°C. Confluent 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 antibodies. 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 glial fibrillary acidic protein antibodies. Confluent cultures of endothelial and smooth muscle cells (~2 × 106 cells) of passage 3 or 4 were used for experiments.

Immunostaining for factor VIII, smooth muscle-specific actin, and glial fibrillary acidic protein was performed by fixing cells on coverslips with acetone for 10 s and subsequently rehydrating in PBS for 20 min. The cells were then washed for 15 min in PBS containing 0.2% BSA, 5% goat serum, and 0.2% Triton X-100. Fixed cells were incubated for 60 min with factor VIII, smooth muscle actin, or glial fibrillary acidic protein antibody (1:50) diluted in PBS containing 10% FCS and 5% goat serum with 0.1% Triton X-100. After five washes in PBS, the secondary FITC-conjugated goat anti-rabbit antibody (1:100) was applied under the same conditions, and cells were washed again in PBS and water. Coverslips were then mounted in Immunomount and examined under an epifluorescent microscope (Leitz Diaplan).

Thromboxane, prostaglandin, and endothelin assays. The effects of 8-iso-PGF2alpha on thromboxane, 6-ketoprostaglandin F1alpha (6-keto-PGF1alpha ), PGE2, and endothelin production were also studied. Retinas were unstimulated or stimulated with 8-iso-PGF2alpha (1 µM) for 15 min after pretreatment for 20 min with saline (equivalent volume of 100 µl in 15 ml bath), indomethacin (10 µM), CGS-12970 (1 µM), BQ-123 (1 µM), phosphoramidon (10 µM), SKF-96365 (20 µM), nifedipine (5 µM), or EGTA (5 mM), and the reaction was stopped with liquid N2. Thromboxane B2 (TxB2, a stable TxA2 metabolite), 6-keto-PGF1alpha (a stable PGI2 metabolite), and PGE2 were determined as previously described (4, 18). Briefly, retinas were suspended in a cold buffer (pH 7.4) of the following composition (mM): 5 Tris · HCl, 1.1 acetylsalicylic acid, 1 EDTA, and 0.045 butyl hydroxytoluene. The tissue was homogenized with a tissue grinder (30,000 rpm, twice for 30 s); proteins were measured in aliquots by the dye-binding method (8). The homogenate was centrifuged at 1,000 g for 10 min at 4°C to remove undisrupted cells and nuclei. The supernatant was rehomogenized and then centrifuged at 28,000 g for 45 min at 4°C to remove membranes and enhance extraction of prostanoids on octadecylsilyl silica columns. The supernatant was dissolved in 100% ethanol and acidified to pH 3 with glacial acetic acid. The samples were applied to the octadecylsilyl silica columns preactivated with methanol and distilled water and subsequently washed with 15% aqueous ethanol and then with petroleum ether. Prostanoids were subsequently eluted with methyl formate and evaporated under vacuum to dryness. TxB2, 6-keto-PGF1alpha , and PGE2 were measured by radioimmunoassay as previously described (4, 18). The recovery of prostanoids was >= 95%, and the interassay variability was <5%. A similar procedure was used to measure prostanoid levels in the culture media of retinovascular endothelial cells and smooth muscle cells stimulated with 8-iso-PGF2alpha (1 µM) for 15 min in the absence or presence of indomethacin (10 µM), CGS-12970 (1 µM), SKF-96365 (20 µM), or nifedipine (5 µM).

For endothelin measurements, retinas were homogenized with a small Potter homogenizer to disperse cells in 2 ml of ice-cold Krebs buffer containing aprotinin (500 kallikrein-inactivating units/ml) to enhance detection of endothelin mostly generated at the cell surface by endothelin-converting enzyme (53); this procedure was adopted because in pilot experiments we failed to detect endothelin in the buffer media without dispersing cells. The homogenate was centrifuged at 1,000 g for 10 min at 4°C. To the supernatant was added an equal volume of 1% trifluoroacetic acid, and then the sample was centrifuged at 10,000 g for 20 min at 4°C. The resulting supernatant was applied to the octadecylsilyl silica columns preactivated with 100% acetonitrile and 1% trifluoroacetic acid. The columns were washed with 1% trifluoroacetic acid, and samples were eluted with 60% acetonitrile in 1% trifluoroacetic acid. Eluates were evaporated under vacuum to dryness. Endothelin was measured by enzyme immunoassay technique with a commercial kit (Peninsula Laboratories, Belmont, CA); specificity of the assay for endothelin-1, -2, and -3 was 100, 7, and 7%, respectively. The intra- and interassay variations were <5%.

Intracellular Ca2+ measurements. Intracellular Ca2+ concentration ([Ca2+]i) was measured using the fluorescent indicator fura 2-AM. Confluent endothelial cells and smooth muscle cells were trypsinized in a solution containing 0.05% trypsin and 0.02% EDTA for 2 min, then 5 ml of HBSS were added. Cells were centrifuged at 250 g for 10 min and resuspended in a buffer containing (in mM) 20 HEPES, 10 D-glucose, 4.6 KCl, 118 NaCl, and 0.5 CaCl2, as well as 1% fetal bovine serum. Cell viability was determined by trypan blue exclusion and was >90%. Fura 2-AM (2 µM) and 0.2% Pluronic F-127 were added to cell suspensions, which were incubated at 37°C for 30 min. The loaded cells were then washed twice and resuspended in HBSS with Ca2+ (2.5 mM) and 1% fetal bovine serum with or without pretreatment for 15 min with SKF-96365 (20 µM), nifedipine (5 µM), or EGTA (5 mM), followed by stimulation with 8-iso-PGF2alpha (1 µM), ATP (1 µM), or U-46619 (1 µM). The [Ca2+]i was determined in 2 ml of fura 2-loaded cell suspension (~2 × 106 cells/ml) continuously stirred and measured by a spectrofluorometer (model LS 50, Perkin-Elmer, Beaconsfield, UK) 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 0.2% Triton X-100 to obtain the maximal fluorescence ratio (Rmax) and to 5 mM EGTA plus 10 µM ionomycin to obtain the minimal fluorescence ratio (Rmin). Autofluorescence was determined by measuring fluorescence from nonloaded cells and subtracting it from the fluorescence produced by fura 2-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 al. (16): [Ca2+]i = Kd [(R - Rmin)/(Rmax - R)](Sf2/Sb2), where Kd (224 nM) is the effective dissociation constant of the fura 2-Ca2+ complex and Sf2/Sb2 is the ratio of fluorescence intensity at 380-nm wavelength in the presence of EGTA to that in the presence of Triton X-100.

Measurement of 8-iso-PGF2alpha in isolated retina subjected to oxidation. Retinas were exposed in vitro to hydroxyl-generating conditions with H2O2 (0.1 mM) and FeCl2 (20 µM) for 30 min in the presence or absence of indomethacin (10 µM) or the free radical scavenger dimethylthiourea (1 mM) (17). Extraction of isoprostanes was performed as described above for thromboxane, reflecting the free active unesterified isoprostanes (36). 8-Iso-PGF2alpha was measured by enzyme immunoassay technique with a commercial kit (Cayman Chemical, Ann Arbor, MI). Briefly, dried samples containing extracted 8-iso-PGF2alpha were reconstituted in 250 µl of 100 mM phosphate buffer (pH 7.4) with 1.5 mM NaN3, 0.4 M NaCl, 1 M EDTA, and 1 g/l BSA. Fifty-microliter samples were placed in microtiter wells precoated with mouse monoclonal anti-rabbit IgG; then 50 µl of acetylcholinesterase linked to 8-isoprostane (tracer) were added, and the samples were incubated for 18 h at room temperature. Unbound reagents were washed five times with 10 mM phosphate buffer (pH 7.4) containing 0.05% Tween 20, and the reaction was developed in 60-90 min with the acetylcholinesterase substrate acetylthiocholine as well as DTNB (Ellman's reagent). Plates were read spectrophotometrically at 405-420 nm to assay formation of 5-thio-2-nitrobenzoic acid generated by the reaction of enzymatically formed thiocholine with DTNB. The specificity for the assay was 100% for 8-isoprostanes, with 85 ± 3% specificity for 8-iso-PGF2alpha and <1% for 8-iso-PGE2 tested in our laboratory (n = 4); cross-reactivity of antibody with TxB2, PGE2, and PGF2alpha was <= 0.1%. The intra- and interassay variability was <= 5%.

Measurement of 8-iso-PGF2alpha in retina of anesthetized piglets subjected to oxidant stress. Oxidant was produced in vivo by subjecting animals to asphyxia followed by reoxygenation, as previously described (9). Piglets were anesthetized with 2% halothane for tracheostomy and catheterization of a femoral vein for drug injection. Animals were ventilated with air by means of a Harvard small animal respirator. Halothane was discontinued, and immediately thereafter piglets were sedated with alpha -chloralose (50 mg/kg bolus followed by 10 mg · kg-1 · h-1) and paralyzed with pancuronium (0.1 mg/kg twice). Animals were kept under a radiant warmer to keep body temperature at 38°C. After 1.5 h of recovery from surgery, piglets were asphyxiated by interruption of ventilation for 5 min. Ventilation was resumed normally for 45 min, and the animals were then killed (120 mg/kg iv pentobarbital sodium) and the eyes were removed. Retinas were collected to measure 8-iso-PGF2alpha , as described above.

Chemicals. L-670596 and CGS-12970 were generous gifts from Merck-Frosst (Pointe-Claire, PQ, Canada) and Ciba-Geigy (Summit, NJ), respectively. The following products were purchased: 8-iso-PGF2alpha (>99% pure), 8-iso-PGF2alpha enzyme immunoassay kit, and U-46619 (Cayman Chemicals); acetylsalicylic acid, ATP, aprotinin, benzamidine, butylated hydroxytoluene, econazole, EDTA, EGTA, FeCl2, H2O2, dimethylthiourea, OPPC, indomethacin, ionomycin, nifedipine, N-(alpha -rhamnopyranosyloxyhydroxyphosphinyl)-Leu-Trp (phosphoramidon), PMSF, soybean trypsin inhibitor (type II-S), Triton X-100, and Tris · HCl (Sigma Chemical, St. Louis, MO); 1-[beta -[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF-96365; BioMol, Plymouth Meeting, PA); endothelin-1 and cyclo(-D-Trp-D-Asp(ONa)Pro-D-Val-Leu) (BQ-123; Research Biochemicals, Natick, MA); N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma MeLeu-D-Trp(COOMe)-D-Nle-ONa (BQ-788), fura 2-AM, and Pluronic F-127 (Calbiochem, La Jolla, CA); TxB2, 6-keto-PGF1alpha , and PGE2 radioimmunoassay kits (Amersham, Oakville, ON, Canada); endothelin enzyme immunoassay kit (Peninsula); endothelium and smooth muscle growth medium (Clonetics); factor VIII antibody, smooth muscle-specific actin antibody, and glial fibrillary acidic protein antibody (Dako, Carpinteria, CA); FITC-conjugated goat anti-rabbit antibody, FCS, goat serum (Jackson Immunoresearch Laboratories, West Grove, PA); all other chemicals (Fisher Scientific, Montreal, PQ, Canada).

Data analysis. Results were analyzed using Student's t-test and a two-way ANOVA, with factoring for concentrations and different treatments. Post-ANOVA comparisons among means were performed using the Tukey-Kramer method (48). Statistical significance was set at P < 0.05. Values are means ± SE.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Vasoconstrictor effects of 8-iso-PGF2alpha , U-46619, and endothelin-1 on retinal arterioles. 8-Iso-PGF2alpha , U-46619, and endothelin-1 caused concentration-dependent constriction of retinal arterioles (Fig. 1A). Maximal efficacy of 8-iso-PGF2alpha was nearly one-half that of U-46619 and endothelin-1. The EC50 of 8-iso-PGF2alpha , U-46619, and endothelin-1 were 5.9 ± 0.5, 159 ± 26, and 0.9 ± 0.7 nM, respectively.


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  		Fig. 1.   Constrictor concentration-response curves to 8-isoprostaglandin F2alpha (8-iso-PGF2alpha ), thromboxane A2 (TxA2) receptor mimetic U-46619, and endothelin-1 on retinal arterioles of piglets in absence (A) and presence of cyclooxygenase inhibitor indomethacin (10 µM), phospholipase A2 blocker oleoyloxyethyl phosphocholine (OPPC, 50 µM), TxA2 synthase inhibitor CGS-12970 (1 µM), and TxA2 receptor antagonist L-670596 (100 nM) (B); ETA receptor antagonist BQ-123 (1 µM), ETB receptor antagonist BQ-788 (25 µM), and endothelin-converting enzyme inhibitor phosphoramidon (10 µM) (C); and L-type voltage-gated Ca2+ channel blocker nifedipine (1 µM) and putative receptor-mediated Ca2+ channel blocker SKF-96365 (20 µM) (D). Values are means ± SE of 4-7 separate experiments. * P < 0.05 compared with effects of 8-iso-PGF2alpha in absence of antagonists (by 2-way ANOVA).

Roles of thromboxane and endothelin in 8-iso-PGF2alpha -induced constriction of retinal arterioles. Retinal vasoconstriction to 8-iso-PGF2alpha was markedly inhibited by the cyclooxygenase blocker indomethacin and the phospholipase A2 inhibitor OPPC, as well as the thromboxane synthase inhibitor CGS-12970 and the thromboxane receptor antagonist L-670596 (Fig. 1B).

Vasoconstriction to 8-iso-PGF2alpha was significantly reduced by the endothelin-converting enzyme inhibitor phosphoramidon (10 µM) and similarly by the ETA-receptor blocker BQ-123 (1 µM; Fig. 1C). The ETB-receptor blocker BQ-788 (even at high concentration, 25 µM) did not affect vasoconstriction to 8-iso-PGF2alpha . Phosphoramidon (10 µM) effectively inhibited endothelin formation (Fig. 2A), and BQ-123 (1 µM) fully prevented constriction in response to endothelin-1 (up to 10 nM).


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  		Fig. 2.   Effects of 8-iso-PGF2alpha (1 µM) on endothelin-1 (ET-1) formation by retina (A) and thromboxane B2 (TxB2) and 6-ketoprostaglandin F1alpha (6-keto-PGF1alpha ) production by retina (B and C) and cultured retinal endothelial cells (D and E). Tissues and cells were pretreated for 20 min with saline or inhibitors as follows: indomethacin (10 µM), CGS-12970 (1 µM), BQ-123 (1 µM), phosphoramidon (10 µM), SKF-96365 (20 µM), nifedipine (5 µM), and EGTA (5 mM). Values are means ± SE of 4-6 experiments. * P < 0.05 compared with basal values; dagger  P < 0.05 compared with saline (+8-iso-PGF2alpha ).

Effects of Ca2+ channel blockers on vasoconstriction produced by 8-iso-PGF2alpha . Because formation of thromboxane and release of endothelin were suspected to contribute to effects of 8-iso-PGF2alpha (on the basis of inhibition of constriction by CGS-12970 and phosphoramidon), we speculated that Ca2+ entry, which is required for the generation and release of these autacoids, may be involved in the action of 8-iso-PGF2alpha . Because L-type voltage-gated Ca2+ channels play a major role in the entry of Ca2+ in smooth muscle and other cell types as well as in vascular contraction (20), their contribution to the 8-iso-PGF2alpha effect was tested using the selective blocker nifedipine. Nifedipine (1 µM) did not modify retinal vasoconstriction to 8-iso-PGF2alpha (Fig. 1D); fivefold higher concentrations of nifedipine also did not alter effects of 8-iso-PGF2alpha but virtually abolished constriction to KCl (20 mM). In contrast, the Ca2+ entry blocker SKF-96365 fully inhibited the vasoconstrictor response to 8-iso-PGF2alpha . Econazole (10 µM), another non-voltage-dependent Ca2+ channel blocker (19), and SKF-96365 inhibited the constrictor effects of 8-iso-PGF2alpha to an equal degree (data not shown).

Effects of 8-iso-PGF2alpha on thromboxane, prostaglandin, and endothelin release. 8-Iso-PGF2alpha stimulated endothelin release (Fig. 2A) from the retina, which was inhibited by pretreatment with phosphoramidon and significantly reduced by SKF-96365. 8-Iso-PGF2alpha also increased production of TxB2 (~7-fold) and, to a lesser extent (by ~2-fold), 6-keto-PGF1alpha and PGE2 (latter not shown) in the retina (Fig. 2, B and C). These effects were markedly diminished by indomethacin, by the Ca2+ chelator EGTA, and by the Ca2+ entry blocker SKF-96365; as expected (5), the thromboxane synthase inhibitor CGS-12970 reduced formation of TxB2, but not 6-keto-PGF1alpha and PGE2. Basal (unstimulated) TxB2 and 6-keto-PGF1alpha formation was not affected by SKF-96365, as previously shown (28), but was reduced by ~80% by indomethacin; CGS-12970 reduced basal TxB2 by ~70%. 8-Iso-PGF2alpha -induced prostanoid production was slightly attenuated by BQ-123 (1 µM) and unaltered by nifedipine (5 µM; Fig. 2, B and C).

The profile of basal prostanoid production in cultured retinal endothelial cells reflected that in whole retinal and oculovascular tissue, exhibiting predominance of 6-keto-PGF1alpha (Fig. 2, D and E), as previously reported (18, 47). 8-Iso-PGF2alpha stimulated formation of TxB2 by ~15-fold and of 6-keto-PGF1alpha and PGE2 (latter not shown) by ~4-fold in cultured retinal endothelial cells. These effects were inhibited by indomethacin and SKF-96365, but not by nifedipine (Fig. 2, D and E). In retinovascular smooth muscle cells, basal TxB2 formation was low (<3 pg · 106 cells-1 · 15 min-1) and was not affected by 8-iso-PGF2alpha ; production of 6-keto-PGF1alpha and PGE2 was also unaltered by 8-iso-PGF2alpha in retinal smooth muscle cells.

Effects of 8-iso-PGF2alpha on intracellular Ca2+ transients in endothelial and smooth muscle cells from retinal vasculature. 8-Iso-PGF2alpha elicited an increase in intracellular Ca2+ in endothelial cells (Fig. 3, A and B). This effect of 8-iso-PGF2alpha on endothelial cells was not significantly affected by nifedipine but was virtually abolished by SKF-96365 and annulled by EGTA and absence of extracellular Ca2+. However, 8-iso-PGF2alpha did not cause an increase in intracellular Ca2+ in smooth muscle cells, whereas ATP was effective (Fig. 3, C and D). Conversely, U-46619 stimulated an increase in intracellular Ca2+ in smooth muscle cells but not in endothelial cells (Fig. 3, B-D).


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  		Fig. 3.   Effects of 8-iso-PGF2alpha and U-46619 on intracellular Ca2+ transients in endothelial and smooth muscle cells from retinal vasculature. Typical tracings and peak increases in intracellular Ca2+ concentrations ([Ca2+]i) are presented in endothelial (A and B) and smooth muscle cells (C and D). Cells were pretreated for 15 min with SKF-96365 (20 µM), nifedipine (5 µM), EGTA (5 mM), or saline with or without extracellular Ca2+ (2.5 mM) before addition of 8-iso-PGF2alpha (1 µM), ATP (1 µM), or U-46619 (1 µM). Values are means ± SE of 4-6 experiments. dagger  P < 0.01 compared with saline (+8-iso-PGF2alpha ).

8-Iso-PGF2alpha formation in retina subjected to oxidation. Finally, generation of 8-iso-PGF2alpha was verified in the retina after in vitro and in vivo oxidant stress. Incubation of retinas with H2O2 and FeCl2 caused a marked increase in 8-iso-PGF2alpha generation, which was prevented by the free radical scavenger dimethylthiourea and unaffected by indomethacin (Fig. 4A). An increase in 8-iso-PGF2alpha concentrations was also detected in the retina of animals subjected to an asphyxic episode followed by reoxygenation (Fig. 4B).


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  		Fig. 4.   8-Iso-PGF2alpha synthesis in retina of piglets subjected to in vitro and in vivo oxidant stress. A: retinas incubated for 30 min without (control) or with H2O2 (100 µM) and FeCl2 (20 µM) with or without 15 min of pretreatment with indomethacin (10 µM) or dimethylthiourea (DMTU, 1 mM). B: 8-iso-PGF2alpha in retinas of piglets subjected to 5-min asphyxic episode followed by 45 min of reoxygenation. Values are means ± SE of 4 experiments. * P < 0.01 compared with control or preasphyxia.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Isoprostanes are stable oxidation products of arachidonic acid produced by a free radical mechanism in numerous tissues (32, 36, 37, 40, 41). One of the F2-isoprostanes that has been shown to be produced in vivo is 8-iso-PGF2alpha (39); in this study we showed that 8-iso-PGF2alpha is produced by the retina when subjected to an oxidant stress in vitro and in vivo (Fig. 4). 8-Iso-PGF2alpha causes constriction of various vascular beds, albeit at different efficacy and potency (7, 21, 26, 27). Although the effects of 8-iso-PGF2alpha have been found to be markedly inhibited by thromboxane receptor blockers (7, 21, 26, 27, 50), binding studies suggest that 8-iso-PGF2alpha does not directly interact with the thromboxane receptor (13, 43, 56) but possibly with distinct binding sites (13, 52). Briefly, the mechanisms of action of 8-iso-PGF2alpha are not clearly defined. The primary purpose of this study was to investigate the effects and potential mechanisms of action of 8-iso-PGF2alpha on retinal vessels. The data suggest that 8-iso-PGF2alpha elicits retinal vasoconstriction by releasing endothelin and more importantly the prostanoid thromboxane from retinal parenchymal and endothelial cells after Ca2+ entry into cells possibly through non-voltage-dependent Ca2+ channels. In this context, it is of relevance that reactive oxygen species can also increase capacitative Ca2+ influx (11), as well as activate cyclooxygenase, and cause vasoconstriction by increasing the synthesis of thromboxane (3, 51). Thus isoprostanes may serve as mediators in peroxidation-induced vasoconstriction.

8-Iso-PGF2alpha caused retinal vasoconstriction with an EC50 in the low nanomolar range and with an efficacy somewhat less than that of the thromboxane mimetic U-46619 and endothelin-1. These vasoconstrictor effects of 8-iso-PGF2alpha were almost completely suppressed by inhibition of synthesis and action of thromboxane and, to a significantly lesser extent, by blockers of endothelin synthesis and of ETA receptors. In addition, 8-iso-PGF2alpha stimulated the formation of endothelin and thromboxane in the retina and in retinal endothelial cells but not in retinovascular smooth muscle cells. These data suggest that thromboxane and endothelin are involved in the vasoconstrictor effect of 8-iso-PGF2alpha . However, the fact that thromboxane synthesis and receptor blockers were more effective than those of endothelin in antagonizing the effects 8-iso-PGF2alpha implies a relatively more important role for thromboxane in the retinal vasoconstriction to 8-iso-PGF2alpha . Along the same lines, although PGI2 (measured by 6-keto-PGF1alpha ) and PGE2 slightly increased in response to 8-iso-PGF2alpha (Fig. 2, C and E), these respective retinal vasodilators and constrictors (4) contributed negligibly to vasomotor effects of 8-iso-PGF2alpha compared with thromboxane, since specific inhibitors of thromboxane action (like those of cyclooxygenase) caused near abolition of 8-iso-PGF2alpha -induced constriction (Fig. 1B).

Equivalent inhibition of the constrictor effect of 8-iso-PGF2alpha by the phospholipase A2 blocker OPPC, the cyclooxygenase inhibitor indomethacin, and the thromboxane synthase inhibitor CGS-12970 (5) (Fig. 1B) suggests that 8-iso-PGF2alpha acts on the synthesis of thromboxane, rather than on its receptors, by stimulating the release of arachidonic acid, which is metabolized by cyclooxygenase into prostanoids of which thromboxane dominates in importance. This suggestion implies that the inhibition of the vasoconstrictor effects of 8-iso-PGF2alpha by the thromboxane receptor antagonist L-670596 is exerted by a blockade of the effects of thromboxane released in response to 8-iso-PGF2alpha . This inference is further supported by the marked increase in the synthesis of thromboxane, compared with other prostanoids, in retina and retinal endothelial cells after stimulation with 8-iso-PGF2alpha (Fig. 2), consistent with previously reported effects of peroxides in the retina (1, 3, 9). Additional evidence that 8-iso-PGF2alpha does not seem to act on the thromboxane receptor is provided by divergent actions of 8-iso-PGF2alpha and the thromboxane mimetic U-46619 on Ca2+ influx in endothelial and smooth muscle cells (Fig. 3, B-D). Hence, it can be inferred that in the retina 8-iso-PGF2alpha seems to interact on a binding site distinct from the thromboxane receptor, concordant with suggestions of others (13, 43, 56).

The relative contribution of the retinal parenchyma per se in generating endothelin and cyclooxygenase products is difficult to clarify because of problems in separating ex vivo tissue parenchyma from its vasculature. Nonetheless, in separate experiments we found that retinas of 1-day-old rats, which are developmentally avascular, are capable of generating thromboxane in response to 8-iso-PGF2alpha ; these observations suggest that vasculature and parenchyma participate in 8-iso-PGF2alpha -induced prostanoid production in the retina. In addition, it should be noted that potentially trapped platelets are an unlikely source of TxB2, because they are not detected histologically in ocular vasculature of perfused animals (9) and, more importantly, platelets do not generate TxB2 in response to 8-iso-PGF2alpha (43).

It is of interest that, in contrast to retinal parenchymal and endothelial cells, smooth muscle cells did not generate prostanoids or exhibit an increase in Ca2+ transients in response to 8-iso-PGF2alpha . These observations would suggest that 8-iso-PGF2alpha might exert little, if any, direct action on retinovascular smooth muscle and that its vasoconstrictor effects are mediated indirectly by release of thromboxane (and endothelin) from retinal parenchymal and endothelial cells (Fig. 2). In contrast to our findings, 8-iso-PGF2alpha has been shown to exert diverse actions on rat aortic smooth muscle cells, including stimulation of DNA, inositol trisphosphate synthesis, and increase in cytosolic Ca2+ (13, 43). The other distinct finding in our study compared with others applies to the predominance of cyclooxygenase dependence of 8-iso-PGF2alpha action. Effects of 8-iso-PGF2alpha have been reported to be unrelated to cyclooxygenase products in kidney and lung (7, 50) and slightly dependent on metabolites of this enzyme in aorta (54). Reasons for disparities between our study in porcine retina and those of others regarding effects of 8-iso-PGF2alpha on smooth muscle cells and cyclooxygenase dependence are not clear but may be due to differences in tissues as well as species; for instance, 8-iso-PGF2alpha constricts bovine, but not ovine, coronary arteries (27).

In addition to thromboxane, endothelin also appears to play a role, albeit to a lesser extent, in the vasoconstrictor response to 8-iso-PGF2alpha . Endothelins are potent vasoconstrictors that can be produced by oxidant stress (10). 8-Iso-PGF2alpha has been shown to stimulate endothelin-1 release from aortic endothelial cells (14). In the present study the vasoconstrictor effects of 8-iso-PGF2alpha were reduced to approximately the same degree by the endothelin-converting enzyme inhibitor phosphoramidon and by the ETA-receptor blocker BQ-123. Furthermore, 8-iso-PGF2alpha induced the release of endothelin from the retina. These findings suggest that vasoconstrictor effects of 8-iso-PGF2alpha are partially mediated by endothelin via the ETA receptor. Because endothelin-1 can release thromboxane (44), it is possible that a part of the endothelin-mediated effects of 8-iso-PGF2alpha is exerted indirectly via thromboxane. Indeed 8-iso-PGF2alpha -induced thromboxane formation was reduced by BQ-123 (Fig. 2B).

Because the release of endothelin and formation of thromboxane are Ca2+-dependent processes, we tested the role of Ca2+ in the action of 8-iso-PGF2alpha . 8-Iso-PGF2alpha elicited an increase in intracellular Ca2+ that was dependent on extracellular Ca2+ (prevented by EGTA as well as by the absence of Ca2+) (Fig. 3). The L-type voltage-gated Ca2+ channels do not seem to be implicated, since nifedipine did not inhibit the effects of 8-iso-PGF2alpha on retinal vessel tone, endothelin and thromboxane release, and intracellular Ca2+ transients. In contrast, the vasomotor effects, the generation of endothelin and thromboxane, and the increase in intracellular Ca2+ by 8-iso-PGF2alpha were significantly inhibited by SKF-96365, a blocker of Ca2+ entry, including that by non-voltage-dependent Ca2+ channels (33, 34).

Non-voltage-gated Ca2+ channels, which comprise receptor-operated channels, are for the most part not well characterized (22). In addition, their physiological role is difficult to elucidate in the absence of selective blockers. SKF-96365 has been reported to inhibit receptor-mediated Ca2+ entry at <= 30 µM; however, at >100 µM, SKF-96365 also blocks voltage-gated Ca2+ channels. In the present study the inhibitory effects of SKF-96365 on 8-iso-PGF2alpha action were observed at <30 µM. Further evidence that non-voltage-gated Ca2+ channels were involved in the vasoconstrictor action of 8-iso-PGF2alpha was obtained in our study with econazole, which can also block Ca2+ influx from non-voltage-dependent Ca2+ channels (19). Altogether, these data suggest that 8-iso-PGF2alpha increases influx of Ca2+ possibly via receptor-operated channels [present in excitable and nonexcitable cells (22, 23)], which in turn leads to stimulation of endothelin release and, more importantly, activation of phospholipase A2 and metabolism of arachidonic acid into prostanoids, among which thromboxane predominates in mediating 8-iso-PGF2alpha -induced retinal vasoconstriction. The involvement of a receptor-operated Ca2+ channel would be consistent with the existence of a distinct 8-iso-PGF2alpha receptor site (13), which remains to be characterized.

In conclusion, this study reveals a retinal vasoconstrictor effect of 8-iso-PGF2alpha by a previously undescribed mechanism. Our results suggest that the effect of 8-iso-PGF2alpha on retinal vasculature is mediated mostly by cyclooxygenase-generated formation of thromboxane and, to a lesser extent, by endothelin, probably through non-voltage-gated cation channels. Because isoprostanes are produced in the retina during oxidant stress (Fig. 4), it is possible that 8-iso-PGF2alpha may contribute to the pathogenesis of ischemia-reperfusion retinal injury such as in retinopathy of prematurity and of diabetes. Along these lines, a role for thromboxane has been proposed in ischemic retinopathies (3, 35).

Perspectives

The abundant content of unsaturated fatty acids in the retina renders this tissue particularly susceptible to peroxidation. In fact, peroxidation exerts a significant role in the genesis of several retinopathies, in particular those that exhibit an ischemic component such as retinopathy of prematurity and of diabetes. Peroxide-induced activation of cyclooxygenase resulting in the generation of thromboxane has been demonstrated to compromise retinal hemodynamics by causing marked vasoconstriction leading to ischemia, which in turn alters retinal function and predisposes to neovascularization. However, the cascade of events leading to this production of thromboxane is not known. The discovery of the stable products of peroxidation, namely, the isoprostanes, shown to evoke constriction in various vascular beds, may conceivably reproduce effects of peroxidation, if it is assumed that they also act through formation of thromboxane. The present findings indeed reveal a novel mechanism of action of 8-iso-PGF2alpha , which elicits a potent retinal vasoconstriction predominantly by causing activation of the cyclooxygenase pathway, resulting in the generation of thromboxane (and separately of lesser importance in the release of endothelin) from retinal parenchymal and endothelial cells after entry of Ca2+ into cells possibly through non-voltage-dependent Ca2+ channels. Because thromboxane has been proposed in ischemic retinopathies, it would be of interest to speculate that 8-iso-PGF2alpha via thromboxane may serve as mediators in peroxidation-induced retinal vasoconstriction by contributing to the pathogenesis of ischemia-reperfusion retinal injury.

    ACKNOWLEDGEMENTS

The authors thank Dr. Jacques Maclouf (Paris, France) for expert assistance in measuring 8-iso-prostaglandins by enzyme immunoassay and Hendrika Fernandez and Daniel Abran from DA Labs for technical support.

    FOOTNOTES

This study was supported by grants from the Medical Research Council of Canada, the Heart and Stroke Foundation of Quebec, the Hospital for Sick Children Foundation, the March of Dimes Birth Defects Foundation, the Fonds de la Recherche en Santé du Québec, and the United Cerebral Palsy Foundation. I. Lahaie is a recipient of a student fellowship from Fight for Sight Research Division of Prevent Blindness America and Fonds pour la Formation de Chercheurs et l'Aide à la Recherche. P. Hardy is recipient of a fellowship award from the Medical Research Council of Canada. S. Chemtob and H. Hasséssian are recipients of scholarships from the Fonds de la Recherche en Santé du Québec.

Address for reprint requests: S. Chemtob, Depts. of Pediatrics, Ophthalmology, and Pharmacology, Research Center of Hôpital Ste Justine, 3175, Chemin Côte Ste Catherine, Montreal, PQ, Canada H3T 1C5.

Received 29 August 1997; accepted in final form 5 January 1998.

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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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