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Am J Physiol Regul Integr Comp Physiol 281: R391-R400, 2001;
0363-6119/01 $5.00
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Vol. 281, Issue 2, R391-R400, August 2001

2,3-Dinor-5,6-dihydro-15-F2t-isoprostane: a bioactive prostanoid metabolite

X. Hou1, L. J. Roberts II2, D. F. Taber3, J. D. Morrow2, K. Kanai3, F. Gobeil Jr.1, M. H. Beauchamp1, S. G. Bernier1, G. Lepage1, D. R. Varma4, and S. Chemtob1,4

1 Departments of Pediatrics and Pharmacology, Centre de Recherche de l'Hôpital Sainte-Justine, Université de Montréal, Montréal, Québec H3T 1C5; 4 Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec H3G 1Y6, Canada; 2 Departments of Pharmacology and Medicine, Vanderbilt University, Nashville, Tennessee 37232; and 3 Department of Chemistry, University of Delaware, Newark, Delaware 19716


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

15-F2t-isoprostane (15-F2t-IsoP), also termed 8-isoprostaglandin F2alpha , is one of a series of prostanoids formed by free radical-mediated peroxidation of arachidonic acid and exerts potent biological actions such as vasoconstriction. We recently demonstrated that 15-F2t-IsoP is metabolized in humans to a major metabolite, 2,3-dinor-5,6-dihydro-15-F2t-IsoP (15-F2t-IsoP-M). 15-F2t-IsoP-M can also potentially be formed as a product of free radical-induced oxidation of the low abundance fatty acid gamma -linolenic acid. We confirmed that 15-F2t-IsoP-M is generated during oxidation of gamma -linolenic acid and explored whether it may exhibit biological activity. 15-F2t-IsoP-M caused marked constriction of porcine surface retinal and intraparenchymal brain microvessels, comparable to that observed with 15-F2t-IsoP. These effects were associated with increased thromboxane A2 (TXA2) formation and were virtually abolished by TXA2-synthase and -receptor inhibitors (CGS-12970 and L-670596). Vasoconstriction induced by either 15-F2t-IsoP or 15-F2t-IsoP-M on perfused ocular choroid was also abrogated by TXA2-synthase inhibition as well as by removal of endothelium. Similar to 15-F2t-IsoP, 15-F2t-IsoP-M evoked vasoconstriction and TXA2 generation by activating Ca2+ influx from nonvoltage-gated channels (SK&F96365 sensitive) in the retina and from both nonvoltage- and N-type voltage-gated Ca2+ channels (omega -conotoxin MVIIA sensitive), respectively, in brain endothelial and astroglial cells; smooth muscle cells were unresponsive to both agents. Cross-desensitization experiments further suggest that 15-F2t-IsoP and 15-F2t-IsoP-M act on the same receptor mechanism. Findings reveal a novel concept by which a beta -oxidation metabolite of 15-F2t-IsoP that can also be formed by nonenzymatic oxidation of gamma -linolenic acid is equivalently bioactive to 15-F2t-IsoP and may prolong the vascular actions of F2-IsoPs.

calcium; thromboxane; peroxidation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

F2-isoprostanes (F2-IsoPs) are prostaglandin (PG) F2-like compounds that are produced in vivo by nonenzymatic free radical-mediated peroxidation of arachidonic acid (36). A few of these IsoPs have been available in synthetic form and found to exhibit biological properties. For instance, 8,12-iso-IsoP F2alpha -III induces cardiomyocyte hypertrophy (27) and 15-F2c-IsoP (12-iso-PGF2alpha ; Ref. 48) can activate the PGF2alpha receptor (26). The IsoP studied most extensively is 15-F2t-IsoP also termed 8-iso-PGF2alpha (48). 15-F2t-IsoP is abundantly generated in vivo (37) and exerts a number of potent biological effects. These include stimulation of endothelial and smooth muscle cell proliferation, endothelin-1 gene and protein expression (12, 56), induction of endothelial barrier dysfunction (16), and, most notably, potent and marked vasoconstriction in numerous vascular beds (4, 19, 20, 24, 25, 28, 33, 36, 49). At present, the precise nature of the receptor site for 15-F2t-IsoP (12) remains to be determined. Nonetheless, the modes of action of 15-F2t-IsoP on vasculature have been studied and are shown to differ between species and vascular beds. For instance, rat 15-F2t-IsoP-induced renal vasoconstriction is unaffected by cyclooxygenase inhibitors (49), but aortic constriction is partly dependent on cyclooxygenase products (51). In the pig retina and brain, 15-F2t-IsoP evokes vasoconstriction by stimulating thromboxane formation from endothelial and astroglial cells (20, 28). In addition to these acute effects, long-lasting in vivo actions of 15-F2t-IsoP in vascular degeneration have recently been suggested (5).

Metabolism is an important means for inactivation of prostanoids, which proceeds through beta -oxidation, omega -oxidation, 15-hydroxy dehydrogenation, and double-bond reduction (41, 44). There are rare exceptions where metabolic products of PGs have been shown to be bioactive, namely metabolites of PGD2, which include 9alpha ,11beta -PGF2 (32), 12-epi-9alpha ,11beta -PGF2 (52), and 13,14-dihydro-15-keto-PGD2 (42). We recently demonstrated that 15-F2t-IsoP is metabolized in humans by processes of the double bond position relative to the COOH terminus (Delta 5) reduction and beta -oxidation to yield a single metabolite 2,3-dinor-5,6-dihydro-15-F2t-IsoP (15-F2t-IsoP-M) (45). Another possible source of 15-F2t-IsoP-M was found to be through free radical-mediated oxidation of the relatively low abundant fatty acid gamma -linolenic acid (23). 15-F2t-IsoP-M has recently been shown to be present in human urine with chronic liver disease in concentrations exceeding those of 15-F2t-IsoP and is further augmented after liver transplantation (ischemia-reperfusion type injury) (6). To date, the molecular origin(s) of 15-F2t-IsoP-M in tissue remain unclear; moreover, its biological activity is unknown. If 15-F2t-IsoP-M was indeed a biologically active compound, it would represent another rare case of a bioactive prostanoid metabolite, but more importantly a novelty in a sense that no other prostanoid metabolite arising from beta -oxidation has so far been shown to be bioactive. Assuming 15-F2t-IsoP-M-induced effects were similar to those of its precursor, this may have important implications by prolonging the biological actions of 15-F2t-IsoP. We, therefore, sought to 1) compare vasomotor effects of 15-F2t-IsoP-M with those of 15-F2t-IsoP previously described on different ocular and cerebral vascular beds (20, 28), 2) investigate the mechanisms of action of the 15-F2t-IsoP-M, and 3) confirm that 15-F2t-IsoP-M can be formed by free radical-induced oxidation of gamma -linolenic acid. Our findings reveal that the effects and mode of actions of 15-F2t-IsoP-M on vascular tissues are essentially the same as those of 15-F2t-IsoP, and 15-F2t-IsoP-M can be formed during oxidation of gamma -linolenic acid.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue preparation. Experiments were performed on pig tissues in accordance with the Guide for the Care and Use of Laboratory Animals provided by the Canadian Council on Animal Care and with the approval of the Animal Care Committee of Hôpital Sainte-Justine. Piglets (2-4 days old) were obtained from Fermes Ménard (L'Ange-Gardien, Québec, Canada). Animals were anesthetized with halothane (~2.5-5%), and an intracardiac injection of India ink (1.5 ml/kg) was given to facilitate visualization of the retinal and brain (neural) microvessels. Animals were killed with pentobarbital sodium (120 mg/kg), and the brain and eyes were removed and placed immediately in ice-cold Krebs buffer (pH 7.4, bubbled with 21% O2, 5% CO2, and 74% N2) of the following composition (in mM): 120 NaCl, 4.5 KCl, 2.5 CaCl2, 1.0 MgSO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, 27 NaHCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, 1.0 KH2PO<UP><SUB>4</SUB><SUP>−</SUP></UP>, and 10 glucose; 1.5 U/ml heparin were added to the buffer. The eyecups and brain slices were pinned securely to a wax base of a 20-ml bath containing Krebs buffer (pH 7.4) at 37°C. The preparations were washed 2-3 times with fresh buffer and allowed to stabilize for 45 min before starting the experiment.

Vasomotor response of retinal and intraparenchymal brain microvessels. Eyecups and brain slices (1-mm thick) exposing, respectively, the retina and brain (intraparenchymal) cortical region were prepared as previously described (2, 7, 20, 28-30) to study retinal surface (100-150 µm) and intraparenchymal brain microvessels (30-50 µm) in situ; these auxotonic preparations (31) minimize vascular injury and reflect better physiological conditions.

Microvessels were visualized and recorded using a video camera (model CCD72, MTI) mounted on a dissecting microscope (model M-400, Nikon), as previously reported (2, 7, 28-30). Vascular diameter was measured using a digital image analyzer (Sigma Scan software, Jandel Scientific, Corte Madera, CA) and repeated three times with a variability of <1%; India ink did not modify vascular responses to constrictors (e.g., U-46619 and phenylephrine) and relaxants (e.g., carbaprostacyclin and sodium nitroprusside) (20). Vascular diameter was recorded before and after topical application of increasing concentrations of 15-F2t-IsoP-M, 15-F2t-IsoP, gamma -linolenic acid, PGF2alpha , and U-46619 [thromboxane A2 (TXA2) mimetic]; in all cases, plateau responses were reached within 10 min after stimulation. 15-F2t-IsoP-M (>99% pure) was chemically synthesized as described by Taber and Kanai (47).

Cross-desensitization experiments were conducted on retinal microvessels. For this purpose, vasoconstrictor responses of 15-F2t-IsoP-M, 15-F2t-IsoP, and U-46619 were tested on tissues that were initially exposed (20-min time period) to different concentrations (0.05, 1, or 10 µM) of either 15-F2t-IsoP-M or 15-F2t-IsoP to obtain desensitization. These responses were compared with those measured parallel to control tissues in which a single maximal concentration (10 µM) of each agent was applied; responses to KCl (30 mM) were also tested to ascertain selectivity of the desensitization. Maximal responses and concentrations of agents producing 50% of the maximal response (EC50) were determined from the concentration-response curves (2). Responses were expressed as percent change in the outer diameter of the vessel from baseline.

We determined if mechanisms involved in 15-F2t-IsoP-M-induced actions are comparable to those of 15-F2t-IsoP (20, 28). Hence, we assessed the contribution of TXA2 receptor-operated and N- as well as L-type voltage-gated Ca2+ channels on effects of 15-F2t-IsoP-M. Tissues were pretreated 30 min with the following agents at known effective concentrations (2, 11, 20, 28): TXA2-synthase inhibitor CGS-12970 (1 µM; Ciba-Geigy, Summit, NJ), TXA2-receptor antagonist L-670596 (0.1 µM; Merck-Frosst, Pointe-Claire, Québec, Canada), putative nonvoltage-dependent Ca2+ entry and receptor-mediated Ca2+ channel blocker SK&F96365 (20 µM; BioMol, Plymouth Meeting, PA) (35), L-type voltage-gated Ca2+ channel blocker nifedipine (5 µM), and N-type voltage-gated Ca2+ channel blocker omega -conotoxin MVIIA (10 µM; Sigma Chemical, St. Louis, MO) (43). Similar experiments were also conducted using U-46619 as a stimulant.

Measurement of vasomotor response of perfused choroid. Vasomotor responses of 15-F2t-IsoP-M and 15-F2t-IsoP were also studied on a perfused nonneural tissue, the ocular choroid, as described in detail (1, 3). The choroid was perfused with Krebs buffer (pH 7.4, 37°C, bubbled with 21% O2, 5% CO2, and 74% N2) at a physiological constant flow rate of ~0.20 ml/min to produce a physiological perfusion pressure of 60 mmHg (2) using a pulsatile minipump (Gilson, France). Perfusion pressure immediately proximal to the eyeball was continuously recorded using a pressure transducer (Perceptor DT, Namic, NY) connected to a Gould multichannel amplifier recorder (TA 240, Gould, OH).

The choroidal vascular bed was perfused for 30 min with Krebs buffer for stabilization of the preparation. In some experiments, the endothelium was removed by infusing air in vasculature that no longer relaxed to acetylcholine but responded normally to endothelium-independent stimulants U-46619 and papaverine (15). 15-F2t-IsoP-M (in Krebs buffer) was infused with or without pretreatment (30 min) with CGS-12970 (1 µM). Vasomotor responses were recorded continuously, and concentration of the agonist was increased every 20 min when responses had reached a plateau.

Astroglial and microvascular endothelial and smooth muscle cell culture. Astrocytes were cultured from brains of newborn pigs (20). 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 1,000 g for 7 min and resuspended in DMEM with 10% fetal calf serum and incubated in air and 5% CO2 at 37°C. Loosely attached macrophages were removed from glial cultures using a rotary shaker 225 rpm for 3 h.

Microvessels from a newborn brain were prepared as previously described (20, 30). Individual endothelial and smooth muscle cells were cultured after suspending microvessels in selective endothelial or smooth muscle growth media (Clonetics) as reported (15, 20, 28). Cells were identified morphologically and by immunoreactivity to Factor VIII, smooth muscle actin, or glial fibrillary acidic protein (Dako, Carpinteria, CA). Cell viability was verified by trypan blue exclusion and was >90%. Confluent cultures of 5-15 passages were used for experiments.

Measurement of thromboxane generation. Thromboxane formation induced by 15-F2t-IsoP-M (0.05-1 µM) (15-min incubation) was studied on brain slices treated or not with CGS-12970 (1 µM), SK&F96365 (20 µM), nifedipine (5 µM), omega -conotoxin MVIIA (10 µM), or EGTA (100 µM). The reaction was stopped with liquid N2. TXB2 (stable TXA2 metabolite) was determined on the homogenized tissue by radioimmunoassay (Amersham, Oakville, ON, Canada) (2, 20, 28).

Ca2+ signals. Intracellular Ca2+ ([Ca2+]i) signals were measured using the fluorescent indicator fura 2-AM (Calbiochem, La Jolla, CA) as reported (20, 28). Briefly, trypsinized cell preparations were resuspended in Hanks' balanced salt solution containing Ca2+ (2.5 mM) and 1% fetal bovine serum. Cell suspension was pretreated (15 min, 37°C) or not with either SK&F96365 (20 µM), nifedipine (5 µM), omega -conotoxin MVIIA (10 µM), L-670596 (0.1 µM), or EGTA (100 µM), and thereafter stimulated with 15-F2t-IsoP-M (1 µM). The [Ca2+]i was measured using 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 fluorescent signal was made using ionomycin (10 mM) and Triton X-100 (0.2%). The [Ca2+]i was calculated as described by Grynkiewicz et al. (13).

Oxidation of gamma -linolenic acid and assay for 15-F2t-IsoP-M. Oxidation of gamma -linolenic acid (NuChek, Elysian, MN) was performed by dissolving 5 mg of the product in 50 µl ethanol added to 5 ml phosphate-buffered saline and incubated with 2,2'-azobis-(2-amido propane) hydrochloride (AAPH; Polysciences, Warrington, PA) (4 mg/ml) at 37°C for 24 h. 15-F2t-IsoP-M was extracted, purified, and analyzed by gas chromatography followed by ion spray mass spectrometry (negative mode) as recently described (38); molecular weights of compounds were determined according to the mass-to-charge ratio (m/z).

Statistics. Results are expressed as means ± SE and analyzed using Student's t-test and two-way ANOVA factoring for concentrations and treatments; comparisons among means were performed using the Tukey-Kramer method. Statistical significance was set at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of 15-F2t-IsoP-M and 15-F2t-IsoP on retinal and brain microvessels. 15-F2t-IsoP-M, 15-F2t-IsoP, PGF2alpha , and U-46619 caused significant constriction of retinal and brain microvessels (Fig. 1). 15-F2t-IsoP-M and 15-F2t-IsoP were similarly effective on both preparations; maximal effects of PGF2alpha and U-46619 were greater than those of the IsoPs. In the retina, the EC50 for 15-F2t-IsoP-M, 15-F2t-IsoP, PGF2alpha , and U-46619 was 12.8 ± 0.6, 14.7 ± 0.9, 15.8 ± 1.1, and 33.1 ± 1.8 nM (n = 5); and in the brain, it was 18.5 ± 2.7, 22.8 ± 3.6, 21.1 ± 2.0, and 49.3 ± 2.2 nM (n = 5), respectively.


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  		Fig. 1.   Effects of 2,3-dinor-5,6-dihydro-15-F2t-isoprostane (15-F2t-IsoP-M), 15-F2t-isoprostane (15-F2t-IsoP), prostaglandin (PG) F2alpha , and U-46619 on vasoconstriction of retinal and brain microvessels. A: in the retina, vasomotor response of surface microvessels (100-150 µm) was evaluated on flat mount preparations. B: in the brain, response of intraparenchymal cortex vessels (30-50 µm) was determined. Constriction is the percent reduction in vascular diameter from basal values. Data are means ± SE of 5 separate experiments.

Role of TXA2 on vascular effects of 15-F2t-IsoP-M. We have previously shown that 15-F2t-IsoP is a strong stimulant of TXA2 formation on ocular and brain vasculature (20, 28). We determined if vascular effects of 15-F2t-IsoP-M are also TXA2 dependent. Retinal and cerebral vasoconstriction evoked by 15-F2t-IsoP-M was abrogated by the TXA2-synthase inhibitor CGS-12970 and by the TXA2-receptor blocker L-670596 (Fig. 2, A and B). Correspondingly, TXB2 levels increased dose dependently after stimulation of retinal and brain preparations with 15-F2t-IsoP-M (Fig. 3).


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  		Fig. 2.   Contribution of thromboxane A2 (TXA2) on vasoconstriction by 15-F2t-IsoP-M in the retina, brain, and choroid. Retinal (A) and brain (B) preparations were pretreated for 30 min with TXA2-synthase inhibitor CGS-12970 (1 µM), TXA2-receptor antagonist L-670596 (0.1 µM), or saline. Data are means ± SE of 4 or 5 separate experiments. *P < 0.01 compared with saline treated (2-way ANOVA). C: choroids were infused with Krebs buffer containing CGS-12970 (1 µM) or not. Deendothelialization of choroid vasculature was performed by infusing air; vasculature no longer relaxed to acetylcholine but responded normally to endothelium-independent constrictor U-46619 and relaxant papaverine. Data are means ± SE of 4 or 5 separate experiments. *P < 0.01 compared with values for choroid with endothelium (2-way ANOVA).



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  		Fig. 3.   Effects of 15-F2t-IsoP-M on thromboxane formation in retinal (A) and brain (B) tissue. Preparations were untreated (basal) or treated with 15-F2t-IsoP-M in the presence or absence of L-670596 (0.1 µM), CGS-12970 (1 µM), SK&F96365 (20 µM), omega -conotoxin MVIIA (10 µM), or nifedipine (5 µM). Data are means ± SE of 3 or 4 separate experiments. *P < 0.05 compared with basal values (ANOVA); dagger P < 0.01 compared with corresponding value for 15-F2t-IsoP-M (1 µM) in the absence of inhibitor (saline); Dagger P < 0.05 compared with value for 15-F2t-IsoP-M (1 µM) in the absence of inhibitor (saline) and when pretreated with CGS-12970 or omega -conotoxin MVIIA.

To assess if this TXA2 dependence also applies to a nonneural tissue and to examine the involvement of endothelium, effects of 15-F2t-IsoP-M were studied on perfused choroid denuded or not denuded of endothelium (1, 3). 15-F2t-IsoP-M caused dose-dependent choroidal vasoconstriction, which was virtually abolished by CGS-12970 as well as by removal of endothelium (Fig. 2C); similar results were seen with 15-F2t-IsoP.

Effects of Ca2+ channel blockers on 15-F2t-IsoP-M-induced TXA2 formation and vasoconstriction. Because removal of endothelium markedly diminishes the TXA2-dependent action of 15-F2t-IsoP-M, this suggested that endothelial cells contribute to the TXA2 formation evoked by 15-F2t-IsoP-M (Fig. 2C). In brain intraparenchymal microvasculature, it is likely perivascular astroglial cells also contribute to TXA2 formation (20). Because enzyme-catalyzed prostanoid formation is Ca2+ dependent through phospholipase A2 requirement, we attempted to identify the type of Ca2+ channel involved in 15-F2t-IsoP-M-induced TXA2 generation. We focused on receptor-operated and N- as well as L-type voltage-gated Ca2+ channels because endothelial cells are not excitable and are mostly devoid of voltage-gated Ca2+ channels (17), whereas astrocytes contain voltage-gated channels, mostly N- and L-types (9, 40, 53). In the retina, TXB2 formation was stimulated by 15-F2t-IsoP-M, and this effect was markedly inhibited by CGS-12970 and by the putative nonvoltage-gated channel blocker SK&F96365, but not by L-670596, the N-voltage-gated Ca2+ channel blocker omega -conotoxin MVIIA, or an L-voltage-gated Ca2+ channel blocker nifedipine (Fig. 3). In the brain, TXB2 generation induced by 15-F2t-IsoP-M was partly inhibited by SK&F96365 and, more significantly, by omega -conotoxin MVIIA, but it was unaffected by nifedipine (Fig. 3).

The relative role of Ca2+ channels involved in 15-F2t-IsoP-M-induced TXB2 formation was also assessed on vasoconstriction. Constriction of retinal surface microvessels (100-150 µm) to 15-F2t-IsoP-M was nearly abolished by SK&F96365 and nifedipine, but it was unaffected by omega -conotoxin MVIIA (Fig. 4A). The 15-F2t-IsoP-M-evoked constriction of intraparenchymal brain microvessels (30-50 µm) was diminished by SK&F96365, reduced more extensively by omega -conotoxin MVIIA, and nearly abrogated by nifedipine. These vasomotor observations are consistent with the relative roles of Ca2+ channels on 15-F2t-IsoP-M-induced TXA2 formation in the retina and brain (Fig. 3).


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  		Fig. 4.   Effects of Ca2+ channel blockers on retinal and brain vasoconstriction in response to 15-F2t-IsoP-M (A) and U-46619 (B). Tissues were prepared as described in Figs. 1-3. Data are means ± SE of 3 or 4 separate experiments. *P < 0.05 compared with values without * (2-way ANOVA).

Effects of 15-F2t-IsoP-M on Ca2+ transients. To further evaluate the effects of 15-F2t-IsoP-M on Ca2+ transients, these were studied on vascular and perivascular cells, specifically on neurovascular endothelial, smooth muscle, and on astroglial cells. 15-F2t-IsoP-M caused an increase in the Ca2+ signal in endothelial cells that was prevented by the nonvoltage-gated channel blocker SK&F96365 and similarly by the Ca2+ chelator EGTA (Fig. 5, A and B); nifedipine, omega -conotoxin MVIIA, and L-670596 had no effect. In astrocytes, Ca2+ transients evoked by 15-F2t-IsoP-M were inhibited by omega -conotoxin MVIIA as well as by EGTA, but they were unaffected by nifedipine, SK&F96365, and L-670596 (Fig. 5, C and D). Smooth muscle cells did not respond to 15-F2t-IsoP-M but did evoke an increase in [Ca2+]i in response to the TXA2 mimetic U-46619, which was blocked by nifedipine (Fig. 5, E and F). Accordingly, vasoconstriction to U-46619 was only abrogated by nifedipine and not by SK&F96365 and omega -conotoxin MVIIA (Fig. 4B).


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  		Fig. 5.   Intracellular calcium ([Ca2+]i) transients in neurovascular endothelial (A and B), astroglial (C and D), and smooth muscle cells (E and F) in response to 15-F2t-IsoP-M as well as of smooth muscle cells to U-46619 (E and F). Cells were pretreated 20 min with saline (control), SK&F96365 (20 µM), omega -conotoxin MVIIA (10 µM), nifedipine (5 µM), L-670596 (0.1 µM), or EGTA (100 µM). [Ca2+]i transients were measured using fura 2-AM (see MATERIALS AND METHODS). down-arrow  Point to moment of addition of 15-F2t-IsoP-M (A and C) and U-46619 (E). Values in histograms (B, D, and F) are means ± SE of 3 or 4 separate experiments. *P < 0.05 compared with all other values without *.

Effects of 15-F2t-IsoP-M and 15-F2t-IsoP after cross-desensitization. Data presented (see Fig. 1) reveal that 15-F2t-IsoP-M and 15-F2t-IsoP are comparably effective and potent on distinct tissues, and their actions are mediated via similar mechanisms. To further test this inference, these compounds (10 µM) as well as U-46619 (1 µM) were tested on retinal preparations initially exposed to different concentrations (0.05-10 µM) of either 15-F2t-IsoP-M or 15-F2t-IsoP, and the results were compared with those obtained on unexposed control tissues. Reciprocal dose-dependent fading of vasomotor response (representative of desensitization) to 15-F2t-IsoP-M and 15-F2t-IsoP was observed, suggesting that these IsoPs may operate at similar receptor sites (Fig. 6, A and B); effects of KCl (30 mM) were not modified (not shown). In contrast, constriction to U-46619 (1 µM) was only slightly diminished; hence, although neurovascular constriction to 15-F2t-IsoP and 15-F2t-IsoP-M is mediated via TXA2, the amount formed appears insufficient to significantly desensitize TXA2 effects in these conditions. Overall, data suggest that 15-F2t-IsoP and 15-F2t-IsoP-M seem to share the same mechanisms that result in TXA2 actions.


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  		Fig. 6.   Retinal vasoconstrictor responses of 15-F2t-IsoP, 15-F2t-IsoP-M, and U-46619 after desensitization with 15-F2t-IsoP (A) or 15-F2t-IsoP-M (B). The responses of agents were tested on tissues initially exposed (20-min time period) to different concentrations (0.05, 1, or 10 µM) of either 15-F2t-IsoP-M or 15-F2t-IsoP, and they were compared with those measured on control tissues. Values are means ± SE of 3 experiments. *P < 0.05, dagger P < 0.01 compared with control responses (without desensitization).

Oxidation of gamma -linolenic acid to 15-F2t-IsoP-M. A small amount of 15-F2t-IsoP-M (5.4 ng/mg gamma -linolenic acid) and other m/z 543 peaks that likely represent isomers of 15-F2t-IsoP-M were detected in the preparation of the polyunsaturated fatty acid gamma -linolenic acid not yet subjected to oxidation with AAPH (Fig. 7A); the formation of isomers of 15-F2t-IsoP-M is analogous to that of multiple isomers of F2-IsoPs during oxidation of arachidonic acid. The amount of 15-F2t-IsoP-M detected after oxidation with AAPH increased markedly to 245.2 ng/mg gamma -linolenic acid (Fig. 7B), confirming formation of 15-F2t-IsoP-M by oxidation of gamma -linolenic acid. Of relevance, gamma -linolenic acid per se produced minimal maximal vessel contraction (4.6 ± 0.4%, n = 3); this may be attributed to the small amount of 15-F2t-IsoP-M present in the preparation (Fig. 7A).


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  		Fig. 7.   Measurement of 15-F2t-IsoP-M in commercial gamma -linolenic acid preparation before (A) and after (B) 24-h oxidation with 2,2'-azobis-(2-amido propane) hydrochloride (4 mg/ml). Top: chromatograms depict the elution of the mass-to-charge ratio (m/z) 547 peak representing the 18O4-labeled 15-F2t-IsoP-M internal standard. Bottom: chromatograms depict an m/z 543 peak with the same retention time as the internal standard; additional m/z 543 peaks likely represent isomers of 15-F2t-IsoP-M formed during oxidation of gamma -linolenic acid. The elution times in A and B differ slightly because the 2 analyses were performed on different days. Note that the arbitrary units of intensity for the m/z 543 ion current chromatogram in B but not in A are ~10-fold higher than that in the m/z 547 ion current chromatogram. Amounts of 15-F2t-IsoP-M are 5.4 (A) and 245.2 ng/mg gamma -linolenic acid (B).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study reveals that 15-F2t-IsoP-M, generated as a metabolite of 15-F2t-IsoP as well as an oxidation product of gamma -linolenic acid, exhibits biological properties that are comparable to those of its precursor, 15-F2t-IsoP, in retinal and cerebral vasculature as well as on astroglial cells. Specifically, 15-F2t-IsoP-M causes constriction of distinct vascular beds with potency similar to that of 15-F2t-IsoP, which is largely mediated via TXA2 generation after enhancing calcium entry. This increased entry of extracellular calcium into cells seems to occur on the larger retinal surface microvessels mostly through nonvoltage-dependent calcium channels, whereas on the smaller penetrating intraparenchymal brain microvessels, it is via both nonvoltage-dependent calcium channels in endothelial cells and N-type voltage-dependent calcium channels in astrocytes. Interestingly, these effects of 15-F2t-IsoP-M on vasomotricity, TXA2 generation, and calcium transients on surface retinal and on intraparenchymal brain microvessels are analogous to those of its precursor 15-F2t-IsoP (20, 28). Moreover, these two compounds seem to share the same mechanisms that lead to the production and subsequent activation of TXA2-mediated effects.

The effects of 15-F2t-IsoP-M on TXA2 formation and vascular contraction on retinal preparations are virtually abolished by the putative nonvoltage-gated calcium channel blocker SK&F96365 (35) and significantly diminished by SK&F96365 and the N-type voltage-gated calcium channel blocker omega -conotoxin MVIIA (43) on brain preparations (Fig. 4). Correspondingly, 15-F2t-IsoP-M-induced calcium signals were inhibited on endothelial cells by SK&F96365 and on astrocytes by omega -conotoxin MVIIA (Fig. 5). 15-F2t-IsoP-M was ineffective on smooth muscle cells. These data suggest that 15-F2t-IsoP-M produces its effects mostly by acting on endothelial cells in ocular vasculature and on both endothelial and astroglial cells in intraparenchymal brain microvasculature, as observed with 15-F2t-IsoP (20, 28).

Because astrocytes are the most abundant cell type in the brain parenchyma, it is reasonable to suggest that astrocytes are the main source of TXA2 formation and contribute most to 15-F2t-IsoP-M-mediated cerebral vasoconstriction; this inference is supported by the relatively greater inhibition of constriction of brain microvessels by omega -conotoxin MVIIA (Fig. 4). The reason for the apparent contrast in retinal and cerebral microvessel dependence on N-type voltage-gated calcium channels in response to 15-F2t-IsoP-M [as well as to 15-F2t-IsoP (20, 28)] could be explained by tissue differences in the expression of these channels (22, 46, 50, 54, 55). In addition, the type of vessel tested differs, such that surface microvessels are larger (100-150 µm) and are therefore not enveloped by astrocytes as is the case for the smaller intraparenchymal microvessels (30-50 µm) (18); hence, the latter are more affected by mediators secreted by astrocytes.

15-F2t-IsoP-M is formed as the major metabolite of 15-F2t-IsoP by processes of one-step beta -oxidation and reduction of the Delta 5 double bond (45). It is of interest that urine levels of 15-F2t-IsoP-M exceed those of 15-F2t-IsoP in vivo (6). In addition, as shown in this study (Fig. 7) as well as recently reported (6), 15-F2t-IsoP-M can be generated by free radical-induced peroxidation of gamma -linolenic acid. At present, the relative contribution of these two pathways in generation of 15-F2t-IsoP-M in vivo remains unknown. Although oxidation of gamma -linolenic acid may be significant in generating 15-F2t-IsoP-M (6), the fact that arachidonic acid is more abundant (23) and the metabolism of 15-F2t-IsoP in humans yields a predominant metabolite, 15-F2t-IsoP-M, rather than a myriad of compounds (45), which is more typical with metabolism of prostanoids, suggests that formation of 15-F2t-IsoP-M in vivo from metabolism of 15-F2t-IsoP may also be important.

In general, the metabolism of prostanoids is rapid and efficient, and the enzymatically derived metabolites of prostanoids are biologically inactive (8, 34, 39). This effective disposition of prostanoids accounts for their very short biological half-life in vivo (10, 14). There are rare known exceptions to this rule. Such is the case for PGD2 metabolites 9alpha ,11beta -PGF2, 12-epi-9alpha ,11beta -PGF2, and 13,14-dihydro-15-keto-PGD2, which have been found to exert biological effects (32, 42, 52). However, the effects of these metabolites differ from those evoked by the parent compound to the extent of being opposite (32, 42). In contrast, 15-F2t-IsoP-M produces biological effects in different tissues that are in essence identical to those of its precursor with regards to action, efficacy, and potency (Figs. 1-6) (20, 28).

In conclusion, the present findings reveal so far undescribed biological properties of 15-F2t-IsoP-M, a beta -oxidation metabolite of prostanoids and a free radical-mediated oxidation product of gamma -linolenic acid. Because of the marked vascular effects of F2-IsoPs, one could speculate that 15-F2t-IsoP-M could prolong the biological actions of 15-F2t-IsoP and possibly contribute in sustaining impaired circulation after an oxidant stress (7, 21).

Perspectives

IsoPGs are free radical-derived prostanoids resulting from peroxidation of arachidonic acid. These abundantly generated peroxidation products reproduce various biological effects evoked by oxidant stresses. One of these stable products that has been available synthetically and studied extensively, 15-F2t-IsoP, was found to be metabolized by processes of Delta 5 reduction and beta -oxidation to yield a single metabolite 15-F2t-IsoP-M (45); beta -oxidation metabolites of prostanoids have so far always been found to be inactive. Interestingly, 15-F2t-IsoP-M can also be generated by peroxidation of gamma -linolenic acid (23 and present study). We hereby disclose that 15-F2t-IsoP-M exhibits biological properties comparable to those of its precursor 15-F2t-IsoP, both of which share similar mechanisms mediated by TXA2. Given the complex and often long-term outcome of oxidant stress, it is possible that 15-F2t-IsoP-M formation may contribute in prolonging the adverse consequences of peroxidation.


    ACKNOWLEDGEMENTS

We thank H. Fernandez for technical assistance.


    FOOTNOTES

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 Quebec, and the Fonds de la Recherche en Santé du Québec. F. Gobeil Jr. and S. Chemtob are recipients, respectively, of fellowship and scientist awards from the Medical Research Council of Canada. X. Hou, M. Beauchamp, and S. Bernier are recipients of fellowships and studentships from the Research Center of Hôpital Ste-Justine.

Address for reprint requests and other correspondence: S. Chemtob, Research Center, Hôpital Sainte-Justine, Dept. of Pediatrics and Pharmacology, 3175 Côte Sainte-Catherine, Montréal, Québec H3T 1C5, Canada (E-mail: sylvain.chemtob{at}umontreal.ca).

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

Received 17 August 2000; accepted in final form 19 March 2001.


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