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 F₂-III induces cardiomyocyte hypertrophy (27) and 15-F_2c-IsoP (12-iso-PGF₂; Ref. 48) can activate the PGF₂ receptor (26). The IsoP studied most extensively is 15-F_2t-IsoP also termed 8-iso-PGF₂ (48). 15-F_2t-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-F_2t-IsoP (12) remains to be determined. Nonetheless, the modes of action of 15-F_2t-IsoP on vasculature have been studied and are shown to differ between species and vascular beds. For instance, rat 15-F_2t-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-F_2t-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-F_2t-IsoP in vascular degeneration have recently been suggested (5).

Metabolism is an important means for inactivation of prostanoids, which proceeds through -oxidation, -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 9,11-PGF₂ (32), 12-epi-9,11-PGF₂ (52), and 13,14-dihydro-15-keto-PGD₂ (42). We recently demonstrated that 15-F_2t-IsoP is metabolized in humans by processes of the double bond position relative to the COOH terminus (⁵) reduction and -oxidation to yield a single metabolite 2,3-dinor-5,6-dihydro-15-F_2t-IsoP (15-F_2t-IsoP-M) (45). Another possible source of 15-F_2t-IsoP-M was found to be through free radical-mediated oxidation of the relatively low abundant fatty acid -linolenic acid (23). 15-F_2t-IsoP-M has recently been shown to be present in human urine with chronic liver disease in concentrations exceeding those of 15-F_2t-IsoP and is further augmented after liver transplantation (ischemia-reperfusion type injury) (6). To date, the molecular origin(s) of 15-F_2t-IsoP-M in tissue remain unclear; moreover, its biological activity is unknown. If 15-F_2t-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 -oxidation has so far been shown to be bioactive. Assuming 15-F_2t-IsoP-M-induced effects were similar to those of its precursor, this may have important implications by prolonging the biological actions of 15-F_2t-IsoP. We, therefore, sought to 1) compare vasomotor effects of 15-F_2t-IsoP-M with those of 15-F_2t-IsoP previously described on different ocular and cerebral vascular beds (20, 28), 2) investigate the mechanisms of action of the 15-F_2t-IsoP-M, and 3) confirm that 15-F_2t-IsoP-M can be formed by free radical-induced oxidation of -linolenic acid. Our findings reveal that the effects and mode of actions of 15-F_2t-IsoP-M on vascular tissues are essentially the same as those of 15-F_2t-IsoP, and 15-F_2t-IsoP-M can be formed during oxidation of -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, 27 NaHCO, 1.0 KH₂PO, 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.

2

Measurement of vasomotor response of perfused choroid. Vasomotor responses of 15-F_2t-IsoP-M and 15-F_2t-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).

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.

Measurement of thromboxane generation. Thromboxane formation induced by 15-F_2t-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), -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), -conotoxin MVIIA (10 µM), L-670596 (0.1 µM), or EGTA (100 µM), and thereafter stimulated with 15-F_2t-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 -linolenic acid and assay for 15-F_2t-IsoP-M. Oxidation of -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-F_2t-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-F_2t-IsoP-M and 15-F_2t-IsoP on retinal and brain microvessels. 15-F_2t-IsoP-M, 15-F_2t-IsoP, PGF₂, and U-46619 caused significant constriction of retinal and brain microvessels (Fig. 1). 15-F_2t-IsoP-M and 15-F_2t-IsoP were similarly effective on both preparations; maximal effects of PGF₂ and U-46619 were greater than those of the IsoPs. In the retina, the EC₅₀ for 15-F_2t-IsoP-M, 15-F_2t-IsoP, PGF₂, 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.

View larger version (20K): [in this window] [in a new window]   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) F2, 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-F_2t-IsoP-M. We have previously shown that 15-F_2t-IsoP is a strong stimulant of TXA₂ formation on ocular and brain vasculature (20, 28). We determined if vascular effects of 15-F_2t-IsoP-M are also TXA₂ dependent. Retinal and cerebral vasoconstriction evoked by 15-F_2t-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-F_2t-IsoP-M (Fig. 3).

View larger version (29K): [in this window] [in a new window]   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).

View larger version (22K): [in this window] [in a new window]   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), -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); P < 0.01 compared with corresponding value for 15-F2t-IsoP-M (1 µM) in the absence of inhibitor (saline); 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 -conotoxin MVIIA.

Effects of Ca²+ channel blockers on 15-F_2t-IsoP-M-induced TXA₂ formation and vasoconstriction. Because removal of endothelium markedly diminishes the TXA₂-dependent action of 15-F_2t-IsoP-M, this suggested that endothelial cells contribute to the TXA₂ formation evoked by 15-F_2t-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-F_2t-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-F_2t-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 -conotoxin MVIIA, or an L-voltage-gated Ca²⁺ channel blocker nifedipine (Fig. 3). In the brain, TXB₂ generation induced by 15-F_2t-IsoP-M was partly inhibited by SK&F96365 and, more significantly, by -conotoxin MVIIA, but it was unaffected by nifedipine (Fig. 3).

View larger version (33K): [in this window] [in a new window]   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-F_2t-IsoP-M on Ca²+ transients. To further evaluate the effects of 15-F_2t-IsoP-M on Ca²⁺ transients, these were studied on vascular and perivascular cells, specifically on neurovascular endothelial, smooth muscle, and on astroglial cells. 15-F_2t-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, -conotoxin MVIIA, and L-670596 had no effect. In astrocytes, Ca²⁺ transients evoked by 15-F_2t-IsoP-M were inhibited by -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-F_2t-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 -conotoxin MVIIA (Fig. 4B).

View larger version (33K): [in this window] [in a new window]   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), -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).  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-F_2t-IsoP-M and 15-F_2t-IsoP after cross-desensitization. Data presented (see Fig. 1) reveal that 15-F_2t-IsoP-M and 15-F_2t-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-F_2t-IsoP-M or 15-F_2t-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-F_2t-IsoP-M and 15-F_2t-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-F_2t-IsoP and 15-F_2t-IsoP-M is mediated via TXA₂, the amount formed appears insufficient to significantly desensitize TXA₂ effects in these conditions. Overall, data suggest that 15-F_2t-IsoP and 15-F_2t-IsoP-M seem to share the same mechanisms that result in TXA₂ actions.

View larger version (48K): [in this window] [in a new window]   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, P < 0.01 compared with control responses (without desensitization).

Oxidation of -linolenic acid to 15-F_2t-IsoP-M. A small amount of 15-F_2t-IsoP-M (5.4 ng/mg -linolenic acid) and other m/z 543 peaks that likely represent isomers of 15-F_2t-IsoP-M were detected in the preparation of the polyunsaturated fatty acid -linolenic acid not yet subjected to oxidation with AAPH (Fig. 7A); the formation of isomers of 15-F_2t-IsoP-M is analogous to that of multiple isomers of F₂-IsoPs during oxidation of arachidonic acid. The amount of 15-F_2t-IsoP-M detected after oxidation with AAPH increased markedly to 245.2 ng/mg -linolenic acid (Fig. 7B), confirming formation of 15-F_2t-IsoP-M by oxidation of -linolenic acid. Of relevance, -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-F_2t-IsoP-M present in the preparation (Fig. 7A).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Measurement of 15-F2t-IsoP-M in commercial -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 -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 -linolenic acid (B).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The present study reveals that 15-F_2t-IsoP-M, generated as a metabolite of 15-F_2t-IsoP as well as an oxidation product of -linolenic acid, exhibits biological properties that are comparable to those of its precursor, 15-F_2t-IsoP, in retinal and cerebral vasculature as well as on astroglial cells. Specifically, 15-F_2t-IsoP-M causes constriction of distinct vascular beds with potency similar to that of 15-F_2t-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-F_2t-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-F_2t-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-F_2t-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 -conotoxin MVIIA (43) on brain preparations (Fig. 4). Correspondingly, 15-F_2t-IsoP-M-induced calcium signals were inhibited on endothelial cells by SK&F96365 and on astrocytes by -conotoxin MVIIA (Fig. 5). 15-F_2t-IsoP-M was ineffective on smooth muscle cells. These data suggest that 15-F_2t-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-F_2t-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-F_2t-IsoP-M-mediated cerebral vasoconstriction; this inference is supported by the relatively greater inhibition of constriction of brain microvessels by -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-F_2t-IsoP-M [as well as to 15-F_2t-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-F_2t-IsoP-M is formed as the major metabolite of 15-F_2t-IsoP by processes of one-step -oxidation and reduction of the ⁵ double bond (45). It is of interest that urine levels of 15-F_2t-IsoP-M exceed those of 15-F_2t-IsoP in vivo (6). In addition, as shown in this study (Fig. 7) as well as recently reported (6), 15-F_2t-IsoP-M can be generated by free radical-induced peroxidation of -linolenic acid. At present, the relative contribution of these two pathways in generation of 15-F_2t-IsoP-M in vivo remains unknown. Although oxidation of -linolenic acid may be significant in generating 15-F_2t-IsoP-M (6), the fact that arachidonic acid is more abundant (23) and the metabolism of 15-F_2t-IsoP in humans yields a predominant metabolite, 15-F_2t-IsoP-M, rather than a myriad of compounds (45), which is more typical with metabolism of prostanoids, suggests that formation of 15-F_2t-IsoP-M in vivo from metabolism of 15-F_2t-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 9,11-PGF₂, 12-epi-9,11-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-F_2t-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-F_2t-IsoP-M, a -oxidation metabolite of prostanoids and a free radical-mediated oxidation product of -linolenic acid. Because of the marked vascular effects of F₂-IsoPs, one could speculate that 15-F_2t-IsoP-M could prolong the biological actions of 15-F_2t-IsoP and possibly contribute in sustaining impaired circulation after an oxidant stress (7, 21).

Perspectives