Proc. Nati. Acad. Sci. USA Vol. 85, pp. 8340-8344, November 1988 Physiological Sciences A4 and B4: Comparison of icosanoids having bronchoconstrictor and vasodilator actions but lacking aggregatory activity (pulmonary parenchymal strips/vascular smooth muscle/platelet-rich plasma/ receptors/ B2) ALLAN M. LEFER*, GREGORY L. STAHL*, DAVID J. LEFER*, MARK E. BREZINSKI*, K. C. NICOLAOUt, C. A. VEALEt, Y. ABEt, AND J. BRYAN SMITH: *Department of Physiology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107; tDepartment of Chemistry, University of Pennsylvania, Philadelphia, PA 19104; and tDepartment of Pharmacology, Temple University School of Medicine, Philadelphia, PA 19140 Communicated by Robert E. Forster, July 28, 1988 (received for review February 1, 1988)

ABSTRACT Lipoxins A4 (LxA4) and B4 (LxB4), two activated protein kinase C in vitro and therefore may serve as lipoxygenase-generated icosanoids of metab- an activator ofintracellular events in smooth muscle cells (6). olism, were found to have a distinct biological profile. Both Secondly, LxA4 has been shown to induce leukocyte lyso- LxA4 and LxB4 slowly contracted pulmonary parenchymal somal membrane leakiness and to release lysosomal hydro- strips isolated from guinea pigs, rabbits, and rats in a concen- lases and superoxide free radicals (7). Immunologically, tration-dependent manner over the range 0.1-1 IAM. This LxA4 and LxB4 block the action of natural killer cells against bronchoconstrictor effect was not associated with release of certain target cells (7). peptide or , nor was it blocked by More recently, LxA4 has been shown to contract pulmo- lipoxygenase inhibitors or thromboxane receptor antagonists, nary smooth muscle (i.e., guinea pig lung) and to relax suggesting it is a direct effect of lipoxins. However, the vascular smooth muscle (8) at concentrations <1 ,uM. Dahlen leukotriene D4 (LTD4) receptor antagonist LY-171883 reduced et al. (8) further showed that LxA4 had no significant the LxA4 response, indicating that LTD4 and LxA4 may share myotropic activity on either guinea pig trachea or guinea pig the same receptor. LxA4 and LxB4 also exerted an endothe- ileum (8). However, virtually nothing is known about the lium-dependent vasorelaxation in guinea pig, rat, and, to a actions of LxB4 in these systems or in blood . lesser extent, rabbit aortic vascular smooth muscle. In contrast Moreover, since the guinea pig is known to be very sensitive to other vasoactive icosanoids, LxA4 and LxB4 failed to to icosanoids (e.g., leukotrienes), additional information on aggregate rat, rabbit, or guinea pig platelets or to inhibit both LxA4 and LxB4 in other laboratory animals (e.g., rat, ADP-induced aggregation. LxA4 also enhanced the release of rabbit) is needed. Finally, very little is known about the liver lysosomal hydrolases in a liver large granule fraction, directness of the responses (i.e., whether lipoxins indicating a lysosomal labilizing action of LxA4. LxA4 and release thromboxane A2, leukotrienes, or other substances), LxB4 share a similar biological profile. It is not clear yet which may exert the alleged action of the lipoxins. whether the lipoxins could be mediators of circulatory or Therefore, the major objectives of this study were to (i) pulmonary disease states. determine the effects of LxB4 in established biological preparations (e.g., pulmonary strips, aortic rings, and plate- The lipoxins are a class of biologically active trihydroxy lets); (ii) compare the LxA4 and LxB4 in three different lipids having a conjugated tetraene and are formed within the laboratory species (i.e., rat, rabbit, and guinea pig); and (iii) arachidonic acid cascade. Lipoxins A and B, the two major establish whether lipoxins act via release of other icosanoids lipoxins, have been described by Serhan et al. (1, 2). These or by endothelium-derived relaxing factor (EDRF). icosanoids are formed by an interesting biosynthetic se- quence first involving oxygenation of arachidonic acid by a 15-lipoxygenase and subsequent oxygenation by a dual func- METHODS tion 5-lipoxygenase involving oxidation and dehydration to During the course of these investigations, adult male animals give a 5,6-epoxytetraene. This epoxide is then opened by of the following species were used: Sprague-Dawley rats attack at the more electrophilic 6-position by an epoxide (225-275 g), Hartley guinea pigs (300-600 g), and New hydrolase yielding lipoxin A (1) or via conjugate attack of the Zealand rabbits (2.5-3.5 kg). All animals were anesthetized hydrolase at the 14-position generating lipoxin B (1). These with sodium pentobarbital (35-40 mg/kg given intraperito- icosanoids have been given the trivial name "lipoxin" (short neally before isolation of organs and tissues). for "lipoxygenation interaction products") because of the Isolated Aortic Rings. After midsternal thoracotomy, the unique interaction of two lipoxygenases in their biosynthesis thoracic aortae were removed and placed in ice-cold Krebs- (1, 2). These biosynthetic events appear to occur in certain Heinseleit (KH) solution consisting of 120 mM NaCl, 4.75 types of leukocytes, including neutrophils and eosinophils mM KCI, 12.5 mM NaHCO3, 1.2 mM KH2PO4, 1.2 mM (2). Recently, the structures of lipoxins A and B have been MgSO4-7H20, 2.5 mM CaCl2-H20 and bubbled with 95% verified by total synthesis (3, 4). Now that the structures and 02/5% CO2 to maintain a pH of 7.3. All excess tissue biosynthesis of the lipoxins are known, their nomenclature surrounding the aortae was carefully removed, and the aortic has been established, and these substances have been termed segments were cut into 2-mm-wide rings. The rings were lipoxin A4 and lipoxin B4 (5). suspended in glass chambers filled with oxygenated KH Very little is known about the biological activity oflipoxins solution at 37°C and subjected to a resting force of 1 g. The A4 (LxA4) and B4 (LxB4). Early findings showed that LxA4 Abbreviations: LxA4, lipoxin A4; LxB4, lipoxin B4; TxA2, throm- The publication costs of this article were defrayed in part by page charge boxane A2; TxB2, thromboxane B2; LTC4, leukotriene C4; LTD4, payment. This article must therefore be hereby marked "advertisement" leukotriene D4; LTE4, leukotriene E4; EDRF, endothelium-derived in accordance with 18 U.S.C. §1734 solely to indicate this fact. relaxing factor. 8340 Downloaded by guest on September 30, 2021 Physiological Sciences: Lefer et al. Proc. Natl. Acad. Sci. USA 85 (1988) 8341 aortic rings were allowed to equilibrate for 60 min until a sorption over 3 orders of magnitude. This procedure was stable baseline was obtained. In some experiments, the done to ensure standardization of lipoxin concentrations, endothelium was removed from the vessel by rubbing it with which ordinarily exhibit a marked loss (>75%) during for- a coarsened polyethylene tube before the rings were sus- mation of the free acid. pended inside the glass chambers as described (9). At the Radloimnmunoassay of Icosanoids. Samples of the bathing start of each experiment, 28 nM 9,11-methanoepoxy- solution were analyzed before and after addition of LxA4 and H2 (U-46619) was administered to each ring LxB4 for leukotriene and thromboxane B2 (TxB2) concen- followed by 1 /LM acetylcholine to verify the completeness of trations. Samples were subjected to radioimmunoassay for the deendothelialization. The rings were then washed with TxB2 by the method of Ingerman-Wojenski et al. (13). The fresh KH solution, and 28 nM U-46619 was added to the TxB2 standard curve was constructed with a lower detection chambers to precontract the rings. Following achievement of limit of 0.075 pmol per ml of TxB2. a peak vascular response, the artery received either vehicle The radioimmunoassay for the peptide leukotrienes C4, D4, (0.9% NaCl), acetylcholine, or LxA4 and LxB4. LxA4, LxB4 and E4 (LTC4, LTD4, and LTE4) was carried out according (0.1-1.1 uM), or their vehicle was then administered to each to the method of Aharony et al. (14). This group has chamber and the responses were recorded for 120 min on a previously described the formation of the antibody that Grass model 7 oscillographic recorder with Grass FT-03 force crossreacts equally with LTC4, LTD4, and LTE4 but not with transducers. endoperoxides, or throm- Pulmonary Parenchymal Strips. Lung parenchymal strips , hydroperoxides, (5 mm x 2 cm) were mounted in water-jacketed organ baths boxanes. The lower limit of detection of this assay was 0.075 containing oxygenated (95% 02/5% CO2) KH solution at pmol/ml. 370C and put under a resting force of 1 g throughout the Statistics. All data in the text and figures are means ± experiment according to the method of Darius et al. (10). The SEM. Individual means were analyzed by analysis of vari- force was continuously monitored by Grass model FT03C ance and verified by Student's t test, with P < 0.05 accepted force transducers connected to a Grass model 7 oscillograph- as the level of significance. ic recorder as described (10). The KH solution was contin- uously gassed with 95% 02/5% CO2 in the tissue bath. After RESULTS an equilibration period of 60 min, an agonist (i.e., LxA4, LxB4, or U-46619) was added to the bathing fluid. Lung strips Effects of Lipoxins on Pulmonary Smooth Muscle. LxA4 and were observed until a peak constriction was obtained. After LxB4 consistently exerted a significant contraction in pul- each response, the lung strip was washed with KH solution monary parenchymal strips, signifying a bronchoconstrictor until tone returned to the original value. effect in small airways. Fig. 1 illustrates the magnitude and Platelet Aggregation. Human, rabbit, or guinea pig blood time course of the bronchoconstrictor effect of LxA4 and was drawn into 1/7th vol of acid/citrate/dextrose (2.5 g of LxB4 in the animal species studied (guinea pigs, rats, and trisodium citrate/1.5 g ofcitric acid/2.0 g ofglucose in 100 ml rabbits). There were significant differences among the pul- ofwater). Platelet-rich plasma was obtained by centrifugation monary strips from the three species in their responsiveness at 180 x g for 15 min at ambient temperature. The platelets to icosanoids. The thromboxane A2 (TxA2) mimetic U-46619 were isolated from the plasma by centrifugation at 800 x g was used as a reference agonist to stimulate pulmonary for 15 min and resuspended in a buffer consisting of 145 mM smooth muscle. As can be seen, this TxA2 mimetic at 28 nM NaCl, 5 mM KCI, 1 mM MgSO4, 0.5 mM NaH2PO4, 5 mM exerted a rapid and significant bronchoconstrictor effect. glucose, 10 mM Hepes (pH 7.4), hirudin (0.01 ,ug/ml), and Rabbit lung strips contracted -80 mg to U-46619, guinea pig apyrase (0.5 ng/ml). lung strips contracted -40 mg, and rat lung strips contracted Liver Lysosomal Membrane Leakiness. Rabbit liver lyso- -20 mg. In contrast to the rapid contraction induced by somes were prepared from liver homogenates (1:5) in 0.25 M U-46619, LxA4 exerted a much slower developing contrac- sucrose containing Tris buffer at pH 7.3 according to the tion requiring 10-15 min to reach its peak. Guinea pig lung method of Bridenbaugh et al. (11). Liver large granule responded best to LxA4, contracting -80 mg to 1.1 uM of fractions were incubated for 30 min at 37°C in the presence LxA4, whereas rat and rabbit pulmonary parenchymal strips of LxA4 (1.1 ,uM), LxB4 (1.1 uM), or their vehicle and were contracted 20-30 mg to 1.1 gM LxA4. assayed for cathepsin D activity according to the method of Anson (12). Pulmonary Parenchymal Strips Preparation ofthe Sodium Salt ofLipoxins. Chemically pure synthetic methyl LxA4 and LxB4 were prepared as described Guinea Pig (3, 4). Stock solutions were kept in benzene at - 78°C. A daily 100r50j aliquot of methyl lipoxin was placed in a test tube and 4r- w benzene was removed under a stream of nitrogen. In a U-46619 refrigerated room (0°C-40C), 20 ,l of tetrahydrofuran and LxA4 LxB4 distilled H20, and 4 equivalents of 1.0 M NaOH were added to the lipoxins. The reaction was followed by high-efficiency 1000 Rat thin-layer chromatography (HETLC) at 0°C-40C. The fluid ' 5 phase of the HETLC was methylene chloride in 5% metha- .2 nol. Complete removal ofthe methyl ester was achieved in 3- S U-46619 LxA4 LxB4 4 hr. 50 After removal of the methyl ester, the remaining tetrahy- Rabbit drofuran was removed under a stream of nitrogen. Physio- 100 logical saline (0.9% NaCI) was then added to the remaining 50 1 L _ _ _ _ lipoxin to make a working solution. The actual working -466w solution concentration was verified spectrophotometrically U-46619 LxA4 LxB4 at 301 nm with extinction coefficients of70,000 and 54,000 for FIG. 1. Typical pulmonary parenchymal response of the three LxA4 and LxB4, respectively. Spectrophotometric cell size species (i.e., guinea pig, rat, and rabbit) to a TxA2 mimetic (U-46619, was calculated to three significant figures with a potassium 28 nM). LxA4 (1.1 1uM) and LxB4 (0.6MuM). Arrows, lipoxin addition; chromate standard. The lipoxins demonstrated a linear ab- solid circles, U-46619 addition; W, washout. Downloaded by guest on September 30, 2021 8342 Physiological Sciences: Lefer et al. Proc. Natl. Acad. Sci. USA 85 (1988)

LxB4 at 0.6 ,M also exerted significant effects in pulmo- Guinea Pig Puhnonary Pwnchymal Strips nary smooth muscle. LxB4 exerted a biphasic response in guinea pig lung strips initially relaxing them and, after 8-10 200 min, the response reversed to a contraction that peaked -20 100 min after addition of the lipoxin to the bath. LxB4 also Propyl gallate exerted a significant contraction of rabbit lung strips but did 0 -W. I Is I LxA4 not contract rat lung strips. Thus, LxB4 (0.6 ,uM) exerts a E2a200 _ LxA4 stronger contraction than LxA4 (1.1 in both guinea pig I- 2Omi -4 ,AM) ToI and rabbit lung strips. Table 1 summarizes these results with IL0 1001 LY-17 1883 LxA4 and LxB4 in the pulmonary strips from the three 0 laboratory animal. species used in this study. In guinea pig w . .______lung strips, application of LxA4 or LxB4 up to three times did a* LxA4 LxA4 not result in loss of activity, whereas rabbit and rat strips 200 [ showed loss ofresponse on repetitive addition ofthe lipoxins, 100 BM-13505 and thus only one lipoxin response could be studied per lung W IL strip isolated from rabbit or rat. 0 _ _ In an attempt to ascertain the directness of the lipoxin LxA4 LxA, response in lung strips, additional experiments were con- ducted with icosanoid blockers and measurement ofpotential FIG. 2. Typical pulmonary parenchymal response to LxA4 (1.1 icosanoid release in the organ bath. Fig. 2 illustrates some of AuM) and various blockers in the guinea pig. Arrows, addition of these effects in guinea pig lung strips. The 5-lipoxygenase compounds; W, washout of compound.-Propyl gallate (a lipoxyge- inhibitor propyl gallate at 50 AM failed to block the LxA4 nase inhibitor, 47 ,uM), LY-171883 (a LTD4 antagonist, 8 AM), and contractile response. This suggests that no lipoxygenase BM-13505 (a TxA2 antagonist, 23 ,uM) were added 6-8 min before product mediates the LxA4 bronchoconstrictor response. LxA4. This is consistent with the failure to detect peptide leuko- Effect of Lipoxins on Vascular Smooth Muscle. LxA4 and trienes in the tissue bath at the peak of the LxA4 response. LxB4 exerted significant effects on isolated vascular smooth In seven strips, the LTC4 plus LTD4 plus LTE4 concentra- muscle, but only in the presence of an intact endothelium. tion was <0.075 pmol/ml, which is the limit of detection of Fig. 4 illustrates representative recordings of the lipoxin this radioimmunoassay. Nevertheless, LY-171883 (8 1AM), a responses in aortic rings isolated from guinea pigs, rats, and LTD4 receptor antagonist, did effectively block the LxA4 rabbits. Aortic rings from all three species contracted signif- response. Thus, LxA4 may activate LTD4 receptors in icantly to U-46619 (i.e., 600-1200 mg of force). Care was pulmonary smooth muscle cells. No attenuation of the LxA4 taken to preserve the integrity of the endothelium, and this bronchoconstrictor effect was observed when the TxA2 was tested by addition of 1 jLM acetylcholine. Rat and rabbit receptor antagonist BM-13505 (20 A.M) was added to the bath. aortic rings responded with a marked relaxation to acetyl- These results are consistent with the measurement ofonly 0.3 choline, indicating a significant release of EDRF. However, ± 0.1 pmol per ml of TxB2, the stable breakdown product of guinea pig aortic rings gave only a modest relaxation to TxA2, in the tissue bath at the peak of the response. In a few acetylcholine. This is not due to a weaker release of EDRF, experiments, a similar lack of thromboxane generation was but rather appears to be due to a lower density of muscarinic observed with LxB4. Thus, LxA4 and LxB4 do not appear to receptors, as shown by the finding that A-23187, a non- contract pulmonary smooth muscle by either releasing TxA2 receptor-mediated releaser of EDRF, produced a marked or activating its receptor on pulmonary smooth muscle cells. relaxation. A similar lack of attenuation of the lipoxin bronchoconstric- LxB4 was a slightly more potent relaxer of aortic smooth tor response at the peak of the LxA4 contraction was muscle than LxA4 in the guinea pig, whereas in the rat, the observed with the platelet-activating factor receptor antago- reverse occurred (i.e., LxA2 > LxB4). In the rabbit aorta, nist CV-6209. neither lipoxin exerted a significant vasorelaxant effect. The bronchoconstrictor response to LxA4 and LxB4 could Thus, the LxA4/LxB4 vasorelaxant ratio was 0.5 for guinea be part of the severe bronchoconstriction ofthe anaphylactic pigs, 1 for rabbits, and 4 for rats. These effects are tabulated shock response in guinea pigs responsible for the sudden death in those guinea pigs sensitized immunologically. We therefore studied the response of LxA4 pulmonary strips *-* Non-sensitized guinea pigs isolated from normal nonsensitized and ovalbumin-sensitized Sensitized guinea pigs guinea pigs. Fig. 3 illustrates these findings and shows that 601- there is no significant difference in the responsiveness of ovalbumin-sensitized guinea pig lung strips over the concen- EA0

tration response curve (0.1-1.1 ,uM) to LxA4. The response 0 appeared to be graded between 0.1 and 0.6 riM. 0. 40 0 Table 1. Bronchoconstrictor effects of LxA4 and LxB4 in pulmonary parenchymal strips 0 // / Contraction, mg 0 20 k Species LxA4 (1.1 ,UM) LxB4 (0.6 AtM) Rabbit 13 + 13 (6) 54 ± 21* (8) 80 ± 9t 63 ± 46 Guinea pig (7) (5) 0.6 1.1 ± t ± 0 0.1 Rat 25 (5) 16 6* (6) LxA4 Concentration (pM) Lung parenchyma were prepared as described in Methods. Num- bers in parentheses represent the number of strips studied. FIG. 3. Concentration curve to LxA4 in ovalbumin-sensitized *P < 0.05 from vehicle. and nonsensitized guinea pig lung parenchymal strips. Each point tP < 0.01 from vehicle. represents the mean of five to eight lung strip responses. Downloaded by guest on September 30, 2021 Physiological Sciences: Lefer et al. Proc. Natl. Acad. Sci. USA 85 (1988) 8343 Aortic Rings With Intact Endothehm DISCUSSION Guinea Pg The lipoxins are newly discovered substances, which have been synthesized only in recent years (3, 4). Very little is 500-OO ACh L.A. w w A-JX1.wLx8w -23187 known about the biological effects ofthese substances. LxA4 U-46619 has been found to induce neutrophils to generate superoxide

f 9 radicals, release elastase, and promote chemotaxis by leu- 1000 Rat kocytes (7). LxA4 also dilates hamster cheek pouch arterioles

. 5001 and contracts guinea pig lung strips without enhancing Veh vascular permeability (8). However, little is known about the .9LL O . wW Rabbt w directness of these pulmonary responses, including the en- aX*1 U-46619 1000r- Rabbit dothelial dependence of the vascular responses, or whether ACh LxA4 exerts platelet aggregatory effects. Moreover, virtually LxE:B, Veh nothing is known about the biological effects of LxB4 except that it inhibits natural killer cell In there 50 6 LxA9 W activity (1). addition, U-46619 are no published reports on species differences to lipoxins in terms of their smooth muscle responses. FIG. 4. Typical response to LxA4 (1.1 AM) and LxB4 (0.6 A.M) We have- shown that LxB4 has a very similar biological in guinea pig, rat, and rabbit aortic rings in the presence of an intact profile to LxA4 in that it (i) contracts pulmonary smooth endothelium. The thromboxane mimetic U-46619 (28 ,uM) was used muscle, (ii) dilates vascular smooth muscle, (iii) fails to to induce vascular tone. Acetylcholine (1 /LM) or A-23187 (960 pM) aggregate platelets, was used to test for the presence of an intact endothelium. W, or (iv) increases lysosomal membrane washout; solid circles and arrows, U-46619 and LxA4 or LxB4 permeability. These findings show that LxA4 and LxB4 have addition, respectively; Veh, vehicle for LxA4 and LxB4 (i.e., 0.9o a different biological profile from the classical bisenoic NaCl). prostaglandins D2 and F2,,, prostacyclin (PGI2), TxA2, as well as the nonpeptide and peptide leukotrienes. Moreover, these in Fig. 5 expressed as percentage relaxation from the peak of effects of LxA4 and LxB4 occur at concentrations at or <1 the U-46619 contraction. These findings illustrate the great gM, so that the lipoxins represent a different class of species variability in responsiveness of aortic rings to the icosanoids. Although the potency ofthe lipoxins is not as high lipoxins. as that of other lipoxygenase products (e.g., leukotrienes), Effect of Lipoxins on Platelet Aggregation. LxA4 or LxB4 we do not know the local concentration of these icosanoids from 0.1 to 25 ,uM was added to platelet suspensions in specific tissues. It remains to be shown whether the containing fibrinogen (160 /hg/ml) and stirred at 1000 rpm at lipoxins are released into biological fluids during disease 370C in an aggregometer. After 2 min, the platelet suspen- states, so that it is not yet known whether they are mediators sions were further challenged with 10,M ADP. Neither an of disease, as are many of the other icosanoids (e.g., TxA2, action of the lipoxins by themselves nor an inhibitory effect LTC4, LTD4). In on ADP-induced platelet aggregation could be detected. investigating the mechanism of the LxA4 and LxB4 effect, we have found that lipoxins do not release biologically Effect ofLipoxins on Liver Lysosomal Membrane Leakiness. significant LxA4 and LxB4 at 1.1,uM increased the rate of leakage of quantities of TxA2 or LTC4, LTD4, or LTE4 in producing its bronchoconstrictor effect in pulmonary paren- rabbit liver lysosomal suspensions over a 30-min incubation chymal strips. We arrived at this conclusion since blockade period. In the case of LxA4, the increase in cathepsin D of thromboxane or leukotriene synthesis with or release was 97% 20% (P < 0.02) in six rabbit livers, and ibuprofen propyl gallate, respectively, did not alter the lipoxin re- was + (P < for LxB4 the increase 101% 26% for five livers sponse, and no detectable levels of TxB2 or LTC4, LTD4, or 0.02). Thus, both lipoxins exert increases in lysosomal LTE4 were found in the tissue bath after addition of LxA4. membrane labilization, an effect that could aggravate cellular We also have established that LxA4 does not activate the injury in ischemic and shock states. thromboxane/endoperoxide receptor in pulmonary smooth muscle since BM-13505, a specific thromboxane receptor Aortic Rings antagonist (9, 15), failed to inhibit the LxA4 response in this tissue. However, the LTD4 receptor antagonist LY-171883 LxA4 LxB4 (16) markedly attenuated the LxA4 response in lung strips 100 despite the lack of blockade by a 5-lipoxygenase inhibitor in the same preparation. However, LY-171883 is also a phos- C 0 80- phodiesterase inhibitor at high concentrations (16). There- fore, we confirmed these findings with a second highly potent 'c and specific LTD4 receptor antagonist, SK&F 104353 (17) 60- and obtained the same results as with LY-171883. We think C these results can best be explained by the postulation that 240- LxA4 and LTD4 share a common receptor, perhaps in a manner analogous to prostaglandins F2. and H2 and TxA2 20- sharing the thromboxane/endoperoxide receptor (18). We have also found that calcium-channel blockers (e.g., nisol- Guinea Rat Rabbit Guinea Rat Rabbit dipine, nimodipine) and the platelet-activating factor recep- 5Pig Pig tor antagonist CV-6209 fail to reverse the bronchoconstrictor response of LxA4. These findings suggest that LxA4 does not FIG. 5. Summary of the vasorelaxant activities ofLxA4 and require calcium transport into cells to induce contraction of LxB4 in the aortic rings of the guinea pig, rat, and rabbit. Bars and pulmonary smooth muscle, but neither do or brackets represent means and SEM, respectively. Numbers in the LTC4 LTD4 bars represent number of vessels studied. Tone was induced by require extracellular calcium to stimulate these tissues (19). U-46619 (28 nM), which gave a peak constrictor response of 485 + In addition, a potent bronchoconstrictor (10, 20), platelet- 77, 531 + 64, and 1471 161 mg in guinea pig, rat, and rabbit aortic activating factor does not seem to mediate the LxA4 response rings, respectively. since CV-6209 failed to alter the LxA4 response. CV-6209 is Downloaded by guest on September 30, 2021 8344 Physiological Sciences: Lefer et al. Proc. Natl. Acad. Sci. USA 85 (1988) a very specific platelet-activating factor receptor antagonist, We gratefully acknowledge the expert technical assistance of which does not block histamine, leukotrienes, or acetylcho- Judith Komlosh and Donna Mulloy during the course of these line (21). In addition, we have shown that prior sensitization investigations. This study was supported by Research Grant HL- 25575 from the National Heart, Lung and Blood Institute of the ofguinea pig pulmonary smooth muscle with ovalbumin does National Institutes of Health. G.L.S. is a National Institutes of not sensitize these cells to LxA4. Although these findings are Health Predoctoral Trainee HL-05799. M.E.B. was supported by the far from conclusive, they do not point to a major role of the Foerderer Foundation. lipoxins in mediating the pulmonary contraction observed in anaphylactic shock in guinea pigs. 1. Serhan, C. N., Hamberg, M. & Samuelsson, B. (1984) Proc. The Natl. Acad. Sci. USA 81, 5335-5339. vascular smooth muscle relaxation in response to 2. Serhan, C. N., Hamberg, M., Samuelsson, B., Morris, J. & LxA4 and LxB4 appear to be fully dependent on the release Wishka, D. G. (1986) Proc. Natl. Acad. Sci. USA 83, 1983- of EDRF, which has recently been identified as nitric oxide 1987. (22, 23). This endothelium-dependent vasodilation is in con- 3. Nicolaou, K. C., Veale, C. A., Webber, S. E. & Katerino- trast to the non-endothelium-dependent dilation produced by poulos, H. (1985) J. Am. Chem. Soc. 107, 7515-7518. other icosanoids (e.g., prostaglandin I2) (24). Previous stud- 4. Nicolaou, K. C. & Webber, S. E. (1986) Synthesis 6, 453-461. ies in vivo 5. Samuelsson, B., Dahlen, S.-E., Lindgren, J. A., Rouzer, C. A. using lipoxins suggested that LxA4 is an endothe- & Serhan, C. N. (1987) Science 237, 1171-1176. lium-dependent vasodilator in the hamster, but the role of 6. Hansson, A., Serhan, C. N., Haeggstrom, J., Ingelman-Sund- released blood-borne vasodilators (e.g., prostaglandin 12) berg, M., Samuelsson, B. & Morris, J. (1986) Biochem. mediating this dilation cannot be ruled out (8). The extent of Biophys. Res. Commun. 134, 1215-1222. the vasorelaxation induced by LxA4 and LxB4 is quite 7. Serhan, C. N., Hamberg, M. & Samuelsson, B. (1985) in considerable and would be expected to produce a marked Prostaglandins, Leukotrienes and Lipoxins, ed. Bailey, J. M. vasodilation if a comparable degree of vasorelaxation oc- (Plenum, New York), pp. 3-16. curred systemically in vivo. This is consistent with the 8. Dahlen, S.-E., Raud, J., Serhan, C. N., Bjork, J. & Samuels- son, B. (1987) Acra Physiol. Scand. 130, 643-647. vasodilation observed by Dahlen et al. (8) in the small 9. -Yanagisawa, A., Smith, J. B., Brezinski, M. E. & Lefer, A. M. arterioles of the hamster cheek pouch and by Badr et al. (25) (1987) Eur. J. Pharmacol. 133, 89-96. in rat renal arteries. Thus, LxA4 appears to dilate both true 10. Darius, H., Lefer, D. J., Smith, J. B. & Lefer, A. M. (1986) resistance vessels as well as large conduit vessels as long as Science 232, 58-60. the endothelium is intact. In our aortic rings, no significant 11. Bridenbaugh, G. A., Flynn, J. T. & Lefer, A. M. (1976) Am. J. vasorelaxation occurred in response to LxA4 or LxB4 when Physiol. 231, 112-119. the endothelium was stripped from the arterial preparation. 12. Anson, M. L. (1936) J. Gen. Physiol. 20, 565-574. 13. Ingerman-Wojenski, C., Silver, M. J., Smith, J. B. & Macarak, LxA4 and LxB4 have been previously shown to exert E. (1981) J. Clin. Invest. 67, 1292-1296. biological activity in smooth muscle preparations in humans, 14. Aharony, D., Dobson, P., Bernstein, P. R., Dusner, E. J., guinea pig (8), and hamster (8), and we have shown effects in Krell, M. D. & Smith, J. B. (1983) Biochem. Biophys. Res. rabbit and rat (i.e., aortic rings). Thus, lipoxins exert effects Commun. 117, 574-579. in a wide variety oflaboratory mammals. However, there are 15. Stegmeier, K., Pill, J. & Patscheke, H. (1986) Naunyn- marked species differences in their responsiveness to LxA4 Schmiedeberg's Arch. Pharmacol. 332, 144. and LxB4. In general, the guinea pig exhibits large responses 16. Fleisch, J. H., Rinkema, L. E., Haisch, K. D., Swansonbean, D., Goodson, T., Ho, P. P. K. & Marshall, W. S. (1985) J. to both LxA4 and LxB4, whereas the rat responds only Pharmacol. Exp. Ther. 233, 148-157. slightly, and the rabbit is intermediate. We are unaware of 17. Hay, D. W. P., Muccitelli, R. M., Tucker, S. S., Vickery- any physiological basis for these species differences at the Clark, L. M., Wilson, K. A., Gleason, J. G., Hall, R. F., present time. Moreover, the lipoxins are much less potent Wasserman, M. A. & Torphy, T. J. (1987) J. Pharmacol. Exp. than the peptide leukotrienes (e.g., LTC4, LTD4) in terms of Ther. 243, 474-481. their pulmonary constrictor effects (26). 18. Halushka, P. V., Mais, D. E. & Saussy, D. L., Jr. (1987) Fed. In summary, both LxA4 and LxB4 exert marked biological Proc. Fed. Am. Soc. Exp. Biol. 46, 149-153. 4 19. Lefer, A. M., Lepran, I., Roth, D. M. & Smith, J. B. (1984) effects at concentrations of ,uM in rat, rabbit, and guinea Pharmacol. Res. Commun. 16, 1141-1150. pig smooth muscle. Both LxA4 and LxB4 contract pulmonary 20. Braquet, P., Touqui, L., Shen, T. Y. & Vargaftig, B. B. (1987) parenchymal strips and relax aortic smooth muscle in all Pharmacol. Rev. 39, 97-145. species studied. 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Commun. 145, 408-414. more responsive to LxA4 and LxB4 than rat or rabbit tissue. 26. Samuelsson, B. (1983) Science 220, 568-575. Downloaded by guest on September 30, 2021