Lipoxins A4 and B4
Total Page：16
File Type：pdf, Size：1020Kb
Proc. Nati. Acad. Sci. USA Vol. 85, pp. 8340-8344, November 1988 Physiological Sciences Lipoxins A4 and B4: Comparison of icosanoids having bronchoconstrictor and vasodilator actions but lacking platelet aggregatory activity (pulmonary parenchymal strips/vascular smooth muscle/platelet-rich plasma/leukotriene receptors/thromboxane 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 arachidonic acid 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 leukotrienes or thromboxane A2, 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 platelets. 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 lipoxin 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 prostaglandin 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 prostaglandins, 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).