Charles Brink

The Lipoxin Receptor ALX: Potent Ligand-Specific and Stereoselective Actions in Vivo

Lipoxins (LXs) and aspirin-triggered LX (ATL) are trihydroxytetraene-containing eicosanoids generated from arachidonic acid that are distinct in structure, formation, and function from the many other proinflammatory lipid-derived mediators. These endogenous eicosanoids have now emerged as founding members of the first class of lipid/chemical mediators involved in the resolution of the inflammatory response. Lipoxin A4 (LXA4), ATL, and their metabolic stable analogs elicit cellular responses and regulate leukocyte trafficking in vivo by activating the specific receptor, ALX. ALX was the first receptor cloned and identified as a G protein-coupled receptor (GPCR) for lipoxygenase-derived eicosanoids with demonstrated cell type-specific signaling pathways. ALX at the level of DNA has sequence homology to the N-formylpeptide receptor and as an orphan GPCR was initially referred to as the N-formylpeptide receptor-like 1. Although LXA4 is the endogenous potent ligand for ALX activation, a number of peptides can also activate this receptor to stimulate calcium mobilization and chemotaxis in vitro. In contrast with LXA4, the counterparts of many of these peptides in vivo remain to be established. The purpose of this review is to highlight the molecular characterization of the ALX receptor and provide an overview of the ALX-LXA4 axis responsible for anti-inflammatory and proresolving signals in vivo. The information in this review provides further support for the initial nomenclature proposition for this GPCR as ALX.

International Union of Pharmacology XXXVII. Nomenclature for Leukotriene and Lipoxin Receptors

The leukotrienes and lipoxins are biologically active metabolites derived from arachidonic acid. Their diverse and potent actions are associated with specific receptors. Recent molecular techniques have established the nucleotide and amino acid sequences and confirmed the evidence that suggested the existence of different G-protein-coupled receptors for these lipid mediators. The nomenclature for these receptors has now been established for the leukotrienes. BLT receptors are activated by leukotriene B4 and related hydroxyacids and this class of receptors can be subdivided into BLT1 and BLT2. The cysteinyl-leukotrienes (LT) activate another group called CysLT receptors, which are referred to as CysLT1 and CysLT2. A provisional nomenclature for the lipoxin receptor has also been proposed. LXA4 and LXB4 activate the ALX receptor and LXB4 may also activate another putative receptor. However this latter receptor has not been cloned. The aim of this review is to provide the molecular evidence as well as the properties and significance of the leukotriene and lipoxin receptors, which has lead to the present nomenclature.

Prostanoid receptors involved in the relaxation of human pulmonary vessels

To characterize the prostanoid receptors on human pulmonary smooth muscle involved in vasodilatations, isolated arteries and veins were contracted with norepinephrine (10 μM) and vessels were subsequently challenged with different prostanoid‐receptor agonists in the absence or presence of selective antagonists. Prostaglandin D2 (PGD2) and the selective DP‐receptor agonist, BW245C, induced relaxations in the contracted human pulmonary venous preparations. The pD2 values were: 6.88±0.11 (n=17) and 7.31±0.12 (n=5), respectively. The relaxant responses induced by PGD2 were reduced by the selective DP‐receptor antagonist, BWA868C, and the estimated pA2 value was 7.84±0.16 (n=4). PGD2 and BW245C did not relax contracted human pulmonary arteries. The selective IP‐receptor agonists, iloprost and cicaprost, both induced relaxations in the contracted human vascular preparations. The pD2 values for iloprost were: 7.84±0.08 (n=6) and 8.25±0.06 (n=4) and for cicaprost: 8.06±0.12 (n=5) and 8.11±0.09 (n=5) in arteries and veins respectively. Prostaglandin E2 (PGE2) and the EP2/EP3‐receptor agonist, misoprostol, partially relaxed the contracted venous preparations and the pD2 values were: 8.10±0.15 (n=15) and 6.24±0.33 (n=3), respectively. These relaxations suggest the presence of an EP receptor in the human pulmonary veins. The contracted human pulmonary arteries did not relax when challenged with PGE2. In human pulmonary venous preparations, the PGE2‐induced relaxations were neither modified by treatment with TP/EP4‐receptor antagonist, AH23848B (10 and 30 μM, n=6), nor by the DP/EP1/EP2‐receptor antagonist, AH6809 (3 μM, n=6). These data suggest that the relaxation induced by prostanoids involved DP‐, IP‐receptors and to a lesser extent an EP‐receptor on human pulmonary venous smooth muscle. In contrast, only the IP‐receptor is involved in the prostanoid induced relaxations on human pulmonary arterial smooth muscle.

The actions of Paf‐acether (platelet‐activating factor) on guinea‐pig isolated heart preparations

1 Paf‐acether (platelet‐activating factor) is a phospholipid capable of stimulating platelets to release their granular contents and cause platelet aggregation. When Paf‐acether was administered to isolated heart preparations from normal guinea‐pigs there was a significant concentration‐dependent reduction in coronary flow and contractile force. The high concentration of Paf‐acether was equally effective in reducing these cardiac parameters in the presence of atropine. 2 The non‐acetylated Paf‐acether analogue, 2‐lyso Paf‐acether, the enantiomer, and a closely related phospholipid 1,α‐lysophosphatidylcholine palmitoyl, did not affect coronary flow and contractile force, indicating the specificity of Paf‐acether. 3 These data demonstrate a potent effect of Paf‐acether on cardiac function. Whether or not these effects are direct or mediated through generation of endogenous mediators remains to be established.

Prostanoid receptors involved in the relaxation of human bronchial preparations.

Iloprost and cicaprost (IP‐receptor agonists) induced relaxations in the histamine‐ (50 μM) contracted human bronchial preparations (pD2 values, 6.63±0.12 and 6.86±0.08; Emax values, 90±04 and 65±08% of the papaverine response for iloprost (n=6) and cicaprost (n=3), respectively). Prostaglandin E2 (PGE2) and misoprostol (EP‐receptor agonist) relaxed the histamine‐contracted human bronchial preparations (pD2 values, 7.13±0.07 and 6.33±0.28; Emax values, 67±04 and 57±08% of the papaverine response for PGE2 (n=14) and misoprostol (n=4), respectively). In addition, both relaxations were inhibited by AH6809 (DP/EP1/EP2‐receptor antagonist; 3 μM; n=5–6). The PGE2‐induced relaxations of human bronchial preparations were not modified by treatment with AH23848B (TP/EP4‐receptor antagonist; 30 μM; n=4). The contracted human bronchial preparations were significantly relaxed by prostaglandin D2 (PGD2) or by BW245C a DP‐receptor agonist. However, these responses did not exceed 40% of the relaxation induced by papaverine. In addition, the relaxations induced by PGD2 were significantly inhibited by treatment with a DP‐receptor antagonist BWA868C (0.1 μM; n=3). These data suggest that the relaxation of human isolated bronchial preparations induced by prostanoids involved IP‐, EP2‐ and to a lesser extent DP‐receptors but not EP4‐receptor.

THE INTERACTION BETWEEN INDOMETHACIN AND CONTRACTILE AGENTS ON HUMAN ISOLATED AIRWAY MUSCLE

1 Concentration‐effect curves to acetylcholine and histamine were produced in fresh human bronchial muscle (2 to 4 h after removal from the patients) and in preparations previously stored at 4°C for 12 h. 2 Sensitivities of fresh human airway muscle preparations to acetylcholine (pD2 value, 5.89 ± 0.03); n = 4) and histamine (pD2 value, 5.41 ± 0.03; n = 13) were similar. There was no significant difference in the sensitivities of stored preparations (acetylcholine: pD2 value, 5.70 ± 0.06; n = 23 and histamine: pD2 value, 5.44 ± 0.07; n = 16) when compared to the fresh preparations. 3 Indomethacin did not significantly change the basal tone in preparations of either fresh or stored human airway muscle. 4 A low concentration of indomethacin (0.17 μm) significantly reduced responsiveness and sensitivity to histamine in stored bronchi but not in fresh bronchi. The acetylcholine concentration‐effect curves were unaltered by exposure to this concentration of indomethacin in either fresh or stored tissues. High concentrations (1.7 μm and 17 μm) depressed the maximal responsiveness of the bronchi to both agonists. 5 These results suggest indirectly that the regulatory role of prostaglandins in human airway muscle may be different from that in other species.

Prostanoid EP1- and TP-receptors involved in the contraction of human pulmonary veins

To characterize the prostanoid receptors (TP, FP, EP1 and/or EP3) involved in the vasoconstriction of human pulmonary veins, isolated venous preparations were challenged with different prostanoid‐receptor agonists in the absence or presence of selective antagonists. The stable thromboxane A2 mimetic, U46619, was a potent constrictor agonist on human pulmonary veins (pEC50=8.60±0.11 and Emax=4.61±0.46 g; n=15). The affinity values for two selective TP‐antagonists (BAY u3405 and GR32191B) versus U46619 were BAY u3405: pA2=8.94±0.23 (n=3) and GR32191B: apparent pKB=8.25±0.34 (n=3), respectively. These results are consistent with the involvement of TP‐receptor in the U46619 induced contractions. The two EP1‐/EP3‐ agonists (17‐phenyl‐PGE2 and sulprostone) induced contraction of human pumonary veins (pEC50=8.56±0.18; Emax=0.56±0.24 g; n=5 and pEC50=7.65±0.13; Emax=1.10±0.12 g; n=14, respectively). The potency ranking for these agonists: 17‐phenyl‐PGE2>sulprostone suggests the involvement of an EP1‐receptor rather than EP3. In addition, the contractions induced by sulprostone, 17‐phenyl‐PGE2 and the IP‐/EP1‐ agonist (iloprost) were blocked by the DP‐/EP1‐/EP2‐receptor antagonist (AH6809) as well as by the EP1 antagonist (SC19220). PGF2α induced small contractions which were blocked by AH6809 while fluprostenol was ineffective. These results indicate that FP‐receptors are not implicated in the contraction of human pulmonary veins. These data suggest that the contractions induced by prostanoids involved TP‐ and EP1‐receptors in human pulmonary venous smooth muscle.

M1 and M3 muscarinic receptors in human pulmonary arteries

1 Acetylcholine (ACh) and the M1 agonists (McN‐A‐343 or PD142505) relaxed human isolated pulmonary arteries which were pre‐contracted with noradrenaline (10 μm). In preparations where the endothelium had been removed ACh induced a contractile response whereas the M1 agonists (McN‐A‐343 or PD142505) had no effect. 2 ACh‐ and McN‐A‐343‐induced relaxations were abolished after treatment of endothelium‐intact preparations with the drug combination NG‐nitro‐L‐arginine (L‐NOARG: 0.1 mM) and indomethacin (1.7 μm). 3 The affinity (pKB value) for pirenzepine was higher in human pulmonary arteries when tissues were relaxed with McN‐A‐343 as compared with ACh (pKB values, 7.71 ± 0.30 (n = 4) and 6.68 ± 0.15 (n = 8), respectively). In addition, the affinity for pFHHSiD against McN‐A‐343‐ and ACh‐induced relaxations was 6.86 ± 0.13 (n = 3) and 7.35 ± 0.11 (n = 9), respectively. 4 The low affinities for methoctramine in human isolated pulmonary arteries with the endothelium either intact or removed, suggested the lack of involvement of M2 and M4 receptors in the ACh responses. 5 Phenoxybenzamine (3 μm: 30 min) abolished both ACh contraction and relaxation in human pulmonary artery. The ACh contraction was present when the phenoxybenzamine treatment was preceded by incubation with pFHHSiD (2 μm) but not with pirenzepine (1 μm). In addition, the ACh relaxation was present when preparations were treated with either pFHHSiD (2 μm) or pirenzepine (1 μm), before exposure to phenoxybenzamine. 6 These results in human isolated pulmonary arteries support the notion that only M3 receptors, on smooth muscle, mediate the ACh‐induced contraction whereas M3 and M1 receptors are involved in the endothelium‐dependent ACh‐induced relaxation.

Evidence that the histamine sensitivity and responsiveness of guinea-pig isolated trachea are modulated by epithelial prostaglandin E2 production.

1 Guinea‐pig isolated tracheal preparations in which the epithelium had been removed exhibited a greater contractile response to histamine (intact: 1.91 ± 0.12g; n = 6 and rubbed: 2.76 ± 0.15g; n = 11; P < 0.001). The histamine sensitivity (pD2 value) of these preparations was also significantly greater (intact: 4.80 ± 0.04 and rubbed: 5.40 ± 0.08; P < 0.01). 2 Indomethacin suppressed the basal tone of both intact and rubbed preparations but was more effective in the former tissues (intact: −0.70 ± 0.14g; n = 22 and rubbed: −0.17 ± 0.05g; n = 12; P < 0.02). 3 Arachidonic acid (AA; 10 μM) suppressed the basal tone of intact tissues but contracted such preparations following indomethacin treatment (1.7 μM; 30 min). However, in rubbed tissues AA (10 μM) induced a contraction which was attenuated following indomethacin treatment. 4 Prostaglandin E2 (PGE2; 0.01 and 0.1 μM) suppressed the basal tone of intact preparations and always evoked contraction of rubbed tissues. Following indomethacin treatment PGE2 (0.01 and 0.1 μM) generally evoked spasm of intact and rubbed tissues while at higher concentrations (1 μM) relaxant effects were observed. 5 Removal of the epithelium did not alter the relaxant effect of PGE2 (pD2 value) on histamine (50 μM)‐contracted tissues (intact: 6.86 ± 0.08 and rubbed: 7.10 ± 0.3; n = 4; P > 0.1). 6 In rubbed preparations treated with indomethacin, PGE2 (0.01 and 0.1 μM) evoked spasm. However, when added to preparations contracted with 5 μM histamine, PGE2 always caused relaxation. 7 The release of immunoreactive PGE2 by rubbed preparations during histamine and/or AA stimulation was significantly less than that produced by intact stimulated tissues. 8 Exogenous PGE2 (0.01‐1 μM) decreased the maximal response and sensitivity of rubbed tracheal preparations to histamine. 9 These results suggest that release of an epithelial derived cyclo‐oxygenase product, namely PGE2, may regulate basal tone, histamine response and sensitivity of the guinea‐pig isolated trachea.

Leukotriene receptors on human pulmonary vascular endothelium.

1 Cysteinyl‐leukotrienes cause contractions and/or relaxations of human isolated pulmonary vascular preparations. Although, the localization and nature of the receptors through which these effects are mediated have not been fully characterized, some effects are indirect and not mediated via the well‐ described LT1 receptor. 2 In human pulmonary veins (HPV) with an intact endothelium, leukotriene D4 (LTD4) induced contraction above basal tone. This response was observed at lower concentrations of LTD4 in the presence of nitric oxide synthase inhibitor Nω‐nitro‐L‐arginine (L‐NOARG). Contractions (in the absence and presence of L‐NOARG) were partially blocked by the LT1 antagonists (MK 571 and ICI 198615). 3 LTD4 relaxed HPV previously contracted with noradrenaline. This relaxation was potentiated by LT1 antagonists, but was abolished by removal of the endothelium. LTD4 also relaxed human pulmonary arteries (HPA) precontracted with noradrenaline but this effect was not modified by LT1 antagonists. 4 The results suggest that contraction of endothelium‐intact HPV by LTD4 is partially mediated via LT1 receptors. Further, in endothelium‐intact HPV, this contraction was opposed by a relaxation induced by LTD4, dependent on the release of nitric oxide, which was mediated, at least in part, via a non‐LTi receptor. In addition, LTD4 relaxation on contracted HPA was not mediated by LT1 receptors. 5 The mechanical effects of LTD4 on human pulmonary vasculature are complex and involve both direct and indirect mechanisms mediated via at least two types of cysteinyl‐leukotriene receptors.