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Nicotinic acid (NA) is commonly used to treat dyslipidemia, but it elicits an adverse effect, termed flushing, which consists of cutaneous vasodilation with associated discomfort. An animal model of NA-induced flushing has been established in mice. As in humans, NA stimulated vasodilation in a dose-dependent manner, was associated with an increase of the vasodilatory prostaglandin (PG) D 2 in plasma and could be blocked by pretreatment with aspirin. Two PGD 2 receptors have been identified: PGD 2 receptor 1 (DP1, also called DP) and PGD 2 receptor 2 (DP2, sometimes termed CRTH2). DP2 does not mediate NA-induced vasodilation; the DP2-specific agonist DK-PGD 2 (13,14-dihydro-15-keto-PGD 2 ) did not induce cutaneous vasodilation, and DP2 −/− mice had a normal vasodilatory response to NA. By contrast, BW245C, a DP1-selective agonist, induced vasodilation in mice, and MK-0524, a DP1-selective antagonist, blocked both PGD 2 - and NA-induced vasodilation. NA-induced vasodilation was also studied in DP1 +/+ , DP1 +/− , and DP1 −/− mice; although NA-induced vasodilation depended almost completely on DP1 in female mice, it depended only partially on DP1 in male mice. The residual NA-induced vasodilation in male DP −/− mice was aspirin-sensitive. Thus, in the mouse, DP1 appears to be an important component involved in NA-induced vasodilation, but other cyclooxygenase-dependent mechanisms also may be involved. A clinical study in healthy men and women demonstrated that treatment with MK-0524 reduced the symptoms of flushing and the increase in skin perfusion after the administration of NA. These studies suggest that DP1 receptor antagonism may be an effective means to suppress NA-induced flushing in humans.

1. The recombinant human prostaglandin D(2) (PGD(2)) receptor, hCRTH2, has been expressed in HEK293(EBNA) and characterized with respect to radioligand binding and signal transduction properties. High and low affinity binding sites for PGD(2) were identified in the CRTH2 receptor population by saturation analysis with respective equilibrium dissociation constants (K(D)) of 2.5 and 109 nM. This revealed that the affinity of PGD(2) for CRTH2 is eight times less than its affinity for the DP receptor. 2. Equilibrium competition binding assays revealed that of the compounds tested, only PGD(2) and several related metabolites bound with high affinity to CRTH2 (K(i) values ranging from 2.4 to 34.0 nM) with the following rank order of potency: PGD(2)>13,14-dihydro-15-keto PGD(2)>15-deoxy-Delta(12,14)-PGJ(2)>PGJ(2)>Delta(12)-PGJ(2)>15(S)-15 methyl-PGD(2). This is in sharp contrast with the rank order of potency obtained at DP : PGD(2)>PGJ(2)>Delta(12)-PGJ(2)>15-deoxy-Delta(12,14)-PGJ(2) >>>13,14-dihydro-15-keto-PGD(2). 3. Functional studies demonstrated that PGD(2) activation of recombinant CRTH2 results in decrease of intracellular cAMP in a pertussis toxin-sensitive manner. Therefore, we showed that CRTH2 can functionally couple to the G-protein G(alphai/o). PGD(2) and related metabolites were tested and their rank order of potency followed the results of the membrane binding assay. 4. By Northern blot analysis, we showed that, besides haemopoietic cells, CRTH2 is expressed in many other tissues such as brain, heart, thymus, spleen and various tissues of the digestive system. In addition, in situ hybridization studies revealed that CRTH2 mRNA is expressed in human eosinophils. Finally, radioligand binding studies demonstrated that two eosinophilic cell lines, butyric acid-differentiated HL-60 and AML 14.3D10, also endogenously express CRTH2.

This study demonstrates the localization of the prostaglandin (PG)D2 receptor (DP) within the mucous-secreting globlet cells of the human colon by in situ hybridization, which suggests a role for DP in mucous secretion. Selective high affinity ligands were used, therefore, to evaluate DP regulation of mucous secretion in LS174T human colonic adenocarcinoma cells. The expression of hDP in LS174T cells was confirmed at the mRNA level by reverse transcriptase-polymerase chain reaction, and at the protein level by radioligand binding assays and signal transduction (cyclic AMP accumulation) assays. PGD2 and the highly selective DP-specific agonist L-644,698 ((4-(3-(3-(3-hydroxyoctyl)-4-oxo-2-thiazolidinyl) propyl) benzoic acid) (racemate)), but not PGE2 competed for [3H]-PGD2-specific binding to LS174T cell membranes (Ki values of 0.4 nM and 7 nM, respectively). The DP-specific agonists PGD2, PGJ2, BW245C (5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropylhydantoin)), and L-644,698 showed similar potencies in stimulating cyclic AMP accumulation (EC50 values: 45–90 nM) and demonstrated the expected rank order of potency. PGE2 also elicited cyclic AMP production in this cell line (EC50 value: 162 nM). The activation of cyclic AMP production by PGD2 and L-644,698, but not PGE2, was inhibited by the selective DP antagonist BW A868C. Thus, PGD2 and L-644,698 act through hDP in LS174T cells. PGD2, L-644,698 and PGE2 (an established mucin secretagogue) potently stimulated mucin secretion in LS174T cells in a concentration-dependent manner (EC50

The contractile and inflammatory actions of the cysteinyl leukotrienes (CysLTs), LTC(4), LTD(4), and LTE(4), are thought to be mediated through at least two distinct but related CysLT G protein-coupled receptors. The human CysLT(1) receptor has been recently cloned and characterized. We describe here the cloning and characterization of the second cysteinyl leukotriene receptor, CysLT(2), a 346-amino acid protein with 38% amino acid identity to the CysLT(1) receptor. The recombinant human CysLT(2) receptor was expressed in Xenopus oocytes and HEK293T cells and shown to couple to elevation of intracellular calcium when activated by LTC(4), LTD(4), or LTE(4). Analyses of radiolabeled LTD(4) binding to the recombinant CysLT(2) receptor demonstrated high affinity binding and a rank order of potency for competition of LTC(4) = LTD(4) LTE(4). In contrast to the dual CysLT(1)/CysLT(2) antagonist, BAY u9773, the CysLT(1) receptor-selective antagonists MK-571, montelukast (Singulair(TM)), zafirlukast (Accolate(TM)), and pranlukast (Onon(TM)) exhibited low potency in competition for LTD(4) binding and as antagonists of CysLT(2) receptor signaling. CysLT(2) receptor mRNA was detected in lung macrophages and airway smooth muscle, cardiac Purkinje cells, adrenal medulla cells, peripheral blood leukocytes, and brain, and the receptor gene was mapped to chromosome 13q14, a region linked to atopic asthma.

1 We characterized the regulation of cyclooxygenase-2 (COX-2) at the mRNA, protein and mediator level in two rat models of acute inflammation, carrageenan-induced paw oedema and mechanical hyperalgesia. 2 Carrageenan was injected in the hind paw of rat at low (paw oedema) and high doses (hyperalgesia). COX-2 and prostaglandin E2 (PGE2) levels were measured by RT-PCR and immunological assays. We also determined the distribution of COX-2 by immunohistochemistry. 3 The injection of carrageenan produced a significant and parallel induction of both COX-2 and PGE2. This induction was significantly higher in hyperalgesia than in paw oedema. This was probably due to the 9 fold higher concentration of carrageenan used to provoke hyperalgesia. 4 Immunohistochemical examination showed COX-2 immunoreactivity in the epidermis, skeletal muscle and inflammatory cells of rats experiencing hyperalgesia. In paw oedema however, only the epidermis showed positive COX-2 immunoreactivity. 5 Pretreatment with indomethacin completely abolished the induction of COX-2 in paw oedema but not in hyperalgesia. 6 These results suggest that multiple mechanisms regulate COX-2 induction especially in the more severe model. In carrageenan-induced paw oedema, prostanoid production have been linked through the expression of the COX-2 gene which suggest the presence of a positive feedback loop mechanism.

1. A human embryonic kidney cell line [HEK 293(EBNA)] stably expressing the human recombinant prostaglandin D2 (PGD2) receptor (hDP) has been characterized with respect to radioligand binding and signal transduction properties by use of prostanoids and prostanoid analogues. Radioligand binding studies included saturation analyses, the effects of nucleotide analogues, the initial rate of ligand-receptor association and equilibrium competition assays. In addition, adenosine 3':5'-cyclic monophosphate (cyclic AMP) generation in response to ligand challenge was also measured, as this is the predominant hDP signalling pathway. 2. L-644,698 ((4-(3-(3-(3-hydroxyoctyl)-4-oxo-2-thiazolidinyl) propyl) benzoic acid) (racemate)) was identified as a novel ligand having high affinity for hDP with an inhibitor constant (Ki) of 0.9 nM. This Ki value was comparable to the Ki values obtained in this study for ligands that have previously shown high affinity for DP: PGD2 (0.6 nM), ZK 110841 (0.3 nM), BW245C (0.4 nM), and BW A868C (2.3 nM). 3. L-644,698 was found to be a full agonist with an EC50 value of 0.5 nM in generating cyclic AMP following activation of hDP. L-644,698 is, therefore, comparable to those agonists with known efficacy at the DP receptor (EC50): PGD2 (0.5 nM), ZK 110841 (0.2 nM) and BW245C (0.3 nM). 4. L-644,698 displayed a high degree of selectivity for hDP when compared to the family of cloned human prostanoid receptors: EP1 (> 25,400 fold), EP2 (approximately 300 fold), EP3-III (approximately 4100 fold), EP4 (approximately 10000 fold), FP (> 25,400 fold), IP (> 25,400 fold) and TP (> 25,400 fold). L-644,698 is, therefore, one of the most selective DP agonists as yet described. 5. PGJ2 and delta12-PGJ2, two endogenous metabolites of PGD2, were also tested in this system and shown to be effective agonists with Ki and EC50 values in the nanomolar range for both compounds. In particular, PGJ2 was equipotent to known DP specific agonists with a Ki value of 0.9 nM and an EC50 value of 1.2 nM.

A cDNA encoding a functional human prostanoid DP (hDP) receptor has been constructed from a genomic clone and a fragment cloned by 3′-rapid amplification of cDNA ends-polymerase chain reaction. The hDP receptor consists of 359 amino acid residues with a predicted molecular mass of 40,276 and has the putative heptahelical transmembrane domains characteristic of G-protein-coupled receptors. The deduced amino acid sequence of the hDP receptor, when compared with all other members of the prostanoid receptor family, shows the highest degree of identity with the hIP and hEP2 receptors, followed by the hEP4 receptor. Radioreceptor binding studies using membranes prepared from mammalian COS-M6 cells transiently transfected with an expression vector containing the DP receptor cDNA showed that the rank order of affinities for prostaglandins and prostaglandin analogs, in competition for [3H]prostaglandin D2 (PGD2) specific binding sites, was as predicted for the DP receptor, with PGD2 PGE2 > PGF2ɑ = iloprost > U46619. The signal transduction pathway of the cloned hDP receptor was studied by transfecting the hDP expression vector into HEK 293(EBNA) cells. Activation of the hDP receptor with PGD2 resulted in an elevation of intracellular cAMP and in mobilization of Ca2+, but did not lead to generation of inositol 1,4,5-trisphosphate. Northern blot analysis of human tissues showed that the hDP receptor has a very discrete tissue distribution and was detectable only in retina and small intestine. In summary, we have cloned and expressed a functional cDNA for the hDP receptor.

Dimethyl sulfoxide-differentiated U937 (dU937) cells express high affinity G-protein-coupled receptors for leukotriene (LT)D4 and LTB4 and, as described here, specific binding sites for LTC4. The specific binding of [3H]LTC4 was of low affinity (KD = 26 nM) and high abundance (Bmax = 33 pmol/mg of protein), as compared to LTD4 and LTB4 receptors. In addition, although [3H]LTC4 specific binding was enhanced by divalent cations, it was not inhibited by nonhydrolyzable GTP analogs. [3H]LTC4 specific binding to dU937 cell membranes does not have, therefore, the characteristics of binding to a G-protein-coupled receptor. Competition for [3H]LTC4 specific binding to dU937 cell membranes by leukotrienes and related analogs, including N-methylated LTC4, as well as glutathione, suggested a dependence on the presence of an arachidonic acid backbone, although varying degrees of saturation were well tolerated, and that the glutathione moiety of LTC4 in particular was important in determining affinity. The possibility that [3H]LTC4 specific binding was to a member of the glutathione S-transferase (GST) family of enzymes, such as LTC4 synthase, cytosolic GST, or microsomal GST, was therefore investigated. [3H]LTC4 specific binding sites could be separated from LTC4 synthase and cytosolic GSTs by differential detergent solubilization, but cofractionated with microsomal GST during solubilization and subsequent anion exchange chromatography. In membranes that were depleted of LTC4 synthase and cytosolic GSTs, 125I-azido-LTC4 (a photoaffinity probe based on LTC4) specifically photolabeled in a cation-dependent manner a 17-kDa polypeptide that was comparable in mass to the microsomal GST polypeptide. Furthermore, [3H]LTC4 bound specifically to purified human microsomal GST with the same characteristics as to the endogenous dU937-cell membrane specific binding sites. The principal [3H]LTC4 specific binding site present in dU937 cells, therefore, is not a G-protein-coupled receptor, LTC4 synthase, or cytosolic GSTs, but is microsomal GST. Finally, the 1:3 stoichiometry of [3H]LTC4 specific binding to purified microsomal GST is consistent with the enzyme functioning as a homotrimer.

Functional cDNA clones coding for three isoforms of the human prostaglandin E receptor EP3 subtype have been isolated from kidney and uterus cDNA libraries. The three isoforms, designated hEP3-I, hEP3-II and hEP3-III, have open reading frames corresponding to 390, 388 and 365 amino acids, respectively. They differ only in the length and amino acid composition of their carboxy-terminal regions, beginning at position 360. The human EP3 receptor has seven predicted transmembrane spanning domains and therefore belongs to the G-protein-coupled receptor family. The rank order of potency for prostaglandins and related analogs in competition for [3H]PGE2 specific binding to membranes prepared from transfected COS cells was comparable for all three isoforms, and as predicted for the EP3 receptor, with PGE2 = PGE1 >> PGF2 alpha = iloprost > PGD2 >> U46619. In addition, the EP3-selective agonist MB28767 was a potent competing ligand with an IC50 value of 0.3 nM, whereas the EP1-selective antagonist AH6909 gave IC50 values of 2-7 microM and the EP2-selective agonist butaprost was inactive. In summary, we have cloned three isoforms of the human EP3 receptor having comparable ligand binding properties.

A cDNA clone coding for a functional human prostanoid FP receptor has been isolated from a uterus cDNA library. The human FP receptor consists of 359 amino acid residues with a predicted molecular mass of 40,060, and has the seven putative transmembrane domains characteristic of G-protein-coupled receptors. Challenge of Xenopus oocytes expressing the FP receptor with 10 nM of either prostaglandin (PG) F2 alpha or the selective FP-receptor agonist fluprostenol resulted in an elevation in intracellular Ca2+. Radioreceptor binding studies using membranes prepared from mammalian COS cells transfected with the FP receptor cDNA showed that the rank order of potency for prostaglandins and prostaglandin analogs in competition for [3H]PGF2 alpha specific binding sites was as predicted for the FP receptor, with PGF2 alpha approximately fluprostenol > PGD2 > PGE2 > U46619 > iloprost. In summary, we have cloned the human prostanoid FP receptor which is functionally coupled to the Ca2+ signalling pathway.

The human acute monocytic leukemia cell line THP-1 has been identified, by radioligand binding, as expressing the leukotriene D 4 receptor at a high level (4000 binding sites per cell), without the need for further cell differentiation. [ 3 H]Leukotriene D 4 -specific binding to THP-1 cell membranes was of high affinity ( K D = 0.47 nM) and saturable, enhanced by divalent cations but inhibited by both monovalent cations and non-hydrolyzable GTP analogs. The cysteinyl leukotrienes competed for [ 3 H]leukotriene D 4 -specific binding with the following rank order of potency: leukotriene D 4 ⪢ leukotriene E 4 〉 leukotriene C 4 In addition, leukotriene D 4 -receptor antagonists from two structural classes, the quinolines MK-571 and L-697,008, and the indole ICI 204,219, displayed nanomolar potency in [ 3 H]leukotriene D 4 competition assays. These data show that [ 3 H]leukotriene D 4 -specific binding to THP-1 cell membranes fulfils the criteria for binding to a leukotriene D 4 receptor regulated through interaction with a G protein. Several novel features of the THP-1 leukotriene D 4 receptor were investigated. Culture of THP-1 cells in the presence of tunicamycin, an inhibitor of N -glycosylation, resulted in a 6-fold decrease in the number of detectable [ 3 H]leukotriene D 4 -specific binding sites. Target-size analysis by radiation inactivation estimated a molecular mass of 65 kDa for the [ 3 H]leukotriene D 4 specific binding site(s) present in THP-1 cell membranes. Together, these results suggest that the human THP-1 cell leukotriene D 4 receptor is a glycosylated protein with a molecular mass of approx. 65 kDa within the membrane environment.

The leukotriene (LT)D4 receptor has been defined as a G-protein-coupled receptor. In order to characterize this receptor, an iodinated, photoactivatable azido derivative of LTD4 (125I-azido-LTD4) has been synthesized for use as a photoaffinity probe. The characteristics of 125I-azido-LTD4 specific binding to guinea pig lung membranes were directly comparable to those of [3H]LTD4 specific binding to this tissue. 125I-Azido-LTD4 specific binding was saturable and of high affinity, enhanced by divalent cations and inhibited by sodium ions, but not potassium ions. 125I-Azido-LTD4 specific binding was also strongly inhibited by the nonhydrolyzable GTP analog, GTP gamma S, with ATP gamma S being 100-fold less potent, suggesting this inhibition was due to selective interaction with a G-protein. The cysteinyl leukotrienes competed for 125I-azido-LTD4 specific binding to guinea pig lung membranes with the following rank order of potency: LTD4 > LTE4 > LTC4, while the non-cysteinyl LTB4 was virtually inactive. Two structurally different LTD4 receptor antagonists, MK-571 and ICI 204,219, also competed for 125I-azido-LTD4 specific binding with nanomolar potency, whereas the leukotriene synthesis inhibitor, MK-886, was 10,000-fold less active. These data are in agreement with 125I-azido-LTD4 binding specifically to a G-protein-coupled LTD4 receptor. Photolysis of 125I-azido-LTD4 under equilibrium binding conditions resulted in the selective radiolabeling of a 45-kDa guinea pig lung membrane protein. The photolabeling of this 45-kDa protein was saturable, modulated by cations and inhibited by nucleotide analogs in an analogous way to 125I-azido-LTD4 specific binding. In addition, the photolabeling of this protein was inhibited in a concentration-dependent manner by all competing ligands, with the same rank order of potency and IC50 values as determined in the 125I-azido-LTD4 binding assay. It is proposed, therefore, that this novel 45-kDa protein is the guinea pig lung LTD4 receptor.

To gain insight into the role of prostanoids in human kidney function, we examined the distribution of cyclooxygenase (COX) 1 and COX-2 by immunofluorescence and immunohistochemistry in human kidneys from adults of various age groups. COX-1 was detected in the collecting ducts, thin loops of Henle and portions of the renal vasculature. COX-2 was detected in the renal vasculature, medullary interstitial cells, and the macula densa. In addition, COX-2 immunoreactivity was noted in afferent arteries and the macula densa of the renal cortex and was more evident in the kidneys of older adults.

1. The recombinant human prostaglandin D(2) (PGD(2)) receptor, hCRTH2, has been expressed in HEK293(EBNA) and characterized with respect to radioligand binding and signal transduction properties. High and low affinity binding sites for PGD(2) were identified in the CRTH2 receptor population by saturation analysis with respective equilibrium dissociation constants (K(D)) of 2.5 and 109 nM. This revealed that the affinity of PGD(2) for CRTH2 is eight times less than its affinity for the DP receptor. 2. Equilibrium competition binding assays revealed that of the compounds tested, only PGD(2) and several related metabolites bound with high affinity to CRTH2 (K(i) values ranging from 2.4 to 34.0 nM) with the following rank order of potency: PGD(2)13,14-dihydro-15-keto PGD(2)15-deoxy-Delta(12,14)-PGJ(2)PGJ(2)Delta(12)-PGJ(2)15(S)-15 methyl-PGD(2). This is in sharp contrast with the rank order of potency obtained at DP : PGD(2)PGJ(2)Delta(12)-PGJ(2)15-deoxy-Delta(12,14)-PGJ(2)13,14-dihydro-15-keto-PGD(2). 3. Functional studies demonstrated that PGD(2) activation of recombinant CRTH2 results in decrease of intracellular cAMP in a pertussis toxin-sensitive manner. Therefore, we showed that CRTH2 can functionally couple to the G-protein G(alphai/o). PGD(2) and related metabolites were tested and their rank order of potency followed the results of the membrane binding assay. 4. By Northern blot analysis, we showed that, besides haemopoietic cells, CRTH2 is expressed in many other tissues such as brain, heart, thymus, spleen and various tissues of the digestive system. In addition, in situ hybridization studies revealed that CRTH2 mRNA is expressed in human eosinophils. Finally, radioligand binding studies demonstrated that two eosinophilic cell lines, butyric acid-differentiated HL-60 and AML 14.3D10, also endogenously express CRTH2.

A cDNA clone encoding the human prostaglandin (PG) E2 receptor EP2 subtype has been isolated from a human lung cDNA library. The 1.9-kilobase pair cDNA, hEP2, encodes for a 488-amino acid protein with a predicted molecular mass of 53,115 and has the seven putative transmembrane domains characteristic of G protein-coupled receptors. The specific binding of [3H]PGE2 to COS cell membranes transfected with the hEP2 cDNA was of high affinity with an equilibrium dissociation constant (Kd) of 1 nM and the rank order of potency for prostaglandins in competition for [3H]PGE2 specific binding was PGE1 = PGE2 >> iloprost > PGF2 alpha > PGD2. In competition studies using more selective prostanoid-receptor agonist and antagonists, the [3H]PGE2 specific binding was competed by MB28767, an EP3 agonist, but not by the EP1-preferring antagonists AH6809 and SC19220, or by the EP2 agonist butaprost. Electrophysiological studies of Xenopus oocytes co-injected with hEP2 and cystic fibrosis transmembrane conductance regulator (cAMP-activated Cl- channel) cDNAs detected PGE2-specific inward Cl- currents, demonstrating that the hEP2 cDNA encoded a functional receptor which produced an increase in cAMP levels. Thus, we have cloned the human EP2 receptor subtype which is functionally coupled to increase in cAMP. Northern blot analysis showed that hEP2 is expressed as a 3.8-kilobase mRNA in a number of human tissues with the highest expression levels present in the small intestine.

1. NK1 and NK2 receptors have been characterized in guinea-pig lung membrane preparations by use of [125I-Tyr8]-substance P and [125I]-neurokinin A binding assays in conjunction with tachykinin-receptor selective agonists ([Sar9Met(O2)11]substance P for NK1 and [beta Ala8]neurokinin A (4-10) for NK2) and antagonists (CP-99,994 for NK1 and SR48968 for NK2). 2. The presence of high affinity, G-protein-coupled NK1 receptors in guinea-pig lung parenchymal membranes has been confirmed. The rank order of affinity for competing tachykinins was as predicted for an NK1 receptor: substance P = [Sar9Met(O2)11]substance Psubstance P-methyl ester = physalaeminneurokinin A = neurokinin B[beta Ala8]neurokinin A (4-10). The novel NK1 antagonist CP-99,994 has a Ki of 0.4 nM at this NK1 site. 3. In order to characterize [125I]-neurokinin A binding to guinea-pig lung, the number of [125I]-neurokinin A specific binding sites was increased 3-4 fold by purification of the parenchymal membranes over discontinuous sucrose gradients. The rank order of affinity determined for NK1- and NK2-receptor agonists and antagonists in competition for these sites showed that the majority (80%) of [125I]-neurokinin A specific binding was also to the NK1 receptor. 4. Under conditions where the guinea-pig lung parenchymal NK1 receptor was fully occupied by a saturating concentration of either [Sar9Met(O2)11]substance P (1 microM) or CP-99,994 (2.7 microM), residual [125I]-neurokinin A specific binding was inhibited in a concentration-dependent manner by both [beta Ala8]neurokinin A and SR48968. This result shows that the NK2 receptor is also present in these preparations. 5. Similar studies using guinea-pig tracheal membranes demonstrated that [125I]-neurokinin A specific binding was composed of a NK1-receptor component (60%), inhibited by both [Sar9Met(02)11]substance P and CP-99,994, and a significant NK2-receptor component, inhibited by both [beta Ala 8]neurokinin A andSR48968.6. In summary, these data demonstrate that guinea-pig lung parenchyma and guinea-pig trachea express both NK1 and NK2 receptors.

Human leukotriene C4 (LTC4) synthase was purified > 10000-fold from dimethylsulfoxide-differentiated U937 cells. Steps included: (a) solubilization of membrane-bound LTC4 synthase from microsomal membranes by the anionic detergent taurocholate; (b) successive anion-exchange chromatography steps in the presence of taurocholate plus Triton X-100 (primary anion exchange) then taurocholate plus n-octyl glucoside (secondary anion exchange); and (c) LTC2-affinity chromatography on a matrix that was constructed by first biotinylating synthetic LTC2 then immobilizing the biotinylated LTC2 on streptavidin agarose. The purification of human LTC4 synthase was enabled by the finding that LTC4 synthase activity in preparations enriched > 500-fold was absolutely dependent on the presence in LTC4 synthase incubation mixtures of divalent cations (specifically Mg2+) and phospholipids (specifically phosphatidylcholine), and that reduced glutathione, which was required at 2-4mM for stabilization of LTC4 synthase, irreversibly inactivated the enzyme when present at ≥ 5 mM during freeze/thaw cycles. The > 10000-fold purified LTC4 synthase preparation was comprised of three polypeptides having molecular masses of 37.1, 24.5 and 18.0 kDa. An 18-kDa polypeptide in both microsomal membranes and in the LTC4-affinity purified fraction was specifically labelled by a radioiodinated LTC4 photoaffinity probe (azido 125I-LTC4). The Km values in the LTC2-affinity purified preparation for reduced glutathione and LTA4 were 1.83mM and 19.6 μM (respectively), closely resembling the Km values in isolated human blood monocytes. The Vmax of LTC2-affinity purified LTC4 synthase was 2–4 μmol LTC4 formed · min−1· mg−1.