Seymour Mong

Characterization of the leukotriene D4 receptor in guinea-pig lung

The effects of monovalent, divalent cations, buffere species and pH dependency on [3H]leukotriene D4 binding to the receptor have been characterized in vitro by using a radioligand binding assay. It was found that Ca2+, Mg2+, Co2+ and Mn2+ enhanced the specific binding. High concentrations of NaCl (150-300 mM) inhibited the specific binding to the receptor. The specific binding was also found to be higher in Pipes buffer (pH 6.5) than in Tris, Hepes and phosphate buffer at pH 7.0-8.0. Conversion of [3H]leukotriene D4 was minimized by inclusion of 1 mM cysteine, glycine in the incubation buffer and maintaining the temperature at 22 degrees C. Under the conditions employed, the dissociation constant (KD) and the receptor density (Bmax) were calculated to be 1.8 +/- 0.9 nM and 2100 +/- 375 fmol/mg protein respectively. The leukotriene antagonist FPL 55712, agonist 5R, 6S-LTD4 and LTE4 competed with the [3H]LTD4 binding to the receptor. Prostaglandins, alpha-, beta-adrenergic and dopaminergic receptor agonists and antagonists did not compete significantly.

Interactions of gold coordination complexes with DNA

The interactions of certain gold(I) and gold(III) complexes with isolated plasmid pBR322 DNA were defined and compared to those of cis-diamminedichloroplatinum(II), CDDP, using an agarose gel electrophoresis assay. Trichloro(pyridine)gold(III) appeared to bind to DNA as evidenced by its ability to produce dose-dependent changes in the electrophoretic mobilities of closed circular, supercoiled, closed circular, relaxed, and open circular plasmid DNAs. These effects suggest that the gold containing complex induces conformational changes in the plasmid as a result of the compound binding to the DNA and the subsequent unwinding of the double helix and shorting of the DNA. Auranofin [(2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranosato-S)-triethyl phosphine gold(I)] did not appear to interact with DNA under any conditions. However, its analog chloro(triethylphosphine) gold(I) interacted with DNA at pH 9.5 in borate buffer and produced electrophoretic mobility changes in pBR322 DNA which were different from those produced by the gold(III) complexes that were evaluated. Binding of chloro(triethylphosphine) gold(I) was inhibited by the co-addition of the thiosugar portion of auranofin suggesting preferential binding of the gold moiety to thiosugar, which results in the production of auranofin (or a sugar containing) gold complex and inhibition of gold binding to DNA. The interactions of a number of gold compounds with DNA were also evidenced by their abilities to inhibit the binding of ethidium bromide to DNA. The results from these studies indicate that: gold containing complexes can bind to, and produce conformational changes in, DNA; gold(I) and gold(III) complexes may interact with DNA via different chemical mechanisms to produce different conformational changes in DNA; and certain coordinating ligands in gold complexes (e.g. Cl, Br and SCN) can be exchanged for binding sites on DNA by gold.

Leukotriene E4 binds specifically to LTD4 receptors in guinea pig lung membranes

High affinity, stereoselective binding sites for [3H]leukotriene E4 ([3H]LTE4) have been identified and characterized in guinea pig lung membranes. [3H]LTE4 bound to these membranes with a pharmacological specificity identical to that previously observed for the [3H]LTD4 receptor in guinea pig lung. [3H]LTE4 specific binding was selectively inhibited by Na+, enhanced by Ca2+, Mg2+ and Mn2+ and modulated by guanine nucleotides. Scatchard analysis of saturation binding data showed a single class of high affinity and saturable binding sites, with a dissociation constant (Kd) of 0.4 +/- 0.2 nM and a density (Bmax) of 430 +/- 50 fmol/mg membrane protein, similar to values observed for the LTD4 receptor in guinea pig lung. The rank order potency of agonist binding to the [3H]LTE4 binding sites was LTD4 greater than LTE4 much greater than LTC4. These results indicate that [3H]LTE4 binds to [3H]LTD4 receptors and suggests that induction of smooth muscle contraction by LTD4 and LTE4 may be mediated by identical mechanisms and receptors in the guinea pig lung.

Identification of specific binding sites for leukotriene C4 in human fetal lung

Specific leukotriene C4 (LTC4)1 binding sites were identified in membrane preparations from human fetal lung. Specific binding of [3H]-LTC4 represented 95 percent of total binding, reached steady-state within 10 minutes and was rapidly reversible upon addition of excess unlabeled LTC4. Binding assays were performed at 4 degrees C under conditions which prevented metabolism of [3H]-LTC4 (80 mM serine-borate, 10 mM cysteine, 10 mM glycine). Under these conditions, greater than 95 percent of the membrane bound radioactivity, as analyzed by high performance liquid chromatography, co-eluted with the LTC4 standard. Computer-assisted analyses of saturation binding data showed a single class of binding sites with a dissociation constant (Kd) of 26 + 6 nM and a density (Bmax) of 84 + 18 pmol/mg protein. Pharmacological specificity was demonstrated by competition studies in which specific binding of [3H]-LTC4 was displaced by LTC4 and its structural analogs with inhibition constants (Ki) of 10 to 30 nM, whereas LTD4, diastereoisomers of LTD1, LTE4 and the end organ antagonist FPL 55712 were 150 to 700 fold less potent competitors than LTC4. These results provide evidence for specific, reversible, saturable, high affinity binding sites for [3H]-LTC4 in human fetal lung membranes.

Leukotriene D4-induced activation of protein kinase C in rat basophilic leukemia cells.

We studied the subcellular distribution of protein kinase C (PKC) in the particulate and cytosolic fractions of rat basophilic leukemia (RBL-1) cells treated with leukotriene D4 (LTD4) and compared these results with those of phorbol myristate acetate (PMA). Consistent with the earlier reports, treatment of RBL-1 cells with PMA resulted in a time- and dose-dependent translocation of PKC from cytosolic to the particulate fractions, sustained for at least 10 min. When RBL-1 cells were treated with LTD4, a small, transient decrease in PKC activity in cytosolic fraction was observed within 7.5 s after LTD4 treatment. This was accompanied by a significant increase of PKC in the particulate fraction. However, at 15 and 30 s, both particulate and cytosolic PKC activities were increased with LTD4 treatment. The activation induced by LTD4 was dose- and time-dependent with maximal effects occurring within 30 s, declining at the later time points. Pretreatment of the cells with 2(R)-hydroxy-3(S)-carboxyethylthio-3-[2-(8-phenyloctyl,pheny l]propanoic acid (SK&F 104353), a high affinity specific LTD4 receptor antagonist, and also with staurosporine, a potent inhibitor of PKC, completely inhibited the LTD4-induced activation of PKC both in the particulate and cytosolic fractions. These results suggest that activation of PKC by LTD4 is different from that elicited by PMA. The ability of SK&F 104353 to block LTD4-induced activation of PKC further suggests that stimulation of PKC might be an important intermediate step in the signal transduction mechanism of the LTD4 receptor in RBL-1 cells.

Binding of leukotriene B4 and its analogs to human polymorphonuclear leukocyte membrane receptors

LTB4-induced proinflammatory responses in PMN including chemotaxis, chemokinesis, aggregation and degranulation are thought to be initiated through the binding of LTB4 to membrane receptors. To explore further the nature of this binding, we have established a receptor binding assay to investigate the structural specificity requirements for agonist binding. Human PMN plasma membrane was enriched by homogenization and discontinuous sucrose density gradient purification. [3H]-LTB4 binding to the purified membrane was dependent on the concentration of membrane protein and the time of incubation. At 20 degrees C, binding of [3H]-LTB4 to the membrane receptor was rapid, required 8 to 10 min to reach a steady-state and remained stable for up to 50 min. Equilibrium saturation binding studies showed that [3H]-LTB4 bound to high affinity (dissociation constant, Kd = 1.5 nM), and low capacity (density, Bmax = 40 pmol/mg protein) receptor sites. Competition binding studies showed that LTB4, LTB4-epimers, 20-OH-LTB4, 2-nor-LTB4, 6-trans-epi-LTB4 and 6-trans-LTB4, in decreasing order of affinity, bound to the [3H]-LTB4 receptors. The mean binding affinities (Ki) of these analogs were 2, 34, 58, 80, 1075 and 1275 nM, respectively. Thus, optimal binding to the receptors requires stereospecific 5(S), 12(R) hydroxyl groups, a cis-double bond at C-6, and a full length eicosanoid backbone. The binding affinity and rank-order potency of these analogs correlated with their intrinsic agonistic activities in inducing PMN chemotaxis. These studies have demonstrated the existence of high affinity, stereoselective and specific receptors for LTB4 in human PMN plasma membrane.

The binding of leukotriene C4 and leukotriene D4 to membranes of a smooth muscles cell line (BC3H1) and evidence that leukotriene induced contraction in these cells is mediated by thromboxane, protein and RNA syntheses

Leukotriene C4 (LTC4) and leukotriene D4 (LTD4) are important mediators of anaphylaxis and induced thromboxane (TxA2) synthesis in vivo and in vitro. The mechanism by which this occurs is not known and the cellular source of leukotriene (LT) induced TxA2 has not been identified. To obtain insights into this problem we have established an in vitro system using the BC3H1 murine smooth muscle cell line. A membrane fraction obtained from these cells contained binding sites for [3H]LTC4 having high specificity and affinity. Binding of [3H]LTC4 was saturable, specific and reversible with a dissociation constant (KD) of 33 +/- 16 nM and a maximum number of binding sites (Bmax) of 25 +/- 6 pmol/mg membrane protein. However, the amount of [3H]LTD4 specifically bound was considerably less than that of [3H]LTC4. LTC4 and LTD4 induced these cells to contract, a process which was blocked by inhibitors of cyclooxygenase and thromboxane synthetase. Using a radioimmunoassay, we have shown that the level of TxB2 (a stable metabolite of TxA2) was increased in response to LTC4 and LTD4 treatment in a dose-dependent manner. Cycloheximide, a protein synthesis inhibitor, and actinomycin D, an inhibitor of RNA synthesis, were found to inhibit both LTC4- and LTD4-induced TxB2 synthesis and cellular contraction. Arachidonic acid, in the absence of LT, increased the levels of TxB2 synthesis and contraction even in the presence of cycloheximide and actinomycin D. These data are consistent with the hypothesis that the rate-limiting step in LT-induced TxB2 synthesis is the formation of free arachidonic acid, a process which requires RNA and protein synthesis.

Differential inhibition of histamine release from mast cells by protein kinase C inhibitors: Staurosporine and K-252a

Pretreatment of rat peritoneal mast cells with either staurosporine or an analog K-252a [(8R*,9S*,11S*)-(-)-9-hydroxyl-9-methoxycarbonyl-8-methyl-2,3, 9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11-atrizadibenzo- [a,g]cycloocta[cde]trinden-1-one] led to a concentration-related inhibition of histamine release when the cells were stimulated with anti-IgE (IC50: staurosporine = 110 nM; K-252a = 100 nM). In contrast, the two protein kinase C (PKC) inhibitors (1-1000 nM) partially (less than 15%) inhibited histamine release induced by compound 48/80 (0.5 to 1 micrograms/mL). Furthermore, prostaglandin E2 (PGE2) synthesis mediated by anti-IgE from rat peritoneal mast cells was also inhibited by staurosporine and K-252a (IC50 = 100 nM). Exposure of anti-arsenate IgE (anti-Ars-IgE) sensitized mouse bone marrow derived mast cells to arsenate-bovine serum albumin (Ars-BSA) led to the release of both histamine (510 +/- 12.6 ng/10(6) cells) and immunoreactive leukotriene C4 (LTC4) (27.0 +/- 2.6 ng/10(6) cells). Both histamine and LTC4 release was inhibited by staurosporine and K-252a with an IC50 of 50 nM for both compounds. We also characterized a 45K molecular weight protein which is phosphorylated by PKC after Ars-BSA or phorbol, 12-myristate, 13-acetate (PMA) stimulation. This protein is phosphorylated in a broken cell preparation in which PKC is activated by phosphatidylserine/Diolein and Ca2+. Peptide mapping by V8 protease of the phosphorylated 45K protein revealed that the 45K protein phosphorylation patterns induced by IgE or PMA or in the broken cell preparation are identical. Pretreatment of 32P-labeled mouse bone marrow derived mast cells with either staurosporine or K-252a led to a concentration-related inhibition of 45K protein phosphorylation induced by PMA or Ars-BSA. This inhibition of protein phosphorylation correlated well with the inhibition of histamine and leukotriene release in bone marrow derived mast cells.

15-acetylthioxy-furodysinin lactone, isolated from a marine sponge Dysidea, SP. is a potent agonist to human leukotriene B4 receptor

A sesquiterpene thioacetate, 15-acetylthioxy-furodysinin (SK&F 105900) has been isolated from the sponge Dysidea SP. This compound can bind specifically to the human peripheral blood polymorphonuclear leukocyte (PMN) and to the differentiated human monocytic leukemic U-937 cell membrane leukotriene B4 (LTB4) receptors with high-affinity. This compound can also promote a concentration-dependent chemotaxis in PMNs and an intracellular calcium mobilization in U-937 cells that can be blocked by the LTB4 receptor antagonist, LY-223982. Furthermore, the calcium mobilization induced by SK&F 105900 can specifically cross-desensitize with the LTB4-induced calcium mobilization. These observations indicate that SK&F 105900 is a novel and specific high-affinity agonist that can bind to the LTB4 receptors and activate the receptor-mediated signal transduction processes in human PMN and U-937 cells.

15-Acetylthioxy-furodysinin lactone, a potent LTB4 receptor partial agonist from a marine sponge of the genus Dysidea

Abstract A novel sesquiterpene thioacetate with high binding-affinity for the human leukotriene B 4 (LTB 4 ) receptor has been isolated from the sponge Dysidea sp. collected in Palau. The structure of the new compound was determined on the basis of spectral data and the compound was prepared semi-synthetically by photo-oxidation of the known metabolite, thiofurodysin acetate.