Biomedical subjects
Publications and source records attributed to J Rokach.
Chemically synthesized 8(S), 15(S)-dihydroxy-5,11-cis-9, 13-trans-eicosatetraenoic acid and 8(R), 15(S)-dihydroxy-5,11-cis-9,13-trans-eicosatetraenoic acid were inactive, in comparison to leukotriene B4, in a human polymorphonuclear leukocyte chemokinetic assay and a rat polymorphonuclear leukocyte aggregation assay.
Radioimmunoassays for leukotriene C4 (LTC4) and for leukotriene B4 (LTB4) have been developed. LTC4 was conjugated with thiolated hemocyanin (Keyhole Limpet) (KLH) using 6-(N-maleimido)hexanoic acid chloride as coupling agent. LTB4 was converted to its hydrazide derivative, via the delta-lactone and the hydrazide was similarly coupled with thiolated KLH using 6-(N-maleimido)hexanoic acid chloride as coupling agent. These conjugates were used to consistently raise high titres of anti-leukotriene antibodies in rabbits. 14,15-[3H]-LTC4 was prepared by total synthesis via two routes. 14,15-[3H]-LTB4 was prepared by total synthesis. The assay for LTC4 recognizes LTC4, LTD4 and LTF4, and to a lesser extent, LTE4 with a detection limit of ca. 0.1 pmoles LTC4 per mL of sample. The assay for LTB4 is highly specific and has a similar detection limit.
The effects of L-640,035 (3-hydroxymethyl-dibenzo [b,f] thiepin-5,5-dioxide) have been studied on pulmonary smooth muscle contraction in vitro and in vivo. When studied in vitro on guinea-pig tracheal chains, L-640,035 produced significant shifts in the dose-response curves to a prostaglandin (PG) endoperoxide analogue (U-44069) (pA2 7.0), PGF2 alpha (pA2 5.9) and PGD2 (pA2 6.5). L-640,035 produced no significant shift in the dose-response curves to leukotriene D4 or histamine and produced a small but statistically significant shift in the dose-response curve to 5-hydroxytryptamine (5-HT) (pA2 5.2). With the exception of PGF2 alpha, Schild analysis did not in general indicate competitive inhibition. The main metabolite of L-640,035, L-636,499, also produced significant parallel shifts in the dose-response curves to U-44069 (pA2 6.0) and PGF2 alpha (pA2 6.0), but with some reduction in the maximal contraction. When L-640,035 was administered intravenously to guinea-pigs, significant inhibition of increases in pulmonary resistance or insufflation pressure induced by U-44069 (ED50 0.16 mg kg-1), leukotriene D4 (ED50 0.25 mg kg-1) and 5-HT (ED50 3.4 mg kg-1) but not histamine (ED50 greater than 10 mg kg-1) was observed. When L-640,035 was administered intravenously to dogs a significant inhibition of increases in pulmonary resistance induced by U-44069 (ED50 0.85 mg kg-1) but not histamine (ED50 greater than 30 mg kg-1) was observed. 5 When L-640,035 was administered by the intraduodenal route to dogs at doses of 3 and 10 mg kg- significant inhibition of increases in pulmonary resistance induced by sodium arachidonate (3 mgkg1 i.v.) was observed with a duration of action of > 255 min. 6 It is concluded that L-640,035 is a novel, relatively selective, and orally active antagonist of the actions of contractile prostanoids on pulmonary smooth muscle.
The effects of biologically prepared leukotriene C4 (LTCb) and leukotriene D4 (LTDb), obtained from rat monocytes stimulated with the calcium ionophore A23187, were compared with those of chemically synthetized leukotrienes (LTCs and LTDs) using two in vitro systems. All four leukotriene preparations (10(-10) to 6 X 10(-6) M) showed equal activity upon human bronchi, inducing slow, sustained contractions. LTCb alone (10(-7) to 6.9 X 10(-7) M) elicited histamine release and enhanced compound 48/80-induced release in a dose-dependent manner from rat mast cells. In contrast, LTDb alone was without effect but inhibited release caused by 48/80. FPL 55712 failed to block the LTCb and LTDb effects on the release process. The synthetic leukotrienes neither caused histamine release nor modulated 48/80-induced release from rat mast cells. We conclude that biologic and synthetic leukotrienes exhibit comparable contractile activity on isolated human bronchi but only biologic preparations modulate histamine release by previously unappreciated substances that isolate with the biologic leukotrienes.
Arachidonic acid metabolism has been explored in preparations of purified human lung mast cells prelabeled with arachidonic acid (AA). Cells were of 83 to greater than 96% purity, and each experiment was performed with four to six different preparations of mast cells. After overnight culture of the purified cells in the presence of 3H-AA, followed by extensive washing in buffer, mast cell uptake of labeled AA was 61.4 +/- 14.8 pmol/10(6) cells with 21 +/- 2.4% of the label in phospholipids, 73 +/- 2.1% in neutral lipids, and 3.6 +/- 0.8% as free AA. Analysis of the distribution of radioactivity in phospholipid classes revealed 51.4 +/- 5.5% of the label in phosphatidylcholine, 14.5 +/- 1.6% in phosphatidylinositol, 12.0 +/- 3.0% in phosphatidylethanolamine, and 9.1 +/- 2.4% in sphingomyelin, with the rest in other phospholipid classes. Challenge of these cells with an optimal concentration of anti-IgE led to the release of 20 +/- 4.0% of cellular histamine and to a reduction in labeled phosphatidylcholine and phosphatidylinositol to 75.5 +/- 8.8% and 84.2 +/- 4.5% of the control levels, respectively, (p less than 0.05); anti-IgE challenge produced no statistically significant change in the quantities of other labeled phospholipids. Activation of human lung mast cells with anti-IgE led to the release of 3.4 +/- 1.3% of the cellular 3H as AA and AA metabolites (1.5 +/- 0.6% as unmetabolized AA) in conjunction with 16 +/- 4.3% of the cellular histamine. Although activation of human lung mast cells with ionophore A23187 caused 70 +/- 1.1% histamine release, a similar quantity of AA and AA metabolites was released (a total of 4.0 +/- 0.8% with 2.3 +/- 1.5% as unmetabolized AA). Analysis of the released metabolites by liquid scintillation spectrometry after high performance liquid chromatography separation showed that approximately equal amounts of metabolites were produced after mast cell activation with anti-IgE and ionophore A23187. In this series of experiments approximately equal amounts of cyclooxygenase and lipoxygenase products were generated.(ABSTRACT TRUNCATED AT 400 WORDS)
We have examined the effects of very pure (greater than 99.8%) chemically synthesized leukotriene B4 of verified structure on the chemotactic and secretory behavior of human polymorphonuclear leukocytes (PMN). The synthetic material is highly chemotactic and shows the same concentration dependence of this activity as does natural LTB4. Synthetic LTB4 is also a weak degranulating agent in cytochalasin B treated PMN. Maximally it released 11%, 17% and 26% as much N-acetyl-beta-D-glucosaminidase, myeloperoxidase and lysozyme as did N-formyl-methionine-leucine-phenylalanine (fMLP). Thus LTB4 differs significantly from other chemotaxins, such as C5a and fMLP, in that it is a poor secretagogue for enzymes of the specific and azurophilic granules of human PMN.
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Leukotriene B4 (LTB4) (I) has been converted to its N-(3-amino-propyl)amide derivative (III) and to its hydrazide derivative (VII) via LTB4 delta-lactone. The amide (III) was coupled with Bovine Serum Albumin using 1,5-difluoro-2,4-dinitrobenzene as coupling agent. The hydrazide (VII), was coupled with Hemocyanin (Keyhole Limpet) (KLH) using 6-N-maleimidohexanoic acid chloride as coupling agent.
Rabbits were immunized with leukotriene C4 (LTC4) coupled to thiolated keyhole limpet hemocyanin (KLH) by using 6-N-maleimidohexanoic acid as a spacer molecule. Immune serum was obtained with 7.9 nmol of LTC4-specific immunoglobulin per milliliter and a mean association constant of 2.1 X 10(9) M-1. A radioimmunoassay was developed that detected 0.1 pmol of LTC4 per 1-ml sample. LTD4 and LTE4, three isomers of LTC4, the sulfones of LTC4, LTD4, and LTE4, and one isomer of LTD4 reacted to varying degrees in the assay. A number of other structurally related compounds, such as LTB4 and 5-HETE, did not react. Conditions were established to determine LTC4 levels in human plasma without loss of LTC4 during sample preparation and without the need for extraction procedures before the measurement of LTC4.
The development of the technique of fast atom bombardment mass spectrometry (FAB-MS) has greatly expanded the capability to analyze non-volatile, complex biochemicals. The structure of leukotriene C4, a slow reacting substance of anaphylaxis, has recently been postulated as 6-glutathionyl-5-hydroxy-7,9,11,14-eicosatetraenoic acid. Even though LTC4 has been synthesized, it has not been possible to obtain direct mass spectrometric confirmation of this structure. FAB-MS has indicated that the biological LTC4 had a molecular weight of 625, identical to that of synthetic LTC4. The abundance of cationized species with one and two sodium atoms was the major difference between these leukotrienes which is probably a result of the difference in salt content of the purified molecules.
Leukotriene F4 (LTF4) and LTF4 sulfone have been synthesized and their biological activities determined in the guinea pig. In vitro LTF4 displayed comparable activity to LTD4 on guinea pig trachea and parenchyma but was less active on the ileum. When injected intravenously into the guinea pig, LTF4 induced a bronchoconstriction (ED50 16 micrograms Kg-1) which was blocked by indomethacin and FPL-55712 and was 50-100 X less potent than LTD4 in this assay. LTF4 sulfone was approximately 2-5 times less active than LTF4 in vitro and in vivo. When injected into guinea pig skin with PGE2 (100 ng); LTF4 and LTF4 sulfone (10-1000 ng) induced changes in vascular permeability. The order of potency in this assay was LTE4 sulfone = LTD4 = LTD4 sulfone greater than LTE4 greater than LTF4 = LTF4 sulfone.
Leukotriene C4 (LTC4) has been conjugated with Bovine Serum Albumin (BSA) and Hemocyanin from Giant Keyhole Limpets (KLH) using 1,5-difluoro-2,4-dinitrobenzene as coupling agent. A second LTC4-KLH conjugate was prepared using a new bifunctional coupling agent, 6-N-maleimidohexanoic acid chloride. Conjugates of some representative LTC4 component parts; S-p-chlorophenacylglutathione with BSA and of 2,4(E),6,9(Z)-pentadecatetraen-1-ol with BSA were also prepared.
The biological activity of synthetic leukotriene C4, D4 and E4 sulfone has been determined in respiratory smooth muscle in vitro and in vivo. The sulfones of LTC4, LTD4 and LTE4 were potent contractile agonists on indomethacin-treated guinea pig tracheal chains with respective pD2-values of 8.2, 8.0 and 7.9. Contractions were submaximal (75-85% of the cholinergic maximum), slow in onset, prolonged in duration, slowly reversed by washing (compared to acetylcholine or histamine) and were partially reversed by 2 muM FPL-55712. The sulfones of LTC4, LTD4 and LTE4 also contracted indomethacin-treated guinea pig parenchyma (respective pD2's of 7.9 8.2 and 7.8) and rat parenchyma (respective pD2's of 7.1, 7.2 and 7.2) but were inactive on rat trachea (0.01-2.0 muM). When administered intravenously to anaesthetized guinea pigs, the sulfones of LTD4, LTE4 and to a lesser degree LTC4 (respective ED50's - 0.5; 2.0 and 4.6 microgram/kg) elicited dose-dependent increases in inflation pressure which were antagonized by FPL-55712 and indomethacin. Leukotriene C4, D4 and E4 sulfones display a qualitatively similar profile of biological activity to that of their corresponding sulfides.
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We have recently described the structure elucidation of slow reacting substance of anaphylaxis S(SRS-A) from lung and of a slow reacting substance (SRS) from basophilic leukaemia cells as 5-hydroxy-6-cysteinylglycinyl-7,9,11,14-eicosatetraenoic acid. The stereochemistry of this molecule has now been shown to be 5(S)-hydroxy- 6(R)-cysteinylghlycinyl-7,9-trans-11,14-ciseicosatetraenoic acid by comparison of the synthetic and natural products and their derivatives using mass spectrometric and HPLC chromatographic techniques. The synthetic and natural compounds are also indistinguishable by their pharmacological properties, their conversion by soybean lipoxygenase, and their UV spectra.
The effects of synthetic leukotriene C-1 (LTC-1) on isolated guinea pig trachea and ileum have been determined and compared to histamine. LTC-1 produced a slow contraction of the trachea and the ileum with pD2 values of 8.7 +/- 0.1 (n = 14) and 8.5 +/- 0.1 (n = 13), respectively. In comparison, the pD2 values for histamine were 5.6 +/- 0.1 (n = 6) and 6.2 +/- 0.1 (n = 6), indicating LTC-1 was 2-3 orders of magnitude more potent. LTC-1 was antagonised by FPL 55712 with pA2 values of 6.9 +/- 0.1 (n = 5) and 6.4 +/- 0.1 (n = 7) on the trachea and ileum, respectively. Incubation with lipoxidase produced a time and enzyme dependent loss of biological activity and a concurrent shift in U.V. absorption spectrum.