Leukotriene D4-induced homologous desensitization of calcium mobilization in rat basophilic leukemia cells

Previous work from our laboratory demonstrated that RBL-1 cells express membrane receptors for leukotriene (LT)D4 and that agonist stimulation of these receptors results in mobilization of intracellular calcium ([Ca++]i). We have used this LTD4 receptor-mediated event to examine the effects of homologous desensitization in RBL-1 cells. Prior exposure of RBL-1 cells to LTD4 resulted in a 40% reduction in the amount of maximal [Ca++]i mobilization. This desensitization was concentration- and time-dependent, with an EC50 of 1 to 3 nM and with 50% of the desensitization occurring after 7.5 min of pretreatment. SKF 104353 (100 nM), a high affinity, LTD4-receptor antagonist, blocked completely LTD4-induced desensitization of RBL-1 cells. The LTD4-induced desensitization was stereospecific, as the (5R,6S)-enantiomer of LTD4 was at least 100 times less potent than LTD4. Pretreatment of RBL-1 cells with LTD4 did not alter the ability of thrombin or ATP to induce [Ca++]i mobilization, suggesting that the desensitization was of the "homologous" type. The density of [3H]LTD4 receptors in RBL-1 cell membranes was decreased 23% by prior treatment of RBL-1 cells with LTD4. These results demonstrate that the [Ca++]i mobilization induced by LTD4 can be desensitized as a result of prior exposure to LTD4 and that the LTD4 receptor and/or second messenger systems can be specifically down-regulated.     

Mechanism of maitotoxin-stimulated phosphoinositide breakdown in HL-60 cells

The marine toxin maitotoxin (MTX) and the chemotactic peptide fMet-Leu-Phe (fMLP) induce the formation of inositol phosphates in HL-60 cells differentiated with dibutyryl cyclic AMP. The increase in [3H]inositol(1,4,5)-trisphosphate is rapid but transient after fMLP stimulation, whereas MTX-induced increase in [3H]inositol(1,4,5)-trisphosphate occurs at a slower rate and is sustained over time. In both cases increases in [Ca++]i, measured with fura-2, parallel the formation of inositol trisphosphate. MTX-mediated stimulation of inositol phosphate formation is inhibited in the absence of calcium, whereas the response to fMLP is not. The calcium ionophore ionomycin stimulates the formation of inositol phosphates in differentiated HL-60 cells. The magnitude of the response is smaller than that obtained with MTX. Ionomycin also induces a rapid but sustained increase of [Ca++]i. In undifferentiated HL-60 cells, neither fMLP nor ionomycin induce significant inositol phosphate formation, and the increase in [Ca++]i elicited by ionomycin is transient. In contrast, the effects of MTX on phosphoinositide breakdown and on [Ca++]i in undifferentiated cells are nearly identical to those elicited by MTX in differentiated cells. In the presence of the intracellular calcium chelator BAPTA, fMLP, ionomycin and MTX still stimulate the generation of inositol phosphates. Guanyl nucleotides and calcium stimulate phospholipase C activity in membrane preparations from differentiated HL-60 cells. fMLP stimulates the enzyme only in the presence of GTP. MTX has no effect on membrane phospholipase C activity.     

The calcium signal and neutrophil activation

The cytosolic free calcium concentration, [Ca2+]i in phagocytic cells (e.g. neutrophils, human leukemic cell line HL-60) is an important determinant of cellular activity. In resting phagocytes [Ca2+]i is low (approximately 100 nM), but in response to occupation of cell surface receptors, it rises to micromolar levels, thereby activating a variety of cellular functions. The increases in [Ca2+]i consist of two components: an immediate that is independent of extracellular Ca2+, and a more delayed that is abolished by the removal of extracellular Ca2+. These two components reflect the involvement of two subcellular structures in intracellular Ca2+ homeostasis: an intracellular Ca2+ store, referred to as the calciosome; and the plasma membrane. The function of the intracellular Ca2(+)-store depends on a Ca2(+)-pump, functionally and immunologically related to the cardiac sarcoplasmic reticulum Ca2(+)-ATPase, a Ca2(+)-storage protein, similar to muscle calsequestrin, and a Ca2(+)-release channel, which is sensitive to inositol 1,4,5-trisphosphate. The Ca2(+)-regulatory function of the plasma membrane depends on a Ca2+ pump, similar to the erythrocyte-type Ca2(+)-ATPase, and a Ca2+ channel; the activity of the Ca2+ channel is closely coupled to phosphatidylinositol turnover.     

Concentration-dependence of acetylcholine-induced changes in calcium and tension in swine trachealis

Acetylcholine (ACh)-induced increases in intracellular calcium concentration ([Ca++]i) and tension were measured simultaneously in swine tracheal smooth muscle strips loaded with the calcium-sensitive fluorescent dye Fura-2. ACh at concentrations greater than or equal to 3 x 10(-8) M induced concentration-dependent increases in tension which reached a maximum at 10(-4) M. Increases in [Ca++]i occurred at all [ACh] tested (10(-8) to 10(-4) M). After addition of ACh at concentrations greater than 3 x 10(-7) M, [Ca++]i increased rapidly to a concentration dependent-peak then declined to a concentration-independent steady state approximately 250 nM above the resting [Ca++]i of 257 +/- 12 nM. There was a steep relationship (slope factor greater than 3) between the peak tension and the peak [Ca++]i reached at each [ACh]. The rate of decline of [Ca++]i to the steady state at [ACh] greater than 73 x 10(-7) M was well correlated with the peak [Ca++]i reached. We conclude that the peak increase in calcium induced by ACh sets the level of tension to be attained and the rate of decline of the transient increase in [Ca++]i. The steady-state [Ca++]i is sufficient for maintenance of tension.     

Mechanism of calcium transport stimulated by chlorothiazide in mouse distal convoluted tubule cells.

Thiazide diuretics inhibit Na+ and stimulate Ca2+ absorption in renal distal convoluted tubules. Experiments were performed on immortalized mouse distal convoluted tubule (MDCT) cells to determine the mechanism underlying the dissociation of sodium from calcium transport and the stimulation of calcium absorption induced by thiazide diuretics. Control rates of 22Na+ uptake averaged 272 +/- 35 nmol min-1 mg protein-1 and were inhibited 40% by chlorothiazide (CTZ, 10(-4) M). Control rates of 36Cl- uptake averaged 340 +/- 50 nmol min-1 mg protein-1 and were inhibited 50% by CTZ. CTZ stimulated 45Ca2+ uptake by 45% from resting levels of 2.86 +/- 0.26 nmol min-1 mg protein-1. Bumetanide (10(-4) M) had no effect on 22Na+, 36Cl-, or 45Ca2+ uptake. Control levels of intracellular calcium activity ([Ca2+]i) averaged 91 +/- 12 nM. CTZ elicited concentration-dependent increases of [Ca2+]i to a maximum of 654 +/- 31 nM at 10(-4) M. CTZ reduced intracellular chloride activity ([Cl-]i), as determined with the chloride-sensitive fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium. The chloride channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 10(-5) M) abolished the effect of CTZ on [Cl-]i. NPPB also blocked CTZ-induced increases of 45Ca2+. Resting membrane voltage, measured in cells loaded with the potential-sensitive dye 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)], averaged -72 +/- 2 mV. CTZ hyperpolarized cells in a concentration-dependent and reversible manner. At 10(-4) M, CTZ hyperpolarized MDCT cells by 20.4 +/- 7.2 mV. Reduction of extracellular Cl- or addition of NPPB abolished CTZ-induced hyperpolarization. Direct membrane hyperpolarization increased 45Ca2+ uptake whereas depolarization inhibited 45Ca2+ uptake. CTZ-stimulated 45Ca2+ uptake was inhibited by the Ca2+ channel blocker nifedipine (10(-5) M). We conclude that thiazide diuretics block cellular chloride entry mediated by apical membrane NaCl cotransport. Intracellular chloride, which under control conditions is above its equilibrium value, exits the cell through NPPB-sensitive chloride channels. This decrease of intracellular chloride hyperpolarizes MDCT cells and stimulates Ca2+ entry by apical membrane, dihydropyridine-sensitive Ca2+ channels.     

Eicosanoids, mesangial contraction, and intracellular signal transduction.

The glomerular mesangial cell is a specialized pericyte with multiple functional capabilities including contraction. Mesangial contraction may reduce the glomerular filtration surface area and hence the ultrafiltration coefficient, Kf. Cultured mesangial cells convert arachidonic acid into biologically active eicosanoids which are either contractile (thromboxane A2 [TxA2], prostaglandin F2 alpha [PGE2 alpha]) or relaxant (PGE2, PGI2). The addition of TxA2 analogues, PGE2 or sulfidopeptide leukotrienes (LTC4 and LTD4) stimulated contraction of cultured mesangial cells with threshold responses at approximately 1 nM and maximum responses at 1 microM. PGE2 and PGI2 antagonized mesangial contraction induced by TxA2 analogues. Contraction was enhanced by inhibiting mesangial cyclooxygenase with nonsteroidal antiinflammatory drugs (NSAID). Contractile eicosanoids stimulated phospholipase C thereby elevating intracellular inositol trisphosphate and cytosolic free Ca2+ concentration ([Ca2+]i). Vasorelaxant prostanoids stimulated adenylate cyclase, increasing intracellular cyclic AMP. We conclude that eicosanoids control mesangial contractility by regulating [Ca2+]i and cAMP. NSAID increase mesangial reactivity by blocking the inhibitory effects of endogenous vasodilator eicosanoids, with potential consequences on glomerular hemodynamics.     

Leukotriene C4 binds to receptors and has positive inotropic effects in bullfrog heart

Leukotriene (LT) C4, LTD4 and LTE4 have positive inotropic effects on contractility of the isolated perfused bullfrog heart. The effects of LTD4 and LTE4 but not LTC4 can be blocked by the mammalian antagonist L-649,923. Characterization of specific binding sites for [3H]LTC4 on membrane preparations from American bullfrog (Rana catesbeiana) ventricle was carried out. Binding assays were done in the presence of serine (5 mM) and borate (10 mM) for 30 min at 23 degrees C. Under these conditions, no metabolism of LTC4 to LTD4 occurred. Specific binding of [3H]LTC4 reached steady state within 10 min, remained constant for 60 min, and was reversible with the addition of 1000-fold excess unlabeled LTC4. Scatchard analysis of the binding data indicated a single class of binding sites with a Kd of 33.9 nM and maximal binding capacity of 51.6 pmol/mg of protein. Competition binding studies revealed an order of potency of LTC4 greater than LTD4 greater than LTE4 with Ki values of 47, 11766 and 32248 nM, respectively. Glutathione and hematin had Ki values of 50566 and 6014 nM, respectively, suggesting that the LTC4 receptor is not a site on glutathione transferase. Two mammalian LTD4 antagonists, L-649,923 and LY171883 failed to inhibit specific binding of [3H]LTC4, suggesting that the LTC4 receptor is distinct from the LTD4 receptor. Guanosine-5'-O-3-thiotriphosphate did not affect specific binding of [3H]LTC4 indicating that, like mammalian LTC4 receptors, a Gi protein is not involved in the transduction mechanism. LTC4 acts on bullfrog hearts through specific membrane receptors and is similar to its mammalian counterpart.     

Action of peptidoleukotrienes on ion transport in rabbit distal colon in vitro.

Serosal but not mucosal addition of the peptidoleukotrienes, leukotriene (LT) C4 (LTC4), LTD4 and LTE4 transiently (maximal response within 2 min) increased short-circuit current (Isc) and transepithelial conductance across stripped rabbit colonic mucosa mounted in Ussing chambers. All three peptidoleukotrienes elicited their responses in the presence of amiloride (10 microM) and were inhibited by serosal addition of the NaCl cotransport inhibitors bumetanide (100 microM) and furosemide (1 mM). The effects of the peptidoleukotrienes on Isc and transepithelial conductance were concentration-dependent with maximal effects occurring at 10 microM. Half-maximal effects were produced at 30 nM for LTC4, 50 nM for LTD4 and 450 nM for LTE4. The secretory responses to both LTD4 and LTE4 were antagonized in a concentration-dependent manner by the LTD4/LTE4 receptor antagonist, SK&F 104353 (2(S)-hydroxy-3-(R)-[(2-carboxyethyl)thio]-3-[2-(8 phenyloctyl)phenyl]propanoic acid). Complete inhibition of the LTD4 and LTE4 effects were observed at 0.1 microM SK&F 104353 and half-maximal effects were achieved at 0.6 nM SK&F 104353. At 10 microM SK&F 104353 only 50% inhibition of the LTC4-induced increase in Isc was observed. These results suggest the peptidoleukotrienes stimulate colonic Cl- secretion by receptor-mediated mechanisms and that receptors for LTC4 are distinct from those mediating the action of LTD4/LTE4.     

Differential effects of calcium channel blockers on leukotriene C4- and D4-induced contractions in guinea pig pulmonary parenchymal strips

Leukotriene (LT)C4 and LTD4-induced contractile effects in guinea pig pulmonary parenchyma were distinguished by their sensitivity to calcium channel blockers. LTC4-induced contractions were inhibited in a noncompetitive manner in the presence of calcium channel blockers, whereas LTD4 contractions were unaffected. In the presence of diltiazem, maximum LTC4-induced (1 X 10(-7) M) contractions were reduced by 24% and the concentration-effect curve was shifted to the right in a nonparallel manner; diltiazem had no significant effect on the LTD4 response. We used this differential sensitivity to calcium channel blockade to permit pharmacological characterization in guinea pig pulmonary parenchyma of the interaction of the competitive LT blocker FPL55712 and the putative LTD4 receptor, LTRd. We showed, using [3H]LTC4, that at least 15% of LTC4 is converted to LTD4 under our experimental conditions. We performed a Schild analysis of the inhibition of LTD4-induced contractions by FPL55712 in the presence of the calcium channel blocker diltiazem (0.67 mM). The Kb derived from this analysis (3.2 X 10(-7) M) agrees closely with the Ki derived for the interaction of FPL55712 and specific LTD4 binding in lung membranes. A Schild analysis of the interaction of FPL55712 and LTC4 in the presence of diltiazem resulted in competitive inhibition with a Kb of 4.7 X 10(-7) M. This apparent competitive inhibition, combined with the similarity of these binding constants, suggests that diltiazem is effective in blocking LTC4-mediated responses and that when these effects are blocked, LTC4 induced contractions are mediated through LTRd. The differential effects of calcium blockade on these two agonists provides evidence for distinct coupling mechanisms for LT receptors in this tissue.     

Molecular heterogeneity of leukotriene receptors: correlation of smooth muscle contraction and radioligand binding in guinea-pig lung

The [3H]leukotriene C4 ([3H]LTC4) and [3H]leukotriene D4 ([3H] LTD4) specific binding sites in guinea-pig lung membranes were characterized and correlated with smooth muscle contractile activities of a series of LTC-, D- and E-type analogs. [3H]LTC4 bound to the specific sites with high affinity (dissociation constant Kd = 15 +/- 5 nM), saturable capacity (maximum binding = 68 +/- 15 pmol/mg of membrane protein), stereoselectivity and specificity. The [3H]LTC4 specific binding sites were detected in the membranes isolated from leukotriene sensitive (e.g., lung and heart) or insensitive (e.g., brain and red blood cells) tissues. [3H] LTD4 also bound to specific sites with high affinity (Kd = 0.20 +/- 0.05 nM), low capacity (maximum binding = 1.1 +/- 0.2 pmol/mg of membrane protein) stereoselectivity and specificity. The [3H] LTD4 specific binding sites were detected in the membranes isolated from lung and trachea. [3H]LTC4 specific binding was inhibited by treatment of the membranes with the sulfhydryl alkylating agent N-ethylmaleimide. [3H]LTD4 specific binding was more sensitive to heat treatment and p-hydroxymercuribenzoate than the [3H]LTC4 specific binding. Radioligand competition activities of the LTD- and LTE-type analogs correlated well with the agonist and antagonist smooth muscle contractile activities. In contrast, the radioligand competition activity of the LTC-type analogs did not correlate with smooth muscle contractile activities. These results indicate that the [3H]LTC4 and [3H]LTD4 specific binding sites in guinea-pig lung membranes are chemically and physically distinct. The [3H]LTD4 specific binding sites represent physiologically and pharmacologically important receptors, and the smooth muscle contraction induced by LTD-, and possible LTE-, type analogs are mediated through the LTD4 receptors.     

Negative inotropic effect of leukotrienes: leukotrienes C4 and D4 inhibit calcium-dependent contractile responses in potassium-depolarized guinea-pig myocardium

The effects of the sulfidopeptide leukotrienes (LTs) on the contractile response of electrically paced guinea-pig right ventricular papillary muscles in vitro were studied. LTs caused a concentration-dependent (1 nM-20 microM) negative inotropic effect; the order of relative potency was LTC4 greater than or equal to LTD4 greater than LTE4. A maximal 30% decrease in contractility occurred with 1 microM LTC4. The LT-induced decrease in contractile force was not mediated by cyclooxygenase products of the arachidonic acid cascade, as it was not influenced by indomethacin (14 microM). On the other hand, the slow-reacting substance-antagonist compound FPL 55712 (480 nM) caused a marked shift to the right of the LTC4 concentration-response curve. Because the negative inotropic effect of LTD4 was attenuated by increasing [Ca++]o, we next assessed the negative inotropic effect of LTs under conditions in which myocardial contractility depends solely on the slow inward Ca++ current. As a model, we used the isoproterenol- or histamine-induced restoration of contractile response in papillary muscles rendered inexcitable by 22 mMK+. LTC4 (16-480 nM) and LTD4 (20-600nM) inhibited isoproterenol- and histamine-induced restoration of contractility in a dose-dependent manner; a maximal 90% inhibition occurred with 0.48 microM LTC4. This effect of LTs was reversed by an elevation in [Ca++]o from 1.8 to 5.4 mM and prevented by FPL 55712 (480 nM). In muscles maintained at 5.4 mM [K+]o, LTC4 (160 and 480 nM) and LTD4 (1 microM) shifted the force-frequency curve (0.1-2 Hz) downwards in a parallel fashion; a similar alteration was obtained by lowering [Ca++]o to 1 mM.     

Leukotriene C4 binds to human glomerular epithelial cells and promotes their proliferation in vitro.

In human and experimental glomerulonephritis, glomerular hypercellularity results both from accumulation of macrophages and proliferation of resident glomerular cells. The recent identification of macrophage-derived factors that stimulate mesangial and epithelial cell proliferation suggests that these factors might contribute to the hypercellularity. To determine the identity of such macrophage-derived growth factors, we studied the effect of leukotrienes (LTs), products that are released from macrophages and leukocytes, on proliferation of human glomerular epithelial cells in culture. Dose-dependent (1-100 nM) stimulation of [3H]thymidine incorporation, an index of cell proliferation, was observed in cells incubated with the sulfidopeptide LTs, LTC4 and LTD4, but not with LTB4. The response was 248 and 172% of control values at 100 nM LTC4 and LTD4, respectively. This effect of LTC4 was abolished by FPL 55712. Subsequent binding studies demonstrated that glomerular epithelial cells possess specific receptors for LTC4. [3H]LTC4 bound rapidly at 8 degrees C to the cells. There was a plateau after 40 min incubation. Maximum specific binding was 70-90% of total binding. Specific binding was totally reversible with addition of an excess of unlabeled LTC4. Analysis of time-course association slopes at two concentrations of [3H]LTC4 and of the competition between a single concentration of [3H]LTC4 and increasing concentrations of unlabelled LTC4 allowed calculation of dissociation constants (Kd) of 220 and 217 nM, respectively. Both LTD4 and LTE4 exhibited ED50 values that were at least one order of magnitude higher than for LTC4. Thus, our findings suggest that LTC4 binds to specific receptors of glomerular epithelial cells, promotes proliferation of these cells, and could contribute to epithelial hypercellularity found in glomerulonephritis.     

Leukotriene D4 receptor-mediated phosphoinositol hydrolysis and calcium mobilization in rat basophilic leukemic cells

A basophilic leukemic cell line from rat (RBL-1) was used to characterize leukotriene D4 (LTD4) receptor-mediated biochemical and pharmacological effects. [3H]LTD4 binding to the plasma membrane enriched preparation was stereo-selective, specific and saturable. Sodium ions and guanine nucleotides specifically regulated [3H]LTD4 binding to the membrane receptors. Leukotriene E4 (LTE4) and high affinity specific antagonists bound to the receptor with a rank-order potency equivalent to that for the LTD4 receptors in guinea pig lung. In the [3]myoinositol labeled RBL-1 cells, LTD4 and LTE4 induced a rapid hydrolysis of [3H]phosphoinositides. The biosynthesis of the [3H]inositol-trisphosphate was rapid and was detectable at 15-sec poststimulation. The biosynthesis of [3H]inositol-monophosphate was stereo-selective and specific and was inhibited specifically by receptor antagonists. In fura-2 loaded RBL-1 cells, LTD4 and LTE4 induced a transient intracellular Ca++ mobilization. Agonist-induced Ca++ mobilization was specific and stereo-selective and was inhibited by specific receptor antagonists. The most (greater than 85%) LTD4-induced immediate response of Ca++ mobilization was from intracellular sources, whereas a small amount (less than 15%) was derived from the extracellular milieu. Both components were stimulated by receptor agonists and inhibited by the receptor antagonists, suggesting that they were regulated by the LTD4 membrane receptors. In addition, the results also suggested that a guanine nucleotide binding protein, insensitive to islet activating protein from Bordetella pertussis (not Gi or Go), was involved in the signal transduction mechanisms for LTD4 receptors in RBL-1 cells. These results suggested that the plasma membrane enriched LTD4 receptor was coupled via an islet activating protein insensitive G protein to a phosphoinositide specific phospholipase C. Agonist binding to the receptor could activate phospholipase C and resulted in phosphoinositide hydrolysis. Diacylglycerol and inositol trisphosphate could function as intracellular messengers that trigger or contribute to calcium mobilization in RBL-1 cells.     

Tissue distribution and functional correlation of [3H]leukotriene C4 and [3H]leukotriene D4 binding sites in guinea-pig uterus and lung preparations

To determine the distribution of leukotriene (LT) C4 and LTD4 receptors and functional significance of each receptor, we used [3H]LTC4 and [3H]LTD4 to measure the binding activity in various tissue homogenates and to correlate the relative ability of the LT agonists to inhibit binding with their contractile responses in guinea-pig uterine or lung parenchymal preparations. Guinea-pig brain contained the highest binding activity of 1 to 2 nM [3H] LTC4 followed by small intestine, heart, lung, kidney, uterus, etc., whereas guinea-pig lung had at least 2.7-fold greater [3H] LTD4 binding activity than any other tissues tested. In the brain and uterine homogenates, the rank order of potency for the compounds in competing with [3H]LTC4 for binding sites was LTC4 much greater than LTD4 greater than LTE4 greater than FPL-55712 = arachidonic acid. The in vitro functional study showed that the ability of the LT agonists to produce uterine contraction was in the order of LTC4 greater than LTD4 greater than LTE4, which is compatible with their relative effect for inhibition of uterine or brain [3H]LTC4 binding. In the lung homogenate, either LTC4, LTD4 or LTE4 inhibited effectively [3H]LTD4 binding and the potency order for the [3H]LTD4 competition study was LTD4 greater than LTE4 greater than LTC4 much greater than FPL-55712 greater than arachidonic acid. These LT agonists also produced lung contraction effectively and the difference among their contractile ability was not significant. We conclude that 1) there are distinct functional LTC4 and LTD4 receptors, 2) activation of the LTC4 receptor could account for the uterine contraction due to the LT agonists and 3) the lung contraction induced by LTC4, LTD4 and LTE4 is at least mediated partly by the LTD4 receptor.     

Metabolism of leukotriene C4, D4 and E4 in allergic inflammation in rats

Using an allergic inflammation model of air pouch type in rats, levels of peptide-leukotriene (LT) C4, D4 and E4 in the pouch fluid were measured chromatographically, and peptide-LT metabolizing activities in the pouch fluid in the anaphylactic phase were examined. 10 min after injection of an antigen (azobenzene arsonate-conjugated acetyl bovine serum albumin) solution into a preformed air pouch on the back of the immunized rats, LTC4 level in the pouch fluid was the highest, followed by LTD4 and LTE4. At 30 min, the order of the level was reversed to LTE4 greater than LTD4 greater than LTC4, and total amount of peptide-LTs (LTC4 + LTD4 + LTE4) was the highest. Supernatant fraction of the pouch fluid collected 30 min after the antigenic challenge, converted [3H]LTC4 into [3H]LTD4, and [3H]LTD4 into [3H]LTE4 in time- and concentration-dependent manner. [3H]LTE4 was not metabolized under these conditions. Heat denaturation of the pouch fluid diminished the conversion of [3H]LTC4 into [3H]LTD4, and [3H]LTD4 into [3H]LTE4. In the granule fraction of purified mast cells, no metabolic activity of [3H]LTs was found. In intact mast cells as well as degranulating mast cells, a small but significant amount of [3H]LTC4 was metabolized into [3H]LTD4 and [3H]LTE4. In contrast, rat serum showed potent metabolizing activities of peptide-LTs. Since plasma exudation into the pouch is very prominent in the anaphylactic phase in this model, peptide-LT metabolizing activities in the pouch fluid are suggested to be attributable to plasma leaked into the pouch during the anaphylactic phase.     

Lipoxygenation of arachidonic acid by differentiated and undifferentiated human promyelocytic HL-60 cells

The human promyelocytic leukemia cell line HL-60 can be induced to differentiate into mature granulocytes by exposure to dimethyl sulfoxide (DMSO), as well as into cells of monocyte-macrophage lineage by exposure to 12-0-tetra-decanoylphorbol-13-acetate (TPA). Incubation of the promyelocytic leukemic HL-60 cells and the two chemically induced differentiated cells with carbon 14-labeled arachidonic acid overnight resulted in the incorporation of label mainly into cellular phospholipids. Stimulation of the labeled cells with Ca2+ ionophore (A23187) resulted in the generation of lipoxygenase products in all the cells. Notably, the DMSO-induced differentiated granulocytic cells biosynthesized predominantly the peptidoeicosatetraenoic acids LTC4 and LTD4 compared with the dihydroxyeicosatetraenoic acid LTB4. On the other hand, the TPA-induced differentiated monocytic-macrophage cells biosynthesized predominantly LTB4 compared with LTC4 and LTD4. The undifferentiated promyelocytic cells lacked the capacity to biosynthesize LTB4, as evidenced in the chemically induced differentiated cells. The monohydroxyeicosatetraenoic acid (HETE) 5-HETE, a metabolite of the 5-lipoxygenase pathway, was a minor component in these cells, and 12-HETE and 15-HETE were barely detectable. These results suggest that the two models of DMSO-induced and TPA-induced differentiated cells may be useful systems for further investigations of arachidonic acid metabolites in granulocytic and monocytic-macrophage functions.     

The bronchopulmonary pharmacology of SK&F 104353 in anesthetized guinea pigs: demonstration of potent and selective antagonism of responses to peptidoleukotrienes

The bronchopulmonary pharmacology of SK&F 104353 [2(S)-hydroxy-3(R)-[2(2-carboxyethyl)thio]-3[2-(8- phenyloctyl)phenyl]-propanoic acid], a potent and selective leukotriene (LT) receptor antagonist in vitro, was assessed in anesthetized, spontaneously breathing guinea pigs. Aerosol administration of SK&F 104353 (5-2000 micrograms/ml x 100 breaths) reduced in a concentration-dependent manner the response to a standard LTD4 challenge (4.33 micrograms/ml x 5 breaths) given 30 min later. Inhalation of a 2000 micrograms/ml solution abolished LTD4-induced bronchoconstriction for at least 2 hr. The i.v. administration of SK&F 104353 10 min before challenge antagonized LTD4-induced bronchoconstriction with an ID50 of 0.55 mumol/kg (0.25 mg/kg). Substantial antagonism of LTD4-induced bronchospasm was observed for at least 60 min after i.v. administration of 5 mumol/kg of SK&F 104353. Infusion of SK&F 104353 at various rates revealed that a steady-state plasma concentration of 0.125 microM (0.06 micrograms/ml) reduced LTD4-induced bronchoconstriction by 60%. In addition to preventing the response to LTD4, i.v. administered SK&F 104353 (10 mumol/kg) rapidly and completely reversed ongoing LTD4-induced bronchoconstriction. SK&F 104353 also was effective when given intraduodenally 1 hr before LTD4 challenge, although the ID50 (32 mumol/kg) was 60-fold greater than the i.v. ID50. Given intragastrically, 100 mumol/kg of SK&F 104353 abolished the response to LTD4 for 1 hr, and reduced the response for 6 hr. SK&F 104353 (20 mumol/kg i.v.) had no effect on the bronchoconstriction induced by aerosolized acetylcholine, histamine or U-44069, but did antagonize the response to LTC4. SK&F 104353 alone did not produce bronchoconstriction when administered by any route or dose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Guinea pig gall bladder contains a single peptide leukotriene receptor which resembles that present in human conducting airway smooth muscle.

The peptide leukotrienes (LTs), LTC4, LTD4 and LTE4, produced concentration-related contraction of isolated strips of guinea pig gall bladder (GPGB) with pD2 values of 8.48 +/- 0.11, 8.26 +/- 0.22 and 7.28 +/- 0.08, respectively. In the presence of propranolol and indomethacin in epithelium-denuded GPGB, pKB values for the LT antagonist ICI198,615 vs. LTC4, LTD4 and LTE4 were, respectively: 8.61 +/- 0.12, 8.89 +/- 0.13 and 8.70 +/- 0.17. Similarly, pKB values for the LT antagonist SKF104,353 were 7.28 +/- 0.19, 7.88 +/- 0.17 and 7.45 +/- 0.17. Previous studies have shown that inhibition of LTC4 metabolism can alter the apparent affinity of the LT, especially LTC4; consequently, metabolism of [3H]LTC4 in chopped GPGB was investigated. [3H]LTC4 was converted rapidly by gamma-glutamyl transpeptidase to [3H]LTD4 with little accumulation of [3H]LTE4. The combination of acivicin (ACI) and reduced glutathione (GSH) provided complete inhibition of gamma-glutamyl transpeptidase. pD2 values for LTC4 in the presence of ACI/GSH were 8.54 +/- 0.16. pKB values for ICI198,615 and SKF104353 in the presence of ACI/GSH were, i.e., 8.42 +/- 0.14 and 7.53 +/- 0.12, respectively, vs. LTC4 and identical to those obtained in the absence of inhibitors. The mechanism(s) underlying LT-induced contraction was evaluated using the calcium channel blockers diltiazem, nifedipine and verapamil. Noncompetitive antagonism was observed against all LTs indicating the involvement of voltage-sensitive calcium channels in the contractile response. Similarly, all LTs increased polyphosphoinositide hydrolysis. These data indicate: 1) GPGB contains a single type of LT receptor similar to that in human airways and 2) LT-induced contraction appears to involve both polyphosphoinositol formation and voltage-dependent Ca++ channels.     

Evidence that peptidoleukotriene is a prerequisite for antigen-dependent thromboxane synthesis in IgG1-passively sensitized guinea pig lungs

Lungs from guinea pigs passively sensitized with an affinity-purified IgG1 antibody produce both leukotriene (LT)D4 and thromboxane (Tx)B2 upon ex vivo antigen challenge. This study was undertaken to determine the possibility of endogenously generated peptido-LTs being a prerequisite for Tx synthesis. In immunoglobulin G1-sensitized lungs, exogenous LTD4 induced TxB2 production with a median effective dose of 4.1 nM, whereas the response to LTE4, LTB4 or platelet-activating factor was relatively weak. Although LTC4 was as potent as LTD4 in stimulating TxB2 generation, LTC4's dose-response curve was shifted significantly to the right by AT-125, an irreversible gamma-glutamyl transpeptidase inhibitor, suggesting that at least a part of LTC4 sensitized lungs with antigen (0.01-30 micrograms/ml ovalbumin) for 20 min precipitated a significant amount of LTD4 production. The levels of LTD4 range from 8 to 26 nM (without taking LTD4 recovery into consideration). This level is 2- to 7-fold greater than the median effective dose value observed with exogenous LTD4. Moreover, pretreatment of sensitized lungs with ICI-198,615 a specific LTD4 antagonist, blocked equally both antigen (IC50 = 0.01 microM)- and LTD4 (IC50 = 0.017 microM)-induced TxB2 production. When sensitized lung fragments were treated with 5 mM AT-125, ICI-198,615 was effective in preventing not only antigen-but also LTC4-dependent production of TxB2 (IC50 = 0.018 and 0.021 microM, respectively). In contrast, neither WEB-2086, a platelet-activating factor antagonist, nor pyrilamine, a histamine antagonist, inhibited antigen and LTD4 responses (IC50 greater than 30 microM). Unlike its effect on antigen response, ICI-198,615 was unable to block Ca2+ ionophore-induced TxB2 production.2     

Endothelin-1 does not modulate O2· release and [Ca2+]i variations in resting or differentiated HL-60 cells

Summary— Endothelin-1 (ET-1) by itself was not an effective stimulus for inducing superoxide (O₂*) generation in human resting or DMSO-differentiated neutrophil-like HL-60 cells. ET-1 (0.01 – 100 nM) was not able to modulate O₂* generation stimulated by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP, EC₅₀ = 4.24 ± 1.63 nM in the absence and 3.16 ± 1.95 nM in the presence of ET-1). Neither did ET-1 (0.01 – 100 nM) promote the mobilization of intracellular calcium ions or modulate fMLP-induced [Ca²⁺]_i increase in this model of human neutrophils. Phosphoramidon, a neutral endopeptidase inhibitor, was not able to reveal any biological (O₂*) or biochemical ([Ca²⁺]_i) response to ET-1 in the absence or in the presence of fMLP in these cells. These results indicate that DMSO-differentiated neutrophil-like HL-60 cells are not sensitive to ET-1 in terms of O₂* generation or [Ca²⁺]_i variations.