Specific Induction of LTC4 Synthase by Retinoic Acid in Rat Basophilic Leukemia-1 Cells

Abstract

Overnight (10–16 h) incubation of retinoic acid (RA), a derivative of vitamin A, specifically induced LTC4 synthase activity (5 to 10-fold), but not LTA4 hydrolase activity in the lysate of rat basophilic leukemia-1 (RBL-1) cells. A time course study revealed that the increase of LTC4 synthase activity was time dependent and that the peak value was obtained after a 24-hour incubation with RA. The induction of enzyme activity was specifically localized to the microsomal fraction. Glutathione (GSH) S-transferase activity measured by using the same cell lysate as an enzyme source and 1-chloro-2,4-dinitrobenzene (DNCB) as a substrate was not influenced by RA treatment, indicating that the induction by RA is specific for membrane-bound LTC4 synthase. The induction of LTC4 synthase may be an important regulatory mechanism of peptide-LT synthesis in allergy and inflammatory diseases.

Inhibition of Leukotriene Production by FK506 in Rat Basophilic Leukemia-1 Cells

“…Mo.) was added to the medium at a final concentration of 0 .1 pg/ml, and cells were incu bated at 37 °C for 16 h to induce LTC4 synthase activity [11,12]. Cells were then distributed into 3.5-cm-diameter petri dishes at a density of 2 x 106/ml.…”

Inhibition of Leukotriene Synthesis by Honokiol in Rat Basophilic Leukemia Cells

“…RBL-1 cells produce LTC4 and LTB4 in amounts sufficiently large to be measured by high-performance liquid chromatography (HPLC) after induction of LTC4 synthase activity by treating with all /raw.s-retinoic acid (RA) at 0.1 ug/ml for 16 h and sub sequent stimulation with A23187 [13][14][15].…”

“…Honokiol (Wako Pure Chemical, Osaka, Japan) was added to the medium at various concentrations, and cells were incubated for 10 min before exposure to 10 pA/ A23187 for 15 min at 37°C. The production o f LTC4 and LTB4 in the medium was measured by HPLC as described previously [14], In brief, the sample was partially purified and concentrated by passing through a Sep Pak Cl 8 cartridge and injected into an HPLC system consisting of a CCPM pump, a UV-8010 absorbance detector (Toso, Japan) and a Novapak CI8 column (Millipore Waters, Milford, Mass., USA). The solvent, acetonitrile:methanol:water:acetic acid [31;17.08:51.24:0.68 (v/v)], adjusted to pH 4.5 with NH4OH, was delivered at 0.8 ml/min, and the elution was monitored at 280 nm.…”

“…Cell lysate was prepared from RA-treated cells in the same way as previously described [14]. Two microliters of 0.1 M AA were added to 198 pi of reaction buffer containing the cell lysate (50 pi), 3 m M glutathione, 0.1 mM ATP, and 2 mM CaCL, and the mixture was incubated at 37°C for 5 min in the absence or presence of various con centrations of honokiol.…”

“…LTA4 (free acid) was prepared from LTA4 methylester as previously described [14]. RA-treated cells (2 X KT/ml) were incubated with LTA4 (free acid; 2 pg) at 37°C for 5 min in the absence or presence o f 10 pAf honokiol.…”