The binding of leukotriene C4 and leukotriene D4 to membranes of a smooth muscle 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.
Clark
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Cook
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Mong
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Crooke
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D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002867:Chromones / Q000494:pharmacology; D016861:Cyclooxygenase Inhibitors; D007700:Kinetics; D051379:Mice; D009119:Muscle Contraction / Q000187:drug effects; D009124:Muscle Proteins / Q000502:physiology*; D009130:Muscle, Smooth / Q000378:metabolism*; D012313:RNA / Q000096:biosynthesis*; D011863:Radioimmunoassay; D013189:SRS-A / Q000378:metabolism*; D013931:Thromboxanes / Q000502:physiology*
D002867:Chromones; D016861:Cyclooxygenase Inhibitors; D009124:Muscle Proteins; D013189:SRS-A; D013931:Thromboxanes; D012313:RNA; C006312:FPL 55712