European Journal of Pharmacology, 147 (1988) 387-396 387 Elsevier EJP 50171 Leukotriene D4-induced activation of protein kinase C in rat basophilic leukemia cells Raju V.K. Vegesna, Seymour Mong * and Stanley T. Crooke Department of Molecular Pharmacology, Smith Kline & French Laboratories, P.O. Box 1539, King of Prussia, PA 19406-0939, U.S.A. Received 7 September 1987, revised MS received 16 December 1987, accepted 22 December 1987 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 D 4 (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 rain. 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,phenyl]propanoic acid (SK&F 104353), a high affinity specific LTD 4 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 LTD 4 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. Leukotriene D4 (LTD4); Protein kinase C (PKC); Leukotriene D4 receptors; Protein kinase C translocation 1. Introduction In a variety of biological systems, the transfer of signals from the cell surface to the cell interior is mediated through the receptor-stimulated rapid breakdown of phosphoinositides (PI) resulting in the generation of second messengers, diacylglycer- ol (DG) and inositol 1,4,5-trisphosphate (IP3) (Berridge and Irvine, 1984; Michell, 1984). The * To whom all correspondence should be addressed: Depart- ment of Molecular Pharmacology (L-108), Smith Kline & French Laboratories, P.O. Box 1539, King of Prussia, PA 19406-0939, U.S.A. DG that is transiently produced during receptor stimulation is a potential activator of Ca 2+, phos- pholipid-dependent protein kinase C (PKC) char- acterized by Nishizuka and his colleagues (Takai et al., 1977). This widespread, multifunctional en- zyme has been suggested to play a vital role in several intracellular control mechanisms (Kikkawa and Nishizuka, 1986). Tumor-promoting phorbol esters such as 4fl-phorbol-12fl-myristate-13a- acetate (PMA) activate PKC directly and can be substituted for DG both in vitro and in vivo (Castagna et al., 1982; Yamanishi et al., 1983). Translocation of PKC from the cytosol to the particulate fractions has been suggested to be an obligatory step in the activation of PKC by tumor 0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)