Arachidonic acid (20:4(n-6)), which is released by cells responding to a wide range of stimuli, may play an important role in intracellular signaling. We now report that incubation of WB cells with 20:4(n-6) resulted in the appearance of several tyrosine-phosphorylated cytosolic proteins. Two of the phosphotyrosine-containing proteins, migrating in SDS-polyacrylamide gels of approximately 43 and 45 kDa, corresponded in mobility to phosphorylated species of the 42- and 44-kDa mitogen-activated protein kinase (MAPK) isoforms. Immunoblots of soluble fractions from unstimulated WB cells with anti-MAPK antibodies revealed the presence of the 42- and 44-kDa isoforms of MAPK. Upon incubation with 20:4(n-6), the mobility of both isoforms was retarded, consistent with their activation by phosphorylation. Chromatography of soluble fractions from these cells on Mono Q columns revealed early and late eluting peaks of myelin basic protein kinase activity, which contained the 42- and 44-kDa MAPK isoforms, respectively. Activation of MAPK was transient, peaking at 5 min, and was detectable at 5 microM 20:4(n-6). Further studies into the mechanisms by which MAPK was activated by 20:4(n-6) strongly suggested the involvement of protein kinase C (PKC). Not only did incubation of WB cells with 20:4(n-6) result in the translocation of PKC alpha, delta, and epsilon to a particulate fraction, it was found that the fatty acid failed to activate MAPK in cells pretreated for 26 h with phorbol 12-myristate 13-acetate, which depleted WB cells of PKC alpha, delta and epsilon. In addition, fatty acids of the n-3 series were effective activators of MAPK. The present study, to our knowledge, is the first to report that polyunsaturated fatty acids can cause the activation of MAPK.

The novel lipid second messenger, ceramide, induced apoptosis in PC 12 cells as determined morphologically by nuclear appearance and internucleosomal DNA fragmentation. Apoptosis was induced by exogenous CVceramide in a dose-and time-dependent manner. Natural ceramide and C 6 -ceramide had a similar effect. This response was specific since the structural analog C 2 -dihydroceramide and other related lipids failed to initiate apoptosis. The apoptotic effect of ceramide also depends critically on cell plating density. Furthermore, the peptide inhibitor of interleukin-lß converting enzyme (ICE)-like proteases, Z-VAD.FMK, completely prevented the nuclear changes induced by ceramide, implicating the involvement of ICE-like protease activation in ceramide-induced apoptosis in PC12 cells.

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Insulin‐like growth factor (IGF) binding to the type 1 IGF receptor (IGF1R) elicits mitogenic effects, promotion of differentiation and protection from apoptosis. This study has systematically measured IGF1R binding affinities of IGF‐I, IGF‐II and 14 IGF analogues to a recombinant high‐affinity form of the IGF1R using BIAcore technology. The analogues assessed could be divided into two groups: (a) those designed to investigate binding of IGF‐binding protein, which exhibited IGF1R‐binding affinities similar to those of IGF‐I or IGF‐II; (b) those generated to probe IGF1R interactions with greatly reduced IGF1R‐binding affinities. The relative binding affinities of IGF‐I analogues and IGF‐I for the IGF1R determined by BIAcore analysis agreed closely with existing data from receptor‐binding assays using cells or tissue membranes, demonstrating that BIAcore technology is a powerful tool for measuring affinities of IGFs for IGF1R. In parallel studies, IGF1R‐binding affinities were related to ability to protect against serum withdrawal‐induced apoptosis in three different assays including Hoechst 33258 staining, cell survival, and DNA fragmentation assays using the rat pheochromocytoma cell line, PC12. In this model system, IGF‐I and IGF‐II at low nanomolar concentrations are able to prevent apoptosis completely. We conclude that ability to protect against apoptosis is directly related to ability to bind the IGF1R.