Wortmannin

A growing body of evidence suggests that mitochondria take up calcium upon receptor (agonist) stimulation and that this contributes to the dynamics of spatiotemporal calcium signaling. We have previously shown that engagement of the high-affinity receptor for immunoglobulin E (FcepsilonRI) stimulates mitochondrial calcium ([Ca2+]m) uptake in mast cells. The present study was undertaken to investigate the mechanisms and biological significance of FcepsilonRI regulation of [Ca2+]m. Antigen stimulated [Ca2+]m uptake in a dose-dependent manner with a minimal effective dose of 0.03-3 ng/ml. This [Ca2+]m uptake took place immediately, reaching its peak within minutes and was inhibited by the src family kinase inhibitor PP1 and phosphatidylinositol-3-kinase inhibitor wortmannin. Analyses using mast cells expressing the wild-type or the mutated type of the FcepsilonRIbeta immunoreceptor tyrosine-based activation motif (ITAM) in which all tyrosine residues were replaced by phenylalanine revealed that the FcepsilonRIbeta ITAM is essential for a sustained [Ca2+]m uptake. The FcepsilonRIbeta ITAM was essential for overall calcium response upon weak FcepsilonRI stimulation (at low antigen concentration), while upon strong stimulation (at high antigen concentration) it appeared necessary selectively to an immediate calcium response that was sensitive to the dihydropyridine receptor (DHPR) antagonist nifedipine and wortmannin but not to the store-operated calcium entry (SOCE) antagonists such as 2-aminoethoxyphenyl borate and SK&F96365.

To elucidate the role of phosphatidylinositol-3-kinase (PI3K) during macrophage activation, we examined the effects of wortmannin, a specific inhibitor of PI3K, on the induction of nitric oxide (NO) synthesis and tumor necrosis factor-alpha (TNF-alpha) secretion from lipopolysaccharide (LPS)-stimulated macrophages. Wortmannin had no effects on NO synthesis and TNF-alpha secretion by itself. Wortmannin markedly potentiated the LPS-induced NO production in a dose-dependent manner. Western blot analysis demonstrated that significantly increased levels of iNOS protein were expressed in LPS-stimulated macrophages treated with wortmannin, compared to those without LPS. Furthermore, enhancement of TNF-alpha secretion was observed in the initiation stage for activation of LPS-stimulated macrophages treated with wortmannin. These results suggest that PI3K plays an important role in transducing the signal that is involved in LPS-induced macrophage activation.

The synergism between insulin and prolactin (PRL) in their effect on protein synthesis in the mammary gland was studied in differentiating mammary epithelial CID-9 cells. Both hormones were needed to induce phosphorylation of PHAS-I which resulted in its dissociation from the eIF-4E translation initiation factor. This step is crucial for the initiation of translation. The induction of PHAS-I phosphorylation was rapid and its rate matched that demonstrated for the JAK2/STAT5a and the binding of STAT5a to its DNA binding motif. However, 120 min was needed for complete phosphorylation of the PHAS-I protein. In the presence of insulin, PRL induced MAP kinase activity, initiated at a comparable rate to that of PHAS-I phosphorylation. However, a line of evidence suggested that although this kinase phosphorylates PHAS-I in vitro, it does not actively participate in its phosphorylation in vivo: (a) the level of insulin needed to enable PRL-induced ERK-1/ERK-2 activation was one order of magnitude higher than that needed for PHAS-I phosphorylation; and (b) PD 098059, a MEK-1 inhibitor, completely inhibited insulin-dependent, PRL-induced ERK-1/ERK-2 activation but had no effect on the PRL-induced PHAS-I phosphorylation.