IMMUNOCYTOCHEMICAL DETECTION OF THE INTRANUCLEAR VARIATIONS OF PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE AMOUNT ASSOCIATED WITH CHANGES OF ACTIVITY AND AMOUNT OF PHOSPHOLIPASE-C BETA(1) IN CELLS EXPOSED TO MITOGENIC OR DIFFERENTIATING AGONISTS

The intracellular localizations of phosphatidylinositol 4,5-bisphospha te (PIP2) and of its hydrolyzing enzyme phospholipase C (PLC; in this case the beta(1) isoform) have been evaluated by electron microscope i mmunocytochemistry in cells exposed to mitogenic or differentiating ag ents. These cells have been previously demonstrated to present a signa l transduction system based on the polyphosphoinositide hydrolysis loc alized at the nuclear level, which can be specifically modulated by ag onists. The results demonstrate that in Swiss 3T3 mouse fibroblasts mi togenically stimulated by insulin-like growth factor I (IGF-I), a rapi d and transient decrease of the PIP2 detectable by immunogold labeling occurs at the nuclear interior. This effect appears due to the activa tion of the PLC beta(1) isozyme already present in the nucleus, since no significant variations of the enzyme amount and distribution can be detected by immunolabeling. However, after 30 min of exposure to IGF- I, when the PLC beta(1) activity is returned to basal level, a slight but significant increase of the enzyme amount is detected both in the nucleus and in the cytoplasm. On the other hand, an increased accumula tion of PIP2 in the nucleus, accompanied by a decrease of the intranuc lear amount of PLC beta(1) isozyme, have been observed in mouse erythr oleukemia Friend cells, induced to erythroid differentiation by dimeth ylsulfoxide (DMSO). These results indicate that quantitative immunocyt ochemistry represents an increment in the available methodologies to i nvestigate the complex regulation of nuclear PI-signalling.