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At subnanomolar concentrations, vasoactive intestinal peptide (VIP) can act as an astroglial mitogen and as a secretagogue for neurotrophic substances released from glia (Brenneman et al.: J Neurosci Res 25:386–394, 1990). Here we report that treatment with subnanomolar (0.1 nM) VIP, that does not produce an increase in intracellular cAMP levels, induced the translocation of protein kinase C (PKC) from the cytoplasm to the nucleus in neonatal cortical astrocytes, as revealed by immunohistochemistry, Western blot analysis, and measurements of the enzyme activity. Western blot analysis of subcellular fractions, using PKC isotype-specific antisera, showed PKC alpha as well as the two novel PKC isotypes, delta and zeta immunoreactivities, whereas PKC beta or gamma immunoreactivities were not detected. PKC alpha was associated predominantly with the cytosolic compartment, while PKC delta was found in the plasma membrane and in nuclear fractions. In contrast, PKC zeta was distributed ubiquitously within the major subcellular fractions. Treatment of the cells with 0.1 nM VIP caused a marked increase in nuclear PKC alpha and, to a lesser extent, PKC delta and PKC zeta immunoreactivities. Western blot analysis showed that a low (1 nM) concentration of phorbol, 12-myristate, 13 acetate also caused the subcellular redistribution of PKC immunoreactivities from the cytoplasm to the nuclear fraction, similar to VIP treatment. Exposure of astrocytes to high concentrations (1 μM) of phorbol, 12-myristate, 13 acetate resulted in the down-regulation of PKC alpha and PKC delta, while distribution of PKC zeta immunoreactivities were only slightly altered. Measurements of Ca2+- and phospholipid-dependent PKC activities also showed a VIP-induced redistribution of PKC activity from the cytoplasmic to the nuclear fractions. These results suggest that PKC may be involved in the signal transduction process elicited by VIP binding to the high affinity VIP receptors present on cortical astrocytes. The observed changes in the nuclear localization of PKC alpha and PKC delta in response to subnanomolar VIP may play a role in mediating the cellular response(s) to this peptide during neurodevelopment. © 1994 Wiley-Liss, Inc.1 |
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