IONIC SIGNALING DURING QUIESCENT FIBROBLAST ACTIVATION

Growth factors are polypeptides that initiate mitogenesis in quiescent
fibroblasts and other cell types. Ultimate control of DNA synthesis
initiated by growth factors must occur in the nucleus, probably by specific
but as yet unknown gene products. However, because growth factors are
initially received by specific receptors at the cell surface, it may
reasonably be hypothesized that cytoplasmic signals are necessary as
informational intermediaries between plasma membrane and nucleus. The
proposed experiments address the possible role of cytoplasmic Ca++ and pH
as cytoplasmic signals for growth factors and certain cell cycle events.
Intracellular free calcium ion concentration ([Ca++]i) will be measured
using the calcium sensitive photoprotein, aequorin; intracellular pH (pHi)
using the fluorescein fluorescence ratio technique. Moreover, several
techniques for loading otherwise impermeant molecules into living cell
cytoplasm will be put to novel experimental use, and a new technique for
bulk-loading will be developed. Some of the questions asked are: 1) is a
rise in [Ca++]i necessary and/or sufficient for initiating DNA synthesis;
2) can DNA synthesis be synergistically activated in quiescent cells by
raising [Ca++]i and activating protein kinase C; 3) does inositol
1,4,5-triphosphate (IP3) release Ca++ from internal stores of living,
quiescent fibroblasts and is IP3 mitogenic; 4) what are the intracellular
sites of growth factor- and IP3-induced changes in [Ca++]i; 5) do changes
in [Ca++]i and pHi occur during fibroblast transit through the cell cycle
and, if so, how do they compare in timing; 6) do experimentally induced
rises in pHi result in initiation of DNA synthesis by quiescent cells; and
7) are there differences between resting and/or growth factor-stimulated
[Ca++]i and pHi of normal versus transformed fibroblasts. The proposed
studies address problems fundamental to understanding the mitogenic
response of mammalian cells, including the chemical identity of possible
cytoplasmic signals for mitogenesis, the concentrations involved and
location in the cytoplasm, whether or not these putative signals are
sufficient and/or necessary by themselves for mitogenesis, and activity of
these signal molecules in transformed cells not responsive to growth
factors.