Membrane depolarization induced DNA synthesis in PU5-1.8 cells. Role of voltage-operated Ca2+ channel and protein kinase C.
Incubation of non-excitable murine macrophage cell line PU5-1.8 in K+(140 mM)-Hepes buffer caused membrane depolarization as measured by bis-oxonol and 3,3'-dipropylthiodicarbocyanine. Depolarization of cell membrane produced an increase of intracellular free Ca2+ concentration ([Ca2+]i). Interestingly, short-term exogenously imposed membrane depolarization in PU5-1.8 cells in the absence of growth factors initiated the incorporation of [3H]thymidine or its analog, 5-bromo-2'-deoxyuridine, as well as the expression of early-response genes such as c-fos and c-myc. The expression of the proto-oncogene products seemed to be dependent on external Ca2+, and the [3H]thymidine incorporation was inhibited by nifedipine and verapamil at similar dosages as for Ca2+ inhibition. Calcium influx and [3H]thymidine incorporation could also be induced by Bay K 8644 or by gramicidin in the Na(+)-Hepes buffer. On the other hand, challenge of cells with sphingosine, staurosporine or 1-(5-isoquinolinesulfonyl)-2-methylpiperazine suppressed the K(+)-mediated DNA synthesis. Furthermore, preincubation of cells with phorbol 12-myristate 13-acetate (PMA) for 15 min in Na(+)-Hepes buffer enhanced the DNA synthesis. Yet, pre-incubation of cells with PMA or 1,2-dioctanoyl-sn-glycerol in K(+)-Hepes buffer suppressed the K(+)-mediated DNA synthesis and membrane depolarization. These observations suggest that a rise of [Ca2+]i is required for the initiation of DNA synthesis, and PKC may be functioning as a modulator with dual action at least on the level of ion conductance.
Kong
S K
SK
Department of Biochemistry, Chinese University of Hong Kong, Shatin, NT.
Choy
Y M
YM
Fung
K P
KP
Lee
C Y
CY
D000818:Animals; D015220:Calcium Channels / Q000378:metabolism*; D002455:Cell Division; D002460:Cell Line; D004247:DNA / Q000096:biosynthesis*; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D008264:Macrophages / Q000166:cytology / Q000378:metabolism*; D008564:Membrane Potentials; D051379:Mice; D008807:Mice, Inbred BALB C; D011188:Potassium / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism
D015220:Calcium Channels; D004247:DNA; D011493:Protein Kinase C; D011188:Potassium