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Membrane depolarization was required to induce DNA synthesis in murine macrophage cell line PU5-1.8
Abstract:
The role of membrane potential (Em) on the initiation of DNA synthesis in murine macrophage cell line PU5-1.8 was investigated with fluorescent probes bis-oxonol and diS-C3-(5). Incubation of PU5-1.8 cells in high K(+)-HEPES buffer or with gramicidin at 37 degrees C for 1h that depolarized the membrane induced [3H]-thymidine incorporation and expression of early response gene such as c-myc and c-fos. When PU5-1.8 cells were treated with a number of agents including fetal calf serum (FCS), lipopolysaccharide (LPS), epidermal growth factor (EGF), N-formyl-methionyl-leucyl-phenylalanine (FMLP) and bradykinin (BK), only FCS caused DNA synthesis and membrane depolarization. Other agents had no effect on these events. The FCS-mediated DNA synthesis in PU5-1.8 cells was inhibited by clamping the membrane potential with valinomycin. Moreover, intracellular alkalinization induced by nigericin at pH 7.9, which is believed to be a permissive signal for mitogenesis, caused membrane depolarization. On the other hand, challenge of cells with phorbol 12-myristate 13 acetate (PMA) suppressed the K(+)-mediated DNA synthesis. However, the treatment of cells with PMA did not change the membrane potential but suppressed the gramicidin-mediated membrane depolarization. These observations suggest that there is a correlation between membrane depolarization and initiation of DNA synthesis in PU5-1.8 cells. PKC may be acting as a modulator in this transducing pathway.
Insights
Membrane depolarization, a change in cell electrical charge, triggers DNA synthesis and gene expression in PU5-1.8 macrophages. This process is linked to mitogenesis and modulated by protein kinase C (PKC).
Area of Science:
- Cell Biology
- Molecular Biology
- Immunology
Background:
- Membrane potential (Em) plays a crucial role in cellular functions.
- The relationship between Em and DNA synthesis initiation in macrophages requires further elucidation.
- Early response genes like c-myc and c-fos are involved in cell proliferation.
Purpose of the Study:
- To investigate the role of membrane potential (Em) in initiating DNA synthesis in murine macrophage cell line PU5-1.8.
- To explore the correlation between membrane depolarization and the expression of early response genes.
- To understand the involvement of signaling pathways, including protein kinase C (PKC), in this process.
Main Methods:
- Utilized fluorescent probes (bis-oxonol and diS-C3-(5)) to measure membrane potential.
- Induced membrane depolarization using high K(+)-HEPES buffer or gramicidin.
- Assessed DNA synthesis via [3H]-thymidine incorporation and measured gene expression (c-myc, c-fos) using various stimuli (FCS, LPS, EGF, FMLP, BK, PMA).
Main Results:
- Membrane depolarization induced by high K(+) or gramicidin stimulated [3H]-thymidine incorporation and c-myc/c-fos expression.
- Fetal calf serum (FCS) was the only agent that induced both DNA synthesis and membrane depolarization.
- Valinomycin inhibited FCS-mediated DNA synthesis, while nigericin-induced alkalinization caused depolarization; PMA modulated depolarization-induced DNA synthesis.
Conclusions:
- A strong correlation exists between membrane depolarization and the initiation of DNA synthesis in PU5-1.8 cells.
- Membrane potential changes are critical for mitogenesis in these macrophages.
- Protein kinase C (PKC) appears to modulate the signal transduction pathway linking membrane potential to DNA synthesis.