Related Experiment Videos
Blockers of platelet-derived growth factor-activated nonselective cation channel inhibit cell proliferation
F Jung1, S Selvaraj, J J Gargus
1Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322.
Abstract:
In serum-deprived G(o)-arrested cells, the addition of serum or growth factors initiates a cascade of events that culminates in DNA synthesis and mitosis. Recently, we showed that in mouse L-M(TK-) fibroblasts a 28-pS nonselective cation channel (NS channel) becomes quiescent at G(o) arrest and rapidly active within seconds of platelet-derived growth factor (PDGF) or serum addition, placing this response very early in the postreceptor signaling cascade. However, lack of specific channel blockers hindered determination of whether channel activation was necessary for mitogenesis. Derivatives of N-phenylanthranilic acid (DCA) have been reported to block a pancreatic nonselective channel. Therefore, using single-channel analysis, we examined the effect of these agents on the L-M(TK-) NS channel. Flufenamic acid and mefenamic acid rapidly produced reversible channel block with an inhibitory constant (Ki) approximately 10 microM. Furthermore, the component of the macroscopic K+ efflux shown to be mediated by the NS channel was blocked with a similar Ki value. DCA effects on cell proliferation were tested by measuring cloning efficiency and growth rate. Both were inhibited over the range of concentration that affected channel activity, and a 50% inhibitory dose of 50-100 microM was determined. This observation further substantiates the hypothesis that NS channel activation forms a necessary component in the transduction of the mitogenic signal from the PDGF receptor.
Insights
Activation of a nonselective cation channel (NS channel) is crucial for cell proliferation following growth factor stimulation. Blocking this channel with N-phenylanthranilic acid derivatives inhibits mitogenesis, confirming its role in signal transduction.
Area of Science:
- Cell biology
- Molecular signaling
- Ion channel physiology
Background:
- Serum or growth factor addition to quiescent cells triggers a cascade leading to DNA synthesis and mitosis.
- A 28-pS nonselective cation channel (NS channel) in mouse fibroblasts rapidly activates upon growth factor stimulation, preceding mitogenesis.
- The necessity of NS channel activation for mitogenesis remained undetermined due to the lack of specific blockers.
Purpose of the Study:
- To investigate the role of the NS channel in mitogenesis.
- To determine if NS channel activation is a necessary step in the signal transduction pathway initiated by platelet-derived growth factor (PDGF).
- To evaluate the effect of N-phenylanthranilic acid derivatives (DCAs) on the NS channel and subsequent cell proliferation.
Main Methods:
- Single-channel patch-clamp analysis was used to assess the effects of flufenamic acid and mefenamic acid on the L-M(TK-) NS channel.
- Macroscopic potassium (K+) efflux mediated by the NS channel was measured.
- Cell proliferation was assessed by measuring cloning efficiency and growth rate in the presence of DCAs.
Main Results:
- Flufenamic acid and mefenamic acid caused rapid, reversible block of the NS channel with an inhibitory constant (Ki) around 10 microM.
- The K+ efflux mediated by the NS channel was inhibited by DCAs with a similar Ki value.
- DCA treatment inhibited both cloning efficiency and growth rate of cells in a dose-dependent manner, with a 50% inhibitory dose of 50-100 microM.
Conclusions:
- NS channel activation is a necessary component in the signal transduction pathway from the PDGF receptor to mitogenesis.
- N-phenylanthranilic acid derivatives effectively block the NS channel and inhibit cell proliferation, providing a tool to study this pathway.
- These findings support the hypothesis that early activation of the NS channel is critical for initiating the mitogenic response.