Figure 4.

KCl signals via a TrkA-independent mechanism. a, KCl does not cause Trk autophosphorylation. NGF-selected sympathetic neurons were washed free of neurotrophin, and then exposed to various concentrations of NGF (ng/ml) or KCl (mM) for 15 min. Cellular lysates were immunoprecipitated with anti-panTrk and probed with antiphosphotyrosine (p-tyr) to visualize Trk autophosphorylation. All samples were normalized for equal amounts of protein. b, Dominant-negative TrkA inhibits NGF-mediated, but not KCl-mediated sympathetic neuron survival. NGF-selected neurons were infected with recombinant adenovirus expressing dnTrkA at titers ranging from 10 to 150 MOI, or with 200 MOI of a control adenovirus expressing the TTA transactivator, and were then switched to 10 ng/ml NGF (10N) or 50 mM KCl (50K) one day later. After 48 h, cell survival was measured by MTT assays. As controls, uninfected sister cultures were either withdrawn from NGF (0N), or maintained in 10 ng/ml NGF or 50 mM KCl. Results are those obtained in one representative experiment performed in triplicate, and represent the mean ± SD. Similar results were obtained in three separate experiments. *Represents those values that are significantly different from 10 ng/ml NGF alone (P < 0.01). c, K252a blocks Trk activation in response to NGF, whereas nifedipine has no effect. NGF-selected neurons were treated with various concentrations of NGF (ng/ml) or KCl (mM) for 15 min in the presence of 200 nm K-252a or 1 μM nifedipine (NIF), and then probed for phosphotyrosine (p-tyr). All samples were normalized for equal amounts of protein. d and e, K252a selectively blocks NGF-mediated sympathetic neuron survival, whereas nifedipine selectively blocks KCl-mediated survival. d, NGF-selected neurons were treated for two days with 200 nm K-252a in the presence of 10 ng/ml NGF (10N) or 50 mM KCl (50K) and survival was measured by MTT assays. Results derive from one representative experiment performed in triplicate and each point is the mean ± SD. Similar results were obtained in three separate experiments. *Indicates those values that are significantly different from 10 ng/ml NGF (P < 0.01). e, Quantitative analysis of TUNEL-labeled sympathetic neurons treated with 200 nm K-252a or 1 μM nifedipine (NIF) in the presence of 10 ng/ml NGF (10N) or 50 mM KCl (50K) for two days. Data are expressed as the percentage of TUNEL-negative cells/total Hoechst-positive nuclei. Each point represents combined data from three separate experiments, each performed in triplicate, and is the mean ± SD. *Indicates those values that are significantly different from 10 ng/ml NGF (*P < 0.01) or 50 mM KCl (**P < 0.01).