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Figure 4 Involvement of calcium (Ca²⁺) and L-type Ca²⁺ channels in 4-aminopyridine (4-AP)-induced ERK activation. (A) Cardiomyocytes were pretreated with ethyleneglycoltetraacetic acid (EG) (1 mmol/l) or BAPTA-AM (BAP) (20 µmol/l) for 30 min and treated with 4-AP (1 mmol/l) for 15 min. Both EG and BAPTA-AM completely abolished 4-AP-induced extracellular signal-regulated kinase (ERK) phosphorylation. (B) The cells were pretreated with the L-type Ca²⁺ channel blocker diltiazem (Dil) (2 µmol/l) and the Na⁺/Ca²⁺ exchanger inhibitor benzamil (BZ) (100 µmol/l) for 5 min, and then treated with 4-AP for 15 min. Diltiazem fully inhibited ERK phosphorylation, whereas BZ did not. (C) The cells were pre-incubated with the nonselective cation channel blocker fulfenamic acid (FA) (100 µmol/l) and then treated with 4-AP for 15 min. Fulfenamic acid was dissolved in ethanol (vehicle). Fulfenamic acid did not attenuate 4-AP-induced ERK activation. (D) The cells were treated with the L-type Ca²⁺ channel opener Bayk8644 (10 µmol/l) for the indicated times. BayK8644 induced phosphorylation of ERK. All experiments were repeated at least five times, and we obtained similar results. (E) Densitometric analysis of the phosphorylation of ERK was shown. Data are presented as the mean value ± SE. *p < 0.01 vs. 4-AP alone. LIF = leukemia inhibitory factor; NS = not significant.