Acute CYM-5442 exposure increases neuronal excitability, but prolonged treatment blocks sensitization to SEW2871, but not to S1P. (A) summarizes the acute effects of 100 nM CYM-5442 on sensory neurons. In one group, CYM-5442 significantly increased the number of APs after only a 4-min exposure (n = 4), whereas the other group appeared to be insensitive to CYM-5442 (n = 4). (B) demonstrates that after a 1-h pretreatment with 100 nM CYM-5442, the S1PR1 selective agonist SEW2871 (100 nM) fails to increase the neuronal excitability (n = 10 control-10 min, n = 8 15 min). (C) shows that in a total of 15 sensory neurons, 10 were insensitive to 1 μM S1P although there was a small but significant increase in the number of APs measured only at the 10-min point. In contrast, five sensory neurons exhibited increased excitability in response to S1P. (D) demonstrates that after normalization of the number of APs to their respective control values, there was no difference in the average number of APs after exposure to 100 nM SEW2871 or in those neurons that appeared to be insensitive to 1 μM S1P. However, there was a significant increase in the number of APs in those sensory neurons that were sensitive to 1 μM S1P. For the 6-min point, the increase measured in the S1P-sensitive neurons was significant compared to all the SEW2871 time points, all the S1P-insensitive times except for the 10-min point, and the S1P-sensitive control. For the 10- and 15-min points, the increase measured in the S1P-sensitive neurons was significant compared to all the SEW2871 and S1P-insensitive time points, as well as the S1P-sensitive control and the 2-min point (P < 0.001 ANOVA Holm-Sidak all-pairs test). AP - action potential, Cont - control, S1P - sphingosine-1-phosphate.