KCl-induced PKA-II activity is cAMP independent but modulated by calpains. (A) The pRII increase induced by KCl (40 mM) was not inhibited by fentanyl (Fent; 10 µM), oxycodone (Oxy; 10 µM), or [Leu⁵]-enkephalin (Enk; 10 µM) in rat sensory neurons. (B and C) Effect of the phosphodiesterase inhibitor IBMX (100 µM, 30-min pretreatment) on the pRII increase induced by 5-HT (250 nM) or KCl (40 mM). Fig. S4, J and K show not-normalized data indicating basal elevation of pRII intensity by IBMX. (D and E) The cAMP antagonist Rp-cAMPS-pAB (10 µM) has no effect on the induction of pRII intensity by KCl (40 mM). (F) Effect of the calpain inhibitor MDL28170 (100 µM, 30 min) on the pRII increase by KCl (40 mM). (G) Time course of pERK1/2 intensity in DRG neurons treated with veratridine (VT; 100 µM) to open VGSCs in comparison to the KCl (40 mM) response. (H) Inhibitory effect of verapamil (VP; 200 µM, 10 min pretreatment) on ERK1/2 phosphorylation induced by depolarization (40 mM KCl). (I) The pERK1/2 response to a low dose of KCl (10 mM) is reinforced by (S)-(-)-Bay K 8644 (2 µM, 10 min). (J) Chelation of extracellular calcium with EGTA (2.5 mM, 30 min) prevents the pERK1/2 increase. (K) The CaMKII inhibitor AIP (1 µM, 30 min) does not inhibit the induction of pERK1/2 by depolarization. (L) Pretreatment with the PKA inhibitor H89 (25 µM, 30 min) reduces the pERK1/2 response to KCl. Values represent means ± SEM; n = 3–4 experiments; >2,000 neurons/condition; two-way ANOVA with Bonferroni’s test; ^§, P < 0.05; ^§§, P < 0.01; ^§§§, P < 0.001 indicate significance levels between KCl-induced pRII signals in the absence or presence of an inhibitor.