cAMP signaling through protein kinase A and Epac2 induces substance P release in the rat spinal cord

Using neurokinin 1 receptor (NK1R) internalization to measure of substance P release in rat spinal cord slices, we found that it was induced by the adenylyl cyclase (AC) activator forskolin, by the protein kinase A (PKA) activators 6-Bnz-cAMP and 8-Br-cAMP, and by the activator of exchange protein activated by cAMP (Epac) 8-pCPT-2-O-Me-cAMP (CPTOMe-cAMP). Conversely, AC and PKA inhibitors decreased substance P release induced by electrical stimulation of the dorsal root. Therefore, the cAMP signaling pathway mediates substance P release in the dorsal horn. The effects of forskolin and 6-Bnz-cAMP were not additive with NMDA-induced substance P release and were decreased by the NMDA receptor blocker MK-801. In cultured dorsal horn neurons, forskolin increased NMDA-induced Ca2+ entry and the phosphorylation of the NR1 and NR2B subunits of the NMDA receptor. Therefore, cAMP-induced substance P release is mediated by the activating phosphorylation by PKA of NMDA receptors. Voltage-gated Ca2+ channels, but not by TRPV1 or TRPA1, also contributed to cAMP-induced substance P release. Activation of PKA was required for the effects of forskolin and the three cAMP analogs. Epac2 contributed to the effects of forskolin and CPTOMe-cAMP, signaling through a Raf - mitogen-activated protein kinase pathway to activate Ca2+ channels. Epac1 inhibitors induced NK1R internalization independently of substance P release. In rats with latent sensitization to pain, the effect of 6-Bnz-cAMP was unchanged, whereas the effect of forskolin was decreased due to the loss of the stimulatory effect of Epac2. Hence, substance P release induced by cAMP decreases during pain hypersensitivity. Diagram of the proposed pathways by which cAMP controls substance P (SP) release. Adenylyl cyclase (AC) produces cAMP, which then activates protein kinase A, Epac1 and Epac2. PKA activates Cav2.2 channels (N type), NMDA receptors (NMDAR) and mTOR, all of which contribute to SP release. Epac2 signals through Rap, Raf-1 kinase, MEK and MAPK, leading to the activation of Cav1, Cav2.1 and Cav3.2 channels (L, P, Q and T types), which also contribute to SP release. Epac1 directly inhibits NK1R internalization. [Display omitted] •cAMP induced substance P release through protein kinase A and Epac2.•Protein kinase A activated NMDA receptors and N type voltage-gated Ca2+ channels.•Epac2 activated L, P, Q and T type voltage-gated Ca2+ channels through MAPK.•In a chronic pain model, signaling through protein kinase