Intrathecal (i.t.) administration of pituitary adenylate cyclase-activating polypeptide (PACAP) induces long-lasting nociceptive behaviors for more than 60 min in mice, while the involvement of PACAP type1 receptor (PAC1-R) has not been clarified yet. The present study investigated signaling mechanisms of the PACAP-induced prolonged nociceptive behaviors. Single i.t. injection of a selective PAC1-R agonist, maxadilan (Max), mimicked nociceptive behaviors in a dose-dependent manner similar to PACAP. Pre- or post-treatment of a selective PAC1-R antagonist, max.d.4, significantly inhibited the nociceptive behaviors by PACAP or Max. Coadministration of a protein kinase A inhibitor, Rp-8-Br-cAMPS, a mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase inhibitor, PD98059 or a c-Jun N-terminal kinase (JNK) inhibitor, SP600125, significantly inhibited the nociceptive behaviors by Max. Immunohistochemistry and immunoblotting analysis revealed that spinal administration of Max-induced ERK phosphorylation and JNK phosphorylation, and also augmented an astrocyte marker, glial fibrillary acidic protein in mouse spinal cord. Furthermore, an astroglial toxin, l-α-aminoadipate, significantly attenuated the development of the nociceptive behaviors and ERK phosphorylation by Max. These results suggest that the activation of spinal PAC1-R induces long-lasting nociception through the interaction of neurons and astrocytes.

BackgroundPituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors are present in the spinal dorsal horn and dorsal root ganglia, suggesting an important role of PACAP–PACAP receptors signaling system in the modulation of spinal nociceptive transmission. We have previously reported that a single intrathecal injection of PACAP or a PACAP specific (PAC1) receptor selective agonist, maxadilan, in mice induced dose-dependent aversive behaviors, which lasted more than 30 min, and suggested that the maintenance of the nociceptive behaviors was associated with the spinal astrocytic activation.ResultsWe found that a single intrathecal administration of PACAP or maxadilan also produced long-lasting hind paw mechanical allodynia, which persisted at least 84 days without affecting thermal nociceptive threshold. In contrast, intrathecal application of vasoactive intestinal polypeptide did not change mechanical threshold, and substance P, calcitonin gene-related peptide, or N-methyl-D-aspartate induced only transient mechanical allodynia, which disappeared within 21 days. Western blot and immunohistochemical analyses with an astrocytic marker, glial fibrillary acidic protein, revealed that the spinal PAC1 receptor stimulation caused sustained astrocytic activation, which also lasted more than 84 days. Intrathecal co-administration of L-α-aminoadipate, an astroglial toxin, with PACAP or maxadilan almost completely prevented the induction of the mechanical allodynia. Furthermore, intrathecal treatment of L-α-aminoadipate at 84 days after the PAC1 stimulation transiently reversed the mechanical allodynia accompanied by the reduction of glial fibrillary acidic protein expression level.ConclusionOur data suggest that spinal astrocytic activation triggered by the PAC1 receptor stimulation contributes to both induction and maintenance of the long-term mechanical allodynia.

Our case series suggest that an acute transient immune response can be directed against small nerve fibers, and that patients so affected can exhibit features of Guillain-Barré syndrome. Muscle Nerve 57: 320-324, 2018.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors are present in the spinal dorsal horn and dorsal root ganglia, suggesting an important role of PACAP signaling systems in the modulation of spinal nociceptive transmission. Previously, we found that intrathecal injection of PACAP or maxadilan, a selective PACAP type I (PAC1) receptor agonist, induced transient aversive responses followed by a long-lasting mechanical allodynia in mice, suggesting that PACAP-PAC1 receptor systems are involved in chronic pain and that selective PAC1 antagonists may become a new class of analgesics. Although several PAC1 antagonists, such as PACAP 6-38, have been reported, all of them are peptide compounds. In the present study, we identified new small-molecule antagonists of the PAC1 receptor using in silico screening and in vitro/vivo pharmacological assays. The identified small-molecule compounds, named PA-8 and PA-9, dose dependently inhibited the phosphorylation of CREB induced by PACAP in PAC1-, but not VPAC- or VPAC-receptor-expressing CHO cells. PA-8 and PA-9 also dose dependently inhibited PACAP-induced cAMP elevation with an IC of 2.0 and 5.6 nM, respectively. In vivo pharmacological assays showed that intrathecal injection of these compounds blocked the induction of PACAP-induced aversive responses and mechanical allodynia in mice. In contrast, the compounds when administered alone exerted neither agonistic nor algesic actions in the in vitro/vivo assays. The compounds identified in the present study are new and the first small-molecule antagonists of the PAC1 receptor; they may become seed compounds for developing novel analgesics.

21 lactate shuttle, protein kinase C, monocarboxylate transporter, glycogenolysis 22 23 Abbreviations used: artificial cerebrospinal fluid (ACSF), adenylate cyclase (AC), 24 astrocyte-neuron lactate shuttle (ANLS), central nervous system (CNS), glial fibrillary 25 acidic protein (GFAP), intrathecal (i.t.), maxadilan (Max), monocarboxylate transporter 26 (MCT), pituitary adenylate cyclase-activating polypeptide (PACAP), protein kinase A 27 (PKA), protein kinase C (PKC), phorbol 12-myristate 13-acetate (PMA), glycogen 28 phosphorylase (PYGB), vasoactive intestinal polypeptide (VIP) 29 30 3 Abstract Previously, we showed that spinal pituitary adenylate cyclase-activating 31 polypeptide (PACAP)/PAC1 receptor signaling triggers long-lasting pain behaviors 32 through astroglial activation. Since astrocyte-neuron lactate shuttle (ANLS) could be 33 essential for long-term synaptic facilitation, we aimed to elucidate a possible 34 involvement of spinal ANLS in the development of the PACAP/PAC1 receptor-induced 35 pain behaviors. A single intrathecal administration of PACAP induced short-term 36 spontaneous aversive behaviors, followed by long-lasting mechanical allodynia. These 37 pain behaviors were inhibited by DAB, an inhibitor of glycogenolysis, and this 38 inhibition was reversed by simultaneous L-lactate application. In the cultured spinal 39 astrocytes, the PACAP-evoked glycogenolysis and lactate secretion were inhibited by a 40 protein kinase C (PKC) inhibitor, and the PKC inhibitor attenuated the PACAP-induced 41 pain behaviors. Finally, an inhibitor for the monocarboxylate transporters blocked the 42 lactate secretion from the spinal astrocytes and inhibited the PACAP-induced pain 43 behaviors. These results suggested that PAC1 receptor-PKC-ANLS signaling is 44 involved in the PACAP-induced pain behaviors.45 46 64 5 neurotransmitters evoke the ANLS activation during any forms of neuronal plasticity. 65 66Pituitary adenylate cyclase-activating polypeptide (PACAP) was originally isolated 67 from ovine hypothalamic extracts based on its ability to stimulate adenylate cyclase in 68 rat anterior pituitary cell cultures (Miyata et al., 1989; Miyata et al., 1990). In normal 69 state, PACAP specific receptor, PAC1 receptor, is particularly abundant in central 70 nervous system (CNS) including spinal dorsal horn (Dickinson et al., 1999; Jongsma et 71 al., 2000; Sakashita et al., 2001; Vaudry et al., 2009; Yokai et al., 2016), where 72 PACAP-immunoreactive fibers are also considerably localized (Moller et al., 1993; 73 Dun et al., 1996a; Dun et al., 1996b; Narita et al., 1996), and PACAP 74 mRNA/immunoreactivity in rat dorsal root ganglia is markedly upregulated in 75 peripheral nerve injury or inflammation (Zhang et al., 1995; Zhang et al., 1998; 76 Jongsma et al., 2003; Mabuchi et al., 2004). These observations coupled with other 77 lines of evidence propose that PACAP/PAC1 receptor system could play an important 78 role in the modulation of spinal nociceptive transmission.79 80 We have previously demonstrated in mice t...

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We have previously showed that spinal pituitary adenylate cyclase-activating polypeptide (PACAP)/PACAP type 1 (PAC1) receptor signaling triggered long-lasting nociceptive behaviors through astroglial activation in mice. Since astrocyte-neuron lactate shuttle (ANLS) could be essential for long-term synaptic facilitation, we aimed to elucidate a possible involvement of spinal ANLS in the development of the PACAP/PAC1 receptor-induced nociceptive behaviors. A single intrathecal administration of PACAP induced short-term spontaneous aversive behaviors, followed by long-lasting mechanical allodynia in mice. These nociceptive behaviors were inhibited by 1,4-dideoxy-1,4-imino-d-arabinitol (DAB), an inhibitor of glycogenolysis, and this inhibition was reversed by simultaneous L-lactate application. In the cultured spinal astrocytes, the PACAP-evoked glycogenolysis and lactate secretion were inhibited by DAB. In addition, a protein kinase C (PKC) inhibitor attenuated the PACAP-induced nociceptive behaviors as well as the PACAP-evoked glycogenolysis and lactate secretion. Finally, an inhibitor for the monocarboxylate transporters blocked the lactate secretion from the spinal astrocytes and inhibited the PACAP- and spinal nerve ligation-induced nociceptive behaviors. These results suggested that spinal PAC1 receptor-PKC-ANLS signaling contributed to the PACAP-induced nociceptive behaviors. This signaling system could be involved in the peripheral nerve injury-induced pain-like behaviors.

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