PACAP-38 but not VIP induces release of CGRP from trigeminal nucleus caudalis via a receptor distinct from the PAC1 receptor

Objective: To investigate if PACAP and VIP have an effect on CGRP release or NOS activity in the trigeminal ganglion and trigeminal nucleus caudalis and if there can be a difference in effect between PACAP and VIP on these two systems. Furthermore, we investigate if PACAP co-localize with CGRP and/or nNOS in the two tissues. Background: The structurally related neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide-38 (PACAP-38) partially share receptors and are both potent vasodilators. However, PACAP-38 but not VIP is an efficient inducer of migraine attacks in migraineurs. Calcitonin gene-related peptide (CGRP) and nitric oxide (NO) are two signaling molecules known to be involved in migraine. Methods: Rat tissue was used for all experiments. Release of CGRP induced by VIP and PACAP in dura mater, trigeminal ganglion (TG) and trigeminal nucleus caudalis (TNC) was quantified by EIA. Regulation of NOS-enzymes caused by VIP and PACAP was investigated in dura mater, TG and TNC by measuring the conversion of L-[H-3]arginine to L-[H-3]citrulline. Co-expression of PACAP, neuronal nitric oxide synthase (nNOS) and CGRP was explored by immunohistochemistry in TG and TNC. mRNA expression studies of VPAC(1), VPAC(2) and PAC(1)-receptors were performed by qRT-PCR. Results: PACAP-38 administered in increasing concentrations caused a concentration-dependent CGRPrelease in the TNC, but not in TG. VIP was without effect in both tissues examined. The PAC(1) receptor agonist maxadilan had no effect on CGRP release and the PAC(1) antagonist M65 did not inhibit PACAP-38 induced CGRP release. PACAP-38 or VIP did not affect NOS activity in homogenates of TG and TNC. Quantitative PCR demonstrated the presence of VPAC(1), VPAC(2) and PAC(1) receptors in TG and TNC. Immunohistochemistry of PACAP and CGRP showed co-expression in TG and TNC. PACAP and nNOS were co-localized in TG, but not in TNC. PACAP was found to co-localize with glutamine synthetase in TG satellite glial cells. Conclusion: PACAP-38 cause release of CGRP from TNC but not from TG. We suggest that the release is not caused via activation of PAC(1), VPAC(1) or VPAC(2) receptors. PACAP has no effect on NOS activity in TG or TNC. In TG PACAP was found in neuronal cells and in satellite glial cells. It co-localized with CGRP and nNOS in the neuronal cells. In TNC PACAP was co-localized with CGRP but not with nNOS. (C) 2014 Elsevier Ltd. All rights reserved.