Characterisation of PACAP-responsive receptor pharmacology and expression in cell models and the spinal cord

Abstract

Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide that is widely expressed and involved in several biological processes, including pain. PACAP exists in two biologically active forms; PACAP-27 and PACAP-38. PACAP is closely related to vasoactive intestinal peptide (VIP) and peptide histidine methionine (PHM). These peptides activate three different class B G protein-coupled receptors; the PAC1, VPAC1 and VPAC2 receptors. To add further complexity, the PAC1 receptor may be alternatively spliced, generating variants that can differ in their agonist or signalling profiles. The PACAP peptide family show promise for the treatment of pain. To target these receptors therapeutically, we need to understand their underlying pharmacology and identify PACAP-responsive receptors at sites involved in pain. However, currently the pharmacological profiles are incomplete and receptor expression in tissue is typically limited to mRNA, which is not sufficient to link the physiological functions to a specific receptor. Therefore, the aim was to fully characterise human PACAP-responsive receptor pharmacology and translate these findings from cell model systems to human spinal cord tissue. Chapter 3 investigated the activation of multiple signalling pathways by all endogenous agonists at all human PACAP-responsive receptor subtypes, including an N-terminal PAC1 receptor splice variant. These receptor profiles were further defined using receptor antagonists. To translate these findings into a relevant endogenous system, chapter 4 examined these responses at PACAP-responsive receptors in rat spinal cord cultures. Chapter 5 validated several antibodies targeting PACAP-responsive receptors, which were then utilised in chapter 6 to translate the functional signalling responses in spinal cord cultures to specific receptor subtypes expressed in the rat and human spinal cord. The blockade of PACAP-responsive receptors in the spinal cord could be beneficial in alleviating pain; therefore, chapter 7 used a proof-of-concept approach to explore the functionality of dual CGRP/PACAP receptor antagonists in cell models. PACAP-responsive receptors were found to exhibit distinct pharmacological profiles but activated signalling in a similar manner. Of particular importance was the differential profile between the PAC1 receptor splice variants, which has significant implications for drug development and linking the physiological functions of an agonist to a specific receptor subtype. Additionally, PACAP-responsive receptor antagonists displayed agonist-dependent responses, an important consideration when screening novel antagonists targeting this family. Endogenous PACAP-responsive receptors in spinal cord cultures exhibited a PAC1 receptor-like pharmacological profile. PAC1 and VPAC2 receptor expression was identified in both rat and human spinal cord. These receptors exhibited differential localisation and therefore, may have diverse roles in pain. Furthermore, linking two peptide antagonists to form a dual CGRP/PACAP receptor antagonist was found to generate potent blockers of signalling for both receptors. This thesis provides new insight into the pharmacological profiles of PACAP-responsive receptors, including their splice variants, and identified their expression at a site important in pain to help facilitate future development of therapeutics targeting this receptor family.