A systematic comparison of intracellular cyclic AMP and calcium signalling highlights complexities in human VPAC/PAC receptor pharmacology

VPAC/PAC receptor activation classically results in cyclic-AMP production, with limited reports evaluating calcium signalling. These studies systematically characterise intracellular cyclic-AMP ([cAMP]i) and calcium ([Ca2+]i) responses in CHO-cells expressing recombinant human (h) VPAC/PAC receptors (hVPAC1R, hVPAC2R, hPAC1R), using two simple, non-radioactive, HT-amenable assays. The rank order of potency (ROP) of the agonists VIP, PACAP-27 and PACAP-38 was similar in both assays for each individual receptor subtype, although potencies (EC50) in the [Ca2+]i assay were approximately 100-fold lower. Importantly, this shift was also evident in SHSY-5Y cells endogenously expressing hPAC1R. Furthermore, [Ala11,22,28]VIP and maxadilan were selective hVPAC1R and hPAC1R agonists, respectively, and although R3P65 had no demonstrable hVPAC2R selectivity, these compounds exhibited comparable reductions in [Ca2+]i EC50 values. In contrast, PG97-269 and PG99-465, putatively selective hVPAC1R and hVPAC2R antagonists, respectively, were marginally less potent in [cAMP]i studies, whereas M65 was equipotent at hPAC1R. Moreover, PG99-465 alone increased [cAMP]i at all three hVPAC/PAC receptor subtypes, with full hVPAC1R and hPAC1R agonism. With equivalent agonist ROPs generated in both assays, [Ca2+]i signalling provides an alternative approach to examine hVPAC/PAC receptor pharmacology. However, these studies underscore the paucity of receptor selective compounds, complexities in comparing drug potencies across assays, and the pleiotropic nature of VPAC/PAC-receptor signalling.