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 ([Ca(2+)](i)) responses in CHO-cells expressing recombinant human (h) VPAC/PAC receptors (hVPAC(1)R, hVPAC(2)R, hPAC(1)R), 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 (EC(50)) in the [Ca(2+)](i) assay were approximately 100-fold lower. Importantly, this shift was also evident in SHSY-5Y cells endogenously expressing hPAC(1)R. Furthermore, [Ala(11,22,28)]VIP and maxadilan were selective hVPAC(1)R and hPAC(1)R agonists, respectively, and although R3P65 had no demonstrable hVPAC(2)R selectivity, these compounds exhibited comparable reductions in [Ca(2+)](i) EC(50) values. In contrast, PG97-269 and PG99-465, putatively selective hVPAC(1)R and hVPAC(2)R antagonists, respectively, were marginally less potent in [cAMP](i) studies, whereas M65 was equipotent at hPAC(1)R. Moreover, PG99-465 alone increased [cAMP](i) at all three hVPAC/PAC receptor subtypes, with full hVPAC(1)R and hPAC(1)R agonism. With equivalent agonist ROPs generated in both assays, [Ca(2+)](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.
Dickson
Louise
L
Astellas CNS Research in Edinburgh, The University of Edinburgh, The Chancellor's Building, 49 Little France Crescent, Edinburgh, EH16 4SB, UK.
Aramori
Ichiro
I
McCulloch
James
J
Sharkey
John
J
Finlayson
Keith
K
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D020013:Calcium Signaling / Q000187:drug effects*; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D006224:Cricetinae; D000242:Cyclic AMP / Q000502:physiology*; D003627:Data Interpretation, Statistical; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D018696:Neuroprotective Agents / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide / Q000494:pharmacology; D051239:Receptors, Vasoactive Intestinal Peptide, Type II / Q000819:agonists / Q000037:antagonists & inhibitors / Q000187:drug effects*; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I / Q000819:agonists / Q000037:antagonists & inhibitors / Q000187:drug effects*; D014162:Transfection; D014660:Vasoactive Intestinal Peptide / Q000031:analogs & derivatives / Q000494:pharmacology
D018696:Neuroprotective Agents; C110307:PG 97-269; D010446:Peptide Fragments; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D051239:Receptors, Vasoactive Intestinal Peptide, Type II; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D014660:Vasoactive Intestinal Peptide; D000242:Cyclic AMP