Partial agonist behaviour depends upon the level of nociceptin/orphanin FQ receptor expression: studies using the ecdysone-inducible mammalian expression system.
(1) Partial agonism is primarily dependent upon receptor density and coupling efficiency. As these parameters are tissue/model dependent, intrinsic activity in different tissues can vary. We have utilised the ecdysone-inducible expression system containing the human nociceptin/orphanin FQ (N/OFQ) peptide receptor (hNOP) expressed in Chinese hamster ovary cells (CHOINDhNOP) to examine the activity of a range of partial agonists in receptor binding, GTPgamma35S binding and inhibition of adenylyl cyclase studies. (2) Incubation of CHOINDhNOP cells with ponasterone A (PON) induced hNOP expression ([leucyl-3H]N/OFQ binding) of 24, 68, 191 and 1101 fmol mg-1 protein at 1, 2, 5 and 10 microm PON, respectively. At 191 fmol mg-1, protein hNOP pharmacology was identical to that reported for other traditional expression systems. (3) pEC50 values for GTPgamma35S binding ranged from 7.23 to 7.72 (2-10 microm PON) for the partial agonist [Phe1psi(CH2-NH)Gly2]N/OFQ(1-13)-NH2 ([F/G]N/OFQ(1-13)-NH2) and 8.12-8.60 (1-10 microm PON) for N/OFQ(1-13)-NH2 and Emax values (stimulation factor relative to basal) ranged from 1.51 to 3.21 (2-10 microm PON) for [F/G]N/OFQ(1-13)-NH2 and 1.28-6.95 (1-10 microm) for N/OFQ(1-13)-NH2. Intrinsic activity of [F/G]N/OFQ(1-13)-NH2 relative to N/OFQ(1-13)-NH2 was 0.3-0.5. [F/G]N/OFQ(1-13)-NH2 did not stimulate GTPgamma35S binding at 1 microm PON, but competitively antagonised the effects of N/OFQ(1-13)-NH2 with a pKB=7.62. (4) pEC50 values for cAMP inhibition ranged from 8.26 to 8.32 (2-10 microm PON) for [F/G]N/OFQ(1-13)-NH2 and 9.42-10.35 for N/OFQ(1-13)-NH2 and Emax values (% inhibition) ranged from 19.6 to 83.2 for [F/G]N/OFQ(1-13)-NH2 and 40.9-86.0 for N/OFQ(1-13)-NH2. The intrinsic activity of [F/G]N/OFQ(1-13)-NH2 relative to N/OFQ(1-13)-NH2 was 0.48-0.97. (5) In the same cellular environment with receptor density as the only variable, we show that the profile of [F/G]N/OFQ(1-13)-NH2 can be manipulated to encompass full and partial agonism along with antagonism.
McDonald
J
J
University Department of Anaesthesia, Critical Care and Pain Management, Leicester Royal Infirmary, Leicester LE1 5WW.
Barnes
T A
TA
Okawa
H
H
Williams
J
J
Calo'
G
G
Rowbotham
D J
DJ
Lambert
D G
DG
D000818:Animals; D016466:CHO Cells; D002452:Cell Count / Q000379:methods; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D011957:Receptors, Opioid / Q000819:agonists* / Q000096:biosynthesis*; D011987:Receptors, Steroid / Q000819:agonists / Q000096:biosynthesis*; D000094942:Nociceptin Receptor
D011957:Receptors, Opioid; D011987:Receptors, Steroid; C055469:ecdysone receptor; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000242:Cyclic AMP; D000094942:Nociceptin Receptor