Pharmacological profile of the cyclic nociceptin/orphanin FQ analogues c[Cys10,14]N/OFQ(1-14)NH2 and c[Nphe1,Cys10,14]N/OFQ(1-14)NH2.
In this study we describe the activity of two cyclic nociceptin/orphanin FQ (N/OFQ) peptides; c[Cys(10,14)]N/OFQ(1-14)NH(2) (c[Cys(10,14)]) and its [Nphe(1)] derivative c[Nphe(1),Cys(10,14)]N/OFQ(1-14)NH(2) (c[Nphe(1),Cys(10,14)]) in native rat and mouse and recombinant human N/OFQ receptors (NOP). Cyclisation may protect the peptide from metabolic degradation. In competition binding studies of rat, mouse and human NOP the following rank order pK(i) was obtained: N/OFQ(1-13)NH(2)(reference agonist)>N/OFQ=c[Cys(10,14)]>>c[Nphe(1)Cys(10,14)]. In GTPgamma(35)S studies of Chinese hamster ovary cells expressing human NOP (CHO(hNOP)) c[Cys(10,14)] (pEC(50) 8.29) and N/OFQ(1-13)NH(2) (pEC(50) 8.57) were full agonists whilst c[Nphe(1)Cys(10,14)] alone was inactive. Following 30 min pre-incubation c[Nphe(1)Cys(10,14)] competitively antagonised the effects of N/OFQ(1-13)NH(2) with a pA(2) and slope factor of 6.92 and 1.01 respectively. In cAMP assays c[Cys(10,14)] (pEC(50) 9.29, E(max) 102% inhibition of the forskolin stimulated response), N/OFQ(1-13)NH(2) (pEC(50) 10.16, E(max) 103% inhibition) and c[Nphe(1)Cys(10,14)] (~80% inhibition at 10 microM) displayed agonist activity. In the mouse vas deferens c[Cys(10,14)] (pEC(50) 6.82, E(max) 89% inhibition of electrically evoked contractions) and N/OFQ(1-13)NH(2) (pEC(50) 7.47, E(max) 93% inhibition) were full agonists whilst c[Nphe(1)Cys(10,14)] alone was inactive. c[Nphe(1)Cys(10,14)] (10 microM) competitively antagonised the effects of N/OFQ(1-13)NH(2) with a pK(B) of 5.66. In a crude attempt to assess metabolic stability, c[Cys(10,14)] was incubated with rat brain membranes and then the supernatant assayed for remaining peptide. Following 60 min incubation 64% of the 1 nM added peptide was metabolised (compared with 54% for N/OFQ-NH(2)). In summary, we report that c[Cys(10,14)] is a full agonist with a small reduction in potency but no improvement in stability whilst c[Nphe(1)Cys(10,14)] displays tissue (antagonist in the vas deferens) and assay (antagonist in the GTPgamma(35)S assay and agonist in cAMP assay) dependent activity.
University Department of Anaesthesia, Critical Care and Pain Management, Leicester Royal Infirmary, Leicester, LE1 5WW, UK.
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M
Kitayama
T A
TA
Barnes
G
G
Carra
J
J
McDonald
G
G
Calo
R
R
Guerrini
D J
DJ
Rowbotham
G
G
Smith
D G
DG
Lambert
D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism; D005576:Colforsin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis; D003545:Cysteine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004558:Electric Stimulation; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism; D008297:Male; D051379:Mice; D009119:Muscle Contraction / Q000187:drug effects; D009270:Naloxone / Q000494:pharmacology; D009292:Narcotic Antagonists; D018847:Opioid Peptides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D011869:Radioligand Assay; D051381:Rats; D017208:Rats, Wistar; D011957:Receptors, Opioid / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D014649:Vas Deferens / Q000187:drug effects / Q000502:physiology; D000097629:Nociceptin
D005576:Colforsin; D000242:Cyclic AMP; D003545:Cysteine; D019204:GTP-Binding Proteins; D009270:Naloxone; D009292:Narcotic Antagonists; D018847:Opioid Peptides; D010649:Phenylalanine; D011957:Receptors, Opioid; D011994:Recombinant Proteins; D000097629:Nociceptin; C479838:(Nphe(1),Arg(14),Lys(15))N-OFQ NH(2)