In this study we describe the activity of two cyclic nociceptin/orphanin FQ (N/OFQ) peptides; c[Cys10,14]N/ OFQ(1–14)NH2 (c[Cys10,14]) and its [Nphe1] derivative c[Nphe1,Cys10,14]N/OFQ(1–14)NH2 (c[Nphe1,Cys10,14]) 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 pKi was obtained: N/OFQ(1– 13)NH2(reference agonist)>N/OFQ=c[Cys10,14]>>c[Nphe1Cys10,14]. In GTPγ35S studies of Chinese hamster ovary cells expressing human NOP (CHOhNOP) c[Cys10,14] (pEC50 8.29) and N/OFQ(1–13)NH2 (pEC50 8.57) were full agonists whilst c[Nphe1Cys10,14] alone was inactive. Following 30 min pre-incubation c[Nphe1Cys10,14] competitively antagonised the effects of N/OFQ(1–13)NH2 with a pA2 and slope factor of 6.92 and 1.01 respectively. In cAMP assays c[Cys10,14] (pEC50 9.29, Emax 102% inhibition of the forskolin stimulated response), N/OFQ(1–13)NH2 (pEC50 10.16, Emax 103% inhibition) and c[Nphe1Cys10,14] (~80% inhibition at 10 μM) displayed agonist activity. In the mouse vas deferens c[Cys10,14] (pEC50 6.82, Emax 89% inhibition of electrically evoked contractions) and N/OFQ(1–13)NH2 (pEC50 7.47, Emax 93% inhibition) were full agonists whilst c[Nphe1Cys10,14] alone was inactive. c[Nphe1Cys10,14] (10 μM) competitively antagonised the effects of N/OFQ(1– 13)NH2 with a pKB of 5.66. In a crude attempt to assess metabolic stability, c[Cys10,14] 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-NH2). In summary, we report that c[Cys10,14] is a full agonist with a small reduction in potency but no improvement in stability whilst c[Nphe1Cys10,14] displays tissue (antagonist in the vas deferens) and assay (antagonist in the GTPγ35S assay and agonist in cAMP assay) dependent activity.
Pharmacological profile of the cyclic nociceptin/orphanin FQ analogues c[Cys(10,14)]N/OFQ(1-14)NH2 and c[Nphe(1),Cys(10,14)]N/OFQ(1-14)NH2
CALO', Girolamo;GUERRINI, Remo;
2003
Abstract
In this study we describe the activity of two cyclic nociceptin/orphanin FQ (N/OFQ) peptides; c[Cys10,14]N/ OFQ(1–14)NH2 (c[Cys10,14]) and its [Nphe1] derivative c[Nphe1,Cys10,14]N/OFQ(1–14)NH2 (c[Nphe1,Cys10,14]) 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 pKi was obtained: N/OFQ(1– 13)NH2(reference agonist)>N/OFQ=c[Cys10,14]>>c[Nphe1Cys10,14]. In GTPγ35S studies of Chinese hamster ovary cells expressing human NOP (CHOhNOP) c[Cys10,14] (pEC50 8.29) and N/OFQ(1–13)NH2 (pEC50 8.57) were full agonists whilst c[Nphe1Cys10,14] alone was inactive. Following 30 min pre-incubation c[Nphe1Cys10,14] competitively antagonised the effects of N/OFQ(1–13)NH2 with a pA2 and slope factor of 6.92 and 1.01 respectively. In cAMP assays c[Cys10,14] (pEC50 9.29, Emax 102% inhibition of the forskolin stimulated response), N/OFQ(1–13)NH2 (pEC50 10.16, Emax 103% inhibition) and c[Nphe1Cys10,14] (~80% inhibition at 10 μM) displayed agonist activity. In the mouse vas deferens c[Cys10,14] (pEC50 6.82, Emax 89% inhibition of electrically evoked contractions) and N/OFQ(1–13)NH2 (pEC50 7.47, Emax 93% inhibition) were full agonists whilst c[Nphe1Cys10,14] alone was inactive. c[Nphe1Cys10,14] (10 μM) competitively antagonised the effects of N/OFQ(1– 13)NH2 with a pKB of 5.66. In a crude attempt to assess metabolic stability, c[Cys10,14] 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-NH2). In summary, we report that c[Cys10,14] is a full agonist with a small reduction in potency but no improvement in stability whilst c[Nphe1Cys10,14] displays tissue (antagonist in the vas deferens) and assay (antagonist in the GTPγ35S assay and agonist in cAMP assay) dependent activity.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
