The pseudopeptide [Nphe(1)]N/OFQ(1-13)NH(2) (Nphe) has been shown to act as a pure, selective and competitive antagonist of nociceptin/orphanin FQ (N/OFQ) receptors in different tissues. However, Nphe displayed a highly variable potency, with pA(2) values ranging from 5.96 to 8.45. In the present study, we show that sodium ions and GTP markedly affect the potency of Nphe in blocking N/OFQ receptors coupled to cyclic AMP inhibition in different cellular systems. In intact N1E-115 neuroblastoma cells, the pA(2) value of Nphe increased from 7.13 to 8.02 when the extracellular sodium concentration was reduced from 138 to 2.5 mM. When N/OFQ inhibition of adenylyl cyclase activity was assayed in cell membranes, 100 mM NaCl decreased the pK(i) value of Nphe from 8.38 to 7.32, but increased that of the nonpeptide N/OFQ receptor antagonist CompB from 8.61 to 8.92. Similar effects of sodium ions on the potencies of Nphe and CompB were observed when the compounds were used to antagonize the N/OFQ inhibition of adenylyl cyclase activity in membranes of the external plexiform layer of the rat olfactory bulb. In the same assay, the increase of GTP concentration from 0.1 to 200 micro M decreased Nphe potency by 8-fold. These data demonstrate that sodium ions and GTP affect the potency of Nphe in a manner similar to that of agonists but not of pure antagonists and suggest that these factors may contribute to the reported variability of Nphe affinity constant.
Sodium ions and GTP decrease the potency of [Nphe1]N/OFQ(1-13) NH2 in blocking nociceptin/orphanin FQ receptors coupled to cyclic AMP in N1E-115 neuroblastoma cells and rat olfactory bulb
OLIANAS, MARIA CONCETTA;ONALI, PIER LUIGI
2003-01-01
Abstract
The pseudopeptide [Nphe(1)]N/OFQ(1-13)NH(2) (Nphe) has been shown to act as a pure, selective and competitive antagonist of nociceptin/orphanin FQ (N/OFQ) receptors in different tissues. However, Nphe displayed a highly variable potency, with pA(2) values ranging from 5.96 to 8.45. In the present study, we show that sodium ions and GTP markedly affect the potency of Nphe in blocking N/OFQ receptors coupled to cyclic AMP inhibition in different cellular systems. In intact N1E-115 neuroblastoma cells, the pA(2) value of Nphe increased from 7.13 to 8.02 when the extracellular sodium concentration was reduced from 138 to 2.5 mM. When N/OFQ inhibition of adenylyl cyclase activity was assayed in cell membranes, 100 mM NaCl decreased the pK(i) value of Nphe from 8.38 to 7.32, but increased that of the nonpeptide N/OFQ receptor antagonist CompB from 8.61 to 8.92. Similar effects of sodium ions on the potencies of Nphe and CompB were observed when the compounds were used to antagonize the N/OFQ inhibition of adenylyl cyclase activity in membranes of the external plexiform layer of the rat olfactory bulb. In the same assay, the increase of GTP concentration from 0.1 to 200 micro M decreased Nphe potency by 8-fold. These data demonstrate that sodium ions and GTP affect the potency of Nphe in a manner similar to that of agonists but not of pure antagonists and suggest that these factors may contribute to the reported variability of Nphe affinity constant.
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