Pharmacological profiles of presynaptic nociceptin/orphanin FQ receptors modulating 5-hydroxytryptamine and noradrenaline release in the rat neocortex

Abstract

1 The pharmacological profiles of presynaptic nociceptin/orphanin FQ (N/OFQ) peptide receptors (NOP) modulating 5-hydroxytryptamine (5-HT) and noradrenaline (NE) release in the rat neocortex were characterized in a preparation of superfused synaptosomes challenged with 10 mM KCl. 2 N/OFQ concentration-dependently inhibited K(+)-evoked [(3)H]-5-HT and [(3)H]-NE overflow with similar potency (pEC(50) approximately 7.9 and approximately 7.7, respectively) and efficacy (maximal inhibition approximately 40%). 3 N/OFQ (0.1 micro M) inhibition of [(3)H]-5-HT and [(3)H]-NE overflow was antagonized by selective NOP receptor antagonists of peptide ([Nphe(1)]N/OFQ(1-13)NH(2) and UFP-101; 10 and 1 microM, respectively) and non-peptide (J-113397 and JTC-801; both 0.1 microM) nature. Antagonists were routinely applied 3 min before N/OFQ. However, a 21 min pre-application time was necessary for J-113397 and JTC-801 to prevent N/OFQ inhibition of [(3)H]-NE overflow. 4 The NOP receptor ligand [Phe(1)psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2) ([F/G]N/OFQ(1-13)NH(2); 3 microM) did not affect K(+)-evoked [(3)H]-NE but inhibited K(+)-evoked [(3)H]-5-HT overflow in a UFP-101 sensitive manner. [F/G]N/OFQ(1-13)NH(2) antagonized N/OFQ actions on both neurotransmitters. 5 The time-dependency of JTC-801 action was studied in CHO cells expressing human NOP receptors. N/OFQ inhibited forskolin-stimulated cAMP accumulation and JTC-801, tested at different concentrations (0.1-10 microM) and pre-incubation times (0, 40 and 90 min), antagonized this effect in a time-dependent manner. The Schild-type analysis excluded a competitive type of antagonism. 6 We conclude that presynaptic NO receptors inhibiting 5-HT and NE release in the rat neocortex have similar pharmacological profiles. Nevertheless, they can be differentiated pharmacologically on the basis of responsiveness to [F/G]N/OFQ(1-13)NH(2) and time-dependent sensitivity towards non-peptide antagonists.

Figure 1

N/OFQ inhibits synaptosomal [³H]-5-HT and [³H]-NE overflow. Concentration–response curves of the inhibitory effects of N/OFQ (0.001–3 μM) on 10 mM K⁺-evoked [³H]-5-HT and [³H]-NE overflow. Significant inhibition of [³H]-5-HT and [³H]-NE overflow was found at 10 nM N/OFQ. Data are expressed as per cent of the K⁺ stimulation and are means±s.e.mean of at least eight experiments.

Figure 2

Selective NOP receptor antagonists prevent N/OFQ inhibition of [³H]-5-HT overflow. Effects of [Nphe¹], UFP-101, J-113397 and JTC-801 on the inhibition of K⁺-evoked [³H]-5-HT overflow induced by N/OFQ (0.1 μM). Antagonists were perfused 3 min before N/OFQ and maintained until the end of experiment. Data are means±s.e.mean of at least eight experiments. ^*P<0.05; different from control. ^aP<0.05; different from N/OFQ.

Figure 3

The NOP receptor ligand [F/G] prevents N/OFQ inhibition of [³H]-5-HT and [³H]-NE overflow. Effect of [F/G] (3 μM) on the inhibition of [³H]-5-HT and [³H]-NE overflow induced by N/OFQ (0.1 μM). [F/G] was applied (in the presence of naloxone 3 μM) 3 min before N/OFQ and maintained until the end of experiment. Data are means±s.e.mean of at least eight experiments. ^*P<0.05; different from control. ^aP<0.05; different from N/OFQ.

Figure 4

Selective peptide NOP receptor antagonists prevent N/OFQ inhibition of [³H]-NE overflow. Effect of [Nphe¹] and UFP-101 on the inhibition of [³H]-NE overflow induced by N/OFQ (0.1 μM). Antagonists were perfused 3 min before N/OFQ and maintained until the end of experiment. Data are means±s.e.mean of at least six experiments. ^*P<0.05; different from control. ^aP<0.05; different from N/OFQ.

Figure 5

Selective non-peptide NOP receptor antagonists prevent N/OFQ inhibition of [³H]-NE overflow. Effect of J-113397 and JTC-801 on the inhibition of [³H]-NE overflow induced by N/OFQ (0.1 μM). Antagonists were applied at different times (3, 9 and 21 min) before N/OFQ and maintained until the end of experiment. Data are means±s.e.mean of at least six experiments. ^*P<0.05; different from control. ^aP<0.05; different from N/OFQ.

Figure 6

N/OFQ inhibits cAMP accumulation in CHO_hNOP cells. The inhibitory effect of N/OFQ (0.001–1 μM) on forskolin-stimulated cyclic AMP accumulation in CHO cells expressing human recombinant NOP receptors was measured in the absence or the presence of the non-peptide NOP receptor antagonist JTC-801 (0.1–10 μM) at different pre-incubation times (0, 40 and 90 min; panel A, B and C, respectively). Data are means±s.e.mean of ⩾3 experiments.