Nocistatin inhibits 5-hydroxytryptamine release in the mouse neocortex via presynaptic Gi/o protein linked pathways

Abstract

Background and purpose: Nocistatin (NST) is a neuropeptide generated from cleavage of the nociceptin/orphanin FQ (N/OFQ) precursor. Evidence has been presented that NST acts as a functional antagonist of N/OFQ, although NST receptor and transduction pathways have not yet been identified. We previously showed that N/OFQ inhibited [(3)H]5-hydroxytryptamine ([(3)H]5-HT) release from mouse cortical synaptosomes via activation of NOP receptors. We now investigate whether NST regulates [(3)H]5-HT release in the same preparation.

Experimental approach: Mouse and rat cerebrocortical synaptosomes in superfusion, preloaded with [(3)H]5-HT and stimulated with 1 min pulses of 10 mM KCl, were used.

Key results: Bovine NST (b-NST) inhibited the K(+)-induced [(3)H]5-HT release, displaying similar efficacy but lower potency than N/OFQ. b-NST action underwent concentration-dependent and time-dependent desensitization, and was not prevented either by the NOP receptor antagonist [Nphe(1) Arg(14),Lys(15)]N/OFQ(1-13)-NH(2) (UFP-101) or by the non-selective opioid receptor antagonist, naloxone. Contrary to N/OFQ, b-NST reduced [(3)H]5-HT release from synaptosomes obtained from NOP receptor knockout mice. However, both N/OFQ and NST were ineffective in synaptosomes pre-treated with the G(i/o) protein inhibitor, Pertussis toxin. NST-N/OFQ interactions were also investigated. Co-application of maximal concentrations of both peptides did not result in additive effects, whereas pre-application of maximal b-NST concentrations partially attenuated N/OFQ inhibition.

Conclusions and implications: We conclude that b-NST inhibits [(3)H]5-HT release via activation of G(i/o) protein linked pathways, not involving classical opioid receptors and the NOP receptor. The present data strengthen the view that b-NST is, per se, a biologically active peptide endowed with agonist activity.

Figure 1

Nociceptin/orphanin FQ (N/OFQ) and bovine nocistatin (b-NST) inhibit [³H]5-HT overflow from cortical synaptosomes. Concentration–response curves describing the effects of N/OFQ (0.001–10 μM) and b-NST (0.001–10 μM) on the K⁺-evoked [³H]5-HT overflow from mouse (a) and rat (b) neocortical synaptosomes. Maximally effective b-NST concentrations were 1 μM (mouse) and 0.1 μM (rat; analysis of variance followed by the Newman–Keuls test for multiple comparisons). Drugs were perfused 9 min before K⁺ and maintained until the end of the experiment. Data are expressed as percentage of the K⁺ stimulation (control) and are means±s.e.m. of 10–20 determinations.

Figure 2

Time dependency of the bovine nocistatin (b-NST) effect. Effect of b-NST (1 μM) on the K⁺-evoked [³H]5-HT overflow in neocortical mouse synaptosomes. b-NST was applied for 6, 9, 12, 15 and 18 min before K⁺ and maintained until the end of the experiment. Data are expressed as percentage of the K⁺ stimulation (control) and are means±s.e.m. of eight experiments. ^*P<0.05, ^**P<0.01; different from control (analysis of variance followed by the Newman–Keuls test for multiple comparisons).

Figure 3

The effect of bovine nocistatin (b-NST) is mediated by neither classical opioid nor N/OFQ opioid peptide (NOP) receptors. (a) Effect of the non-selective opioid receptor antagonist naloxone (1 μM) and the NOP selective receptor antagonist [Nphe¹, Arg¹⁴, Lys¹⁵]N/OFQ(1–13)-NH₂ (UFP-101; 1 μM) on the inhibition of K⁺-evoked [³H]5-HT overflow induced by b-NST (1 μM) in neocortical mouse synaptosomes. Antagonists were perfused 3 min before b-NST and maintained until the end of the experiment. (b) Effect of b-NST (1 μM) and N/OFQ (0.1 μM) on the K⁺-evoked [³H]5-HT overflow from neocortical synaptosomes obtained from wild-type (NOP^+/+) and NOP receptor knockout (NOP^−/−) mice. Data are expressed as percentage of the K⁺ stimulation (control) and are means±s.e.m. of 8–12 determinations. ^*P<0.05; ^**P<0.01, different from control (analysis of variance followed by the Newman–Keuls test for multiple comparisons).

Figure 4

The effects of bovine nocistatin (b-NST) and nociceptin/orphanin FQ (N/OFQ) are prevented by Pertussis toxin (PTX). Effect of pretreatment with PTX (5 nM) on the inhibition of K⁺-evoked [³H]5-HT overflow induced by b-NST (1 μM) and N/OFQ (0.1 μM) in mouse neocortical synaptosomes. Data are expressed as percentage of the K⁺ stimulation (control) and are means±s.e.m. of 8–12 determinations. ^*P<0.05; different from control (analysis of variance followed by the Newman–Keuls test for multiple comparisons).

Figure 5

Nociceptin/orphanin FQ (N/OFQ) and bovine nocistatin (b-NST) interacted in modulating [³H]5-HT overflow. Effect of co-application (a) or pre-application (b) of maximal b-NST (1 μM) and N/OFQ (0.1 μM) concentrations on the K⁺-evoked [³H]5-HT overflow. (a) Drugs were co-applied 9 min before K⁺ and maintained until the end of the experiment. (b) b-NST and N/OFQ were applied 18 and 9 min before K⁺, respectively. Data are expressed as percentage of the K⁺ stimulation (control) and are means±s.e.m. of 12 (a) or 10 (b) determinations. ^*P<0.05, ^**P<0.01; different from control. °P<0.05; different from N/OFQ (analysis of variance followed by the Newman–Keuls test for multiple comparisons).