Direct and indirect inhibition by nociceptin/orphanin FQ on noradrenaline release from rodent cerebral cortex in vitro

The modulation exerted by nociceptin/orphanin FQ (NC) on noradrenaline (NE) release in rodent cerebral cortex slices and synaptosomes was studied. Rat, mouse and guinea-pig cortical slices and synaptosomes were preincubated with 0.1 μM [3H]-NE and superfused. NE release was evoked by 2 min of electrical (3 Hz) stimulation in slices and by 1 min pulse of 10 mM KCl in synaptosomes. In rat cortical slices, 0.01–3 μM NC reduced the evoked [3H]-NE efflux (Emax−54%), with a bell-shaped concentration-response curve, which regained its monotonic nature in the presence of either 0.1 μM naloxone (NX) or 30 μM bicuculline. In synaptosomes, the NC effect curve was sygmoidal in shape and reached a plateau at 1 μM concentration. In the rat, both 1 μM [Phe1ψ(CH2-NH)Gly2]NC(1-13)NH2 and 10 μM [Nphe1]NC(1-13)NH2 (NPhe) antagonised NC-induced inhibition, without per se modifying [3H]-NE efflux. The effects of 0.3–1 μM NC concentrations were partially prevented by 1 μM NX; 1 μM D-Phe-Cys-Thr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) was also an effective antagonist, but 0.1 μM norbinaltorphimine was not. In the mouse cerebral cortex, NC-induced inhibition of NE release (pEC50 6.87, Emax−61%, in the slices) was prevented by Nphe but was NX-insensitive. In guinea-pig cortical slices, NC effect (pEC50 6.22, Emax−38%) was prevented by Nphe, but was NX-insensitive. These findings demonstrate that NC inhibits NE release from rodent cerebral cortex via presynaptically located ORL1 receptors. In the rat, μ opioid and GABAA receptors are involved as well. Keywords: Nociceptin/orphanin FQ, ORL1 receptors, noradrenaline release, naloxone, (Nphe1)nociceptin(1-13)NH2, rodent cerebral cortex, slices, synaptosomes, GABA, species differences Introduction Since the discovery of the opioid receptor-like 1 (ORL1) receptor (Mollereau et al., 1994; Bunzow et al., 1994) and its endogenous ligand nociceptin/orphanin FQ (NC) (Meunier et al., 1995; Reinscheid et al., 1995), a great effort has been made to assess its physiological role, and its involvement in pathological states, in order to predict pharmacological applications for both agonists and antagonists (for reviews see Henderson & McKnight, 1997; Meunier, 2000; Calo et al., 2000b; Mogil & Pasternak, 2001). Despite the high degree of structural homology between the classical opioid receptors and ORL1, which has been proposed as a fourth opioid receptor (Cox, 2000), neither opioid agonists nor antagonists display high affinity for ORL1 and most NC actions are naloxone (NX)-insensitive (Henderson & McKnight, 1997). However, NX has been shown to effectively antagonize some of the NC effects, both in vivo (Rossi et al., 1996; Pomonis et al., 1996; Konya et al., 1998) and in vitro (Madamba et al., 1999). In agreement with its transduction mechanism, namely inhibition of adenylylcyclase and of calcium entry as well as activation of a potassium conductance (Hawes et al., 2000; Harrison & Grandy, 2000), NC has been shown to directly inhibit the release of various neurotransmitters, both in the central nervous system and in periphery (Schlicker & Morari, 2000; Giuliani et al., 2000). In this context, the inhibitory effect of NC on 5-hydroxytryptamine release from rat cerebral cortex has recently been reported (Siniscalchi et al., 1999a; Sbrenna et al., 2000). AS far as NC modulation on noradrenaline (NE) release is concerned, two detailed studies, carried out in mouse brain slices (Schlicker et al., 1998; Werthwein et al., 1999) and a third study comparing in vitro and in vivo nociceptin-amide effects (Okawa et al., 2001) have been published. In the present work, these studies have been extended checking the existence of indirect NC effects involving other neurotransmitter systems. Moreover, the question of the presynaptic location of the receptors responsible for NC actions has been addressed by employing the synaptosomal preparation. Finally, since both quantitative and qualitative discrepancies in neuropeptide receptor binding and effects have been reported (Yoburn et al., 1991; Hall et al., 1993; Benyhe et al., 1999), we checked for possible differences between various rodent species – namely the rat, the mouse and the guinea-pig – as regards the inhibitory NC effect on NE release. Some of the present data have been preliminarily reported in abstract form (Siniscalchi et al., 1999b).