Identification of the G-protein-coupled ORL1 receptor in the mouse spinal cord by [35S]-GTPgammaS binding and immunohistochemistry

Abstract

1 Although the ORL1 receptor is clearly located within the spinal cord, the functional signalling mechanism of the ORL1 receptor in the spinal cord has not been clearly documented. The present study was then to investigate the guanine nucleotide binding protein (G-protein) activation mediated through by the ORL1 receptor in the mouse spinal cord, measuring the modulation of guanosine-5'-o-(3-[35S]-thio) triphosphate ([35S]-GTPgammaS) binding by the putative endogenous ligand nociceptin, also referred as orphanin FQ. We also studied the anatomical distribution of nociceptin-like immunoreactivity and nociceptin-stimulated [35S]-GTPgammaS autoradiography in the spinal cord. 2 Immunohistochemical staining of mouse spinal cord sections revealed a dense plexus of nociceptin-like immunoreactive fibres in the superficial layers of the dorsal horn throughout the entire length of the spinal cord. In addition, networks of fibres were seen projecting from the lateral border of the dorsal horn to the lateral grey matter and around the central canal. 3 In vitro [35S]-GTPgammaS autoradiography showed high levels of nociceptin-stimulated [35S]-GTPgammaS binding in the superficial layers of the mouse dorsal horn and around the central canal, corresponding to the areas where nociceptin-like immunoreactive fibres were concentrated. 4 In [35S]-GTPgammaS membrane assay, nociceptin increased [35S]-GTPgammaS binding of mouse spinal cord membranes in a concentration-dependent and saturable manner, affording maximal stimulation of 64.1+/-2.4%. This effect was markedly inhibited by the specific ORL1 receptor antagonist [Phe1Psi (CH2-NH) Gly2] nociceptin (1 - 13) NH2. None of the mu-, delta-, and kappa-opioid and other G-protein-coupled receptor antagonists had a significant effect on basal or nociceptin-stimulated [35S]-GTPgammaS binding. 5 These findings suggest that nociceptin-containing fibres terminate in the superficial layers of the dorsal horn and the central canal and that nociceptin released in these areas may selectively stimulate the ORL1 receptor to activate G-protein. Furthermore, the unique pattern of G-protein activation in the present study provide additional evidence that nociceptin is distinct from the mu-, delta- or kappa-opioid system.

Figure 1

Photomicrographs of a mouse cervical spinal cord labelled with nociceptin-antisera or antisera pre-absorbed with the peptide. (A) Low magnification showing nociceptin-like immunoreactivity is concentrated in dense networks of fibres in the superficial layers (I and II) and in the lateral border of the dorsal horn. A few nociceptin-like immunoreactivity fibres can be seen around the central canal (cc). (B) A higher magnification showing nociceptin-like immunoreactivity fibres occupying layers I and II. (C) A higher magnification showing dense networks of nociceptin-like immunoreactivity fibres in the lateral border of the dorsal horn. (D) A section of thoracic spinal cord processed with nociceptin-antisera pre-absorbed with the peptide overnight. Positive labelling is not seen in this section. Calibration bar: 500 μm for A and D; 50 μm for B and C.

Figure 2

Representative sections at the level of the mouse lumbar spinal cord showing the distributions of nociceptin-stimulated [³⁵S]-GTPγS binding. Sections were incubated with 2 mM GDP, then [³⁵S]-GTPγS (20 pM) with 2 mM GDP and 10 μM nociceptin. Basal binding was assessed in the absence of nociceptin (A). Nociceptin stimulated high levels of [³⁵S]-GTPγS binding in the superficial layers of the mouse dorsal horns and around the central canal as compared to basal (B).

Figure 3

(A) Influence of GDP concentration (0.1–30 μM) on the binding of [³⁵S]-GTPγS to mouse spinal cord membranes in the absence or presence of 10 μM nociceptin. Incubations were performed at 25°C for 2 h in the presence of 50 pM [³⁵S]-GTPγS. Data are expressed as the mean±s.e.mean of the per cent total [³⁵S]-GTPγS binding in the absence of GDP. Comparable results were obtained from more than three independent sets of experiments. (B) Nociceptin (10 μM)-stimulated [³⁵S]-GTPγS binding to mouse spinal cord membranes, expressed as the mean±s.e.mean of the per cent stimulation over basal [³⁵S]-GTPγS binding at each concentration of GDP. (C) Concentration-effect curve of nociceptin-stimulated [³⁵S]-GTPγS binding in the mouse spinal cord. Data are expressed as the mean±s.e.mean of the per cent stimulation over basal [³⁵S]-GTPγS binding in the presence of 30 μM GDP and absence of nociceptin.

Figure 4

Effect of the selective ORL1 receptor antagonist [Phe¹Ψ (CH₂-NH) Gly₂] nociceptin (1–13) NH₂ (0.3–10 μM) on nociceptin-stimulated (A) and basal (B) [³⁵S]-GTPγS binding in the mouse spinal cord. Incubations were performed at 25°C for 2 h in the presence of 50 pM [³⁵S]-GTPγS and 30 μM GDP. Data are expressed as the mean±s.e.mean of the per cent stimulation over basal [³⁵S]-GTPγS binding in the absence of agonist and/or antagonist. Comparable results were obtained from more than three independent sets of experiments. ^*P<0.01 vs nociceptin alone.

Figure 5

Effect of the selective ORL1 receptor antagonist [Phe¹Ψ (CH₂-NH) Gly²] nociceptin (1–13) NH₂ on [³⁵S]-GTPγS binding by each selective opioid μ-(DAMGO), δ-(DPDPE)- or κ-(U50,488H) receptor agonist. Assay was performed with 10 μM of each opioid receptor agonist in the presence or absence of 10 μM [Phe¹Ψ (CH₂-NH) Gly²] nociceptin (1–13) NH₂. Data are expressed as the mean±s.e.mean of the per cent stimulation over basal [³⁵S]-GTPγS binding in the absence of agonist and/or antagonist. Comparable results were obtained from more than three independent sets of experiments.

Figure 6

(A) The lack of effect of μ-(CTOP), δ-(NTI) and κ-(nor-BNI) opioid receptor antagonists on [³⁵S]-GTPγS binding by nociceptin. Assay was initiated by incubations with 1 μM nociceptin in the presence or absence of 1 μM of each antagonist. Data are expressed as the mean±s.e.mean of the per cent stimulation over basal [³⁵S]-GTPγS binding in the absence of agonist and/or antagonist. Comparable results were obtained from more than three independent sets of experiments. (B) Effect of several G-protein-coupled receptor antagonists on [³⁵S]-GTPγS binding by nociceptin. Atropine: muscarinic acetylcholine (M) receptor antagonist; Haloperidole: dopamine (DA) receptor antagonist; Phaclofen: GABA_B receptor antagonist; Propranolol: β-adrenergic receptor antagonist; Yohimbine: α₂-adrenergic receptor antagonist.