Identification of the G-protein-coupled ORL1 receptor in the mouse spinal cord by [35S]-GTPgammaS binding and immunohistochemistry.
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.
Narita
M
M
Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin, WI 53226, USA.
Mizoguchi
H
H
Oji
D E
DE
Dun
N J
NJ
Hwang
B H
BH
Nagase
H
H
Tseng
L F
LF
D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer / Q000494:pharmacology; D000818:Animals; D001285:Atropine / Q000494:pharmacology; D001345:Autoradiography; D001418:Baclofen / Q000031:analogs & derivatives / Q000494:pharmacology; D001667:Binding, Competitive / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D020881:Enkephalin, D-Penicillamine (2,5)- / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000494:pharmacology; D006220:Haloperidol / Q000494:pharmacology; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008297:Male; D008566:Membranes / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008813:Mice, Inbred ICR; D009271:Naltrexone / Q000031:analogs & derivatives / Q000494:pharmacology; D009292:Narcotic Antagonists; D018847:Opioid Peptides / Q000032:analysis / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D011433:Propranolol / Q000494:pharmacology; D011957:Receptors, Opioid / Q000032:analysis* / Q000378:metabolism; D013004:Somatostatin / Q000031:analogs & derivatives / Q000494:pharmacology; D013116:Spinal Cord / Q000737:chemistry / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D015016:Yohimbine / Q000494:pharmacology; D000094942:Nociceptin Receptor; D000097629:Nociceptin
D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; D001285:Atropine; D001418:Baclofen; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D020881:Enkephalin, D-Penicillamine (2,5)-; D019204:GTP-Binding Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006153:Guanosine Diphosphate; D006220:Haloperidol; D009271:Naltrexone; D009292:Narcotic Antagonists; D018847:Opioid Peptides; D010446:Peptide Fragments; D011433:Propranolol; D011957:Receptors, Opioid; D013004:Somatostatin; D013462:Sulfur Radioisotopes; D015016:Yohimbine; D000094942:Nociceptin Receptor; D000097629:Nociceptin; C114149:nociceptin (1-13)-NH2, Phe(1)-psi(CH2-NH)-Gly(2)-; C049352:phenylalanyl-cyclo(cysteinyltyrosyl-tryptophyl-ornithyl-threonyl-penicillamine)threoninamide; C052329:phaclofen; C051844:norbinaltorphimine; C055382:naltrindole; C000722287:Oprl1 protein, mouse