Characterization of Specific [3H]Nociceptin Binding in Rat Brain and Spinal Cord.

Abstract

Nociceptin (orphanin FQ), a heptadecapeptide isolated from rat and porcine brain, has been identified as the endogenous ligand of opioid receptor-like ORL1 receptor, an orphan G protein-coupled receptor. 1,2) The sequences of human, mouse and rat complementary DNA for the ORL1 receptor indicate a significant degree of homology with the 'classical opioid' receptors (m, d and k). [3][4][5][6][7] In spite of this homology with opioid receptors, potent opioid ligands fail to bind to ORL1 receptors. Nociceptin itself has high homology with opioid peptides, particularly dynorphin A, the endogenous ligand of the k-opioid receptor, but this peptide exhibits very low binding affinity for opioid receptors. 8) Pharmacological studies have shown that nociceptin may be involved in a wide variety of physiological functions: pain and analgesia, 9) opioid tolerance, 10) learning and memory, 11) locomotion, 12) feeding, 13) stress and anxiety 14) and neuronal differentiation 15) in the central nervous system. It may also produce effects on the cardiovascular system, 16,17) urogenital tract 18,19) and immune system 20) in the periphery. It is of interest to note that local microinjection of nociceptin into brain and spinal cord of rats and mice produces opposite effects on the nociceptive response. 21) In relation to these effects, the ORL1 receptor has been suggested to be present in the brain and spinal cord following in situ hybridization analysis, 4,6) and in peripheral tissues such as the intestine, vas deferens, liver and spleen. 5) Radioligand binding studies of the ORL1 receptor have been performed in brain tissues of rats, humans, mice, frogs and Chinese hamster ovary (CHO) cells expressing ORL1 receptors. [22][23][24][25] However, to our knowledge, little information has been published on ORL1 receptor characteristics in the spinal cord. It is considered that this tissue may play a significant role in the appearance of allodynia and antinociception by nociceptin. Thus, the aim of the present study was to characterize specific [ 3 H]nociceptin binding simultaneously in rat brain and spinal cord. Further, the distribution of ORL1 receptors in the rat brain was examined. Tissue Preparation Male Sprague-Dawley rats (200-300 g) at 7-8 weeks of age were sacrificed by decapitation and the whole brain and spinal cord were removed. To study the distribution of specific binding of [ 3 H]nociceptin, cerebral cortex, corpus striatum, hippocampus, thalamus, pons/medulla oblongata, midbrain and cerebellum were dissected. The tissues (whole brain and spinal cord) were homogenized in 19 volumes of ice-cold 50 mM Tris-HCl buffer (pH: 7.4) containing 2 mM EDTA, 0.1 mM ( p-amidinophenyl)methanesulfonyl fluoride hydrochloride ( p-APMSF) (buffer A) 26) using a Polytron homogenizer, and the homogenate was centrifuged at 500ϫg for 10 min. The supernatant was centrifuged at 40000ϫg for 15 min. The pellet was finally suspended in ice-cold buffer A containing 2 mg/ml bovine serum albumin (buffer B) and used in [ 3 H]nociceptin binding assays. Al...

Selective κ Opioid Antagonists nor-BNI, GNTI and JDTic Have Low Affinities for Non-Opioid Receptors and Transporters

“…Nor-BNI showed negligible affinity, consistent with prior reports [29], [30], as did GNTI. Surprisingly, despite its high affinity and lack of efficacy, JDTic only weakly inhibited the response to N/OFQ in a functional assay (inhibition of cyclic AMP production: pA 2  = 7.1, 95% CI = 5.9 to 8.3, Figure 4A).…”

“…No binding was detected at 10 µM to the glucocorticoid receptor NR3C1 [28]. Nor-BNI's affinity for the opioid-like nociceptin/orphanin FQ receptor (NOP) is also negligible; one study found low affinity ( K i  = 780 nM) [29], while another detected no binding at 10 µM [30].…”

The nociceptin/orphanin FQ receptor system as a target to alleviate cancer‐induced bone pain in rats: Model validation and pharmacological evaluation

“…Determination of the binding affinity of nociceptin at the NOP receptor has been notoriously difficult and reported K i values vary widely (0.03-2 nM) due to differences in binding assays and unusual binding properties of nociceptin (see Dooley & Houghten, 2000). Furthermore, differences may exist in the binding affinity of nociceptin in the spinal cord and brain (Kusaka, Yamada & Kimura, 2001). Here, full efficacy was observed after spinal application of 10-μg (~5.…”