Pharmacological characterization of a nociceptin receptor from zebrafish (Danio rerio)

Abstract

The nociceptin receptor (NOP) and its endogenous ligand, nociceptin/orphanin FQ (OFQ), are involved in a wide range of biological functions, such as pain, anxiety, learning, and memory. The zebrafish has been proposed as a candidate to study the in vivo effects of several drugs of abuse and to discover new pharmacological targets. We report the cloning, expression, and pharmacological characterization of a NOP receptor from zebrafish (drNOP). The full-length cDNA codes a protein of 363 residues, which shows high sequence similarity to other NOPs. Phylogenetic analysis indicates that NOPs are broadly conserved during vertebrate evolution, and that they stand for the most divergent clade of the opioid/OFQ receptor family. Expression studies have revealed that drNOP mRNA is highly expressed in the central nervous system, and low expression levels are also found in peripheral tissues such as gills, muscle, and liver. Pharmacological analysis indicates that drNOP displays specific and saturable binding for 4, H]nociceptin, with a K d Z0 . 20G0 . 02 nM and a B max Z1703G81 fmol/mg protein. 35 S] GTPgS stimulation studies showed that drNOP receptor is functional, as nociceptin is able to fully activate the receptor and DYN A behaves as a partial agonist (50% stimulation). Our results indicate that drNOP receptor displays mixed characteristics of both NOP and k opioid receptors. Hence, drNOP, which has retained more of the likely ancestral features, bridges the gap between nociceptin and opiate pharmacology.

Behavioral screening for neuroactive drugs in zebrafish

“…Nevertheless, the systematic analysis of the binding activities of small molecules on zebrafish receptor subtypes will be needed to enhance the quality of drug library annotation for zebrafish small molecule screens. Such efforts include recent in vitro analyses that show conserved pharmacology of zebrafish opioid receptors (Gonzalez-Nunez et al, 2007;de Velasco et al, 2009), the nociceptin receptor (Rivas-Boyero et al, 2011), the zebrafish M2 muscarinic receptor (Hsieh and Liao, 2002), melanocortin receptors (Ringholm et al, 2002), the androgen receptor (Hossain et al, 2008), and bradykinnin receptors (Duner et al, 2002;Bromee et al, 2005). Radiolabel-binding studies of small molecule agonists/antagonists in zebrafish brain membranes or slices are also useful, especially when coupled with competitive binding assays, as shown in studies on muscarinic acetylcholine receptors (Williams and Messer, 2004), GABA-A and GABA-B receptors (Renier et al, 2007), H2 and H3 histamine receptors (Peitsaro et al, 2000;Peitsaro et al, 2007), and adrenergic receptors (Ruuskanen et al, 2005).…”

Construction of an Affordable and Easy-to-Build Zebrafish Facility

“…It is also suggested that the chrome rack be cleaned and dried at this point to prevent rust and build up. If signs of contamination are observed, individual tanks are disinfected with a 2% bleach solution 8 . General health is monitored with special attention to signs of infection (swelling, skin ulcers, exolpthalmia, color off, etc.…”

Effects of Nociceptin and Nocistatin on Uterine Contraction

“…Expression studies have revealed a low expression of NOP-R mRNA in zebrafish peripheral tissues, such as gills, muscle, and liver (Rivas-Boyero et al, 2011). A higher level of N/OFQ was observed in hepatic carcinoma tissue, as compared with the normal hepatic tissue from the same subject.…”