Identification of dynorphin a from zebrafish: a comparative study with mammalian dynorphin A.

We report the cloning and molecular characterization of the zfPDYN. The complete open reading frame for this propeptide is comprised in two exons that are localized on chromosome 23. zfPDYN cDNA codes for a polypeptide of 252 amino acids that contains the consensus sequences for four opioid peptides: an Ile-enkephalin, the neo-endorphins, dynorphin A and dynorphin B. Upon comparison between zebrafish (zfDYN A) and mammalian dynorphin A (mDYN A) it has been stated that these two peptides only differ in two amino acids: the Leu(5) is replaced by Met(5) and the Lys(13) by Arg(13). Taking into consideration that mDYN A is able to bind to the three mammalian opioid receptors, we have compared the pharmacological profile of zfDYN A and mDYN A on the zebrafish opioid receptors. By means of radioligand binding techniques, we have established that these two dynorphins bind and activate all of the cloned opioid receptors from zebrafish (delta-, mu- and kappa-like), although with different affinities. zfDYN A and mDYN A displace [(3)H]-diprenorphine binding with K(i) values on the nanomolar range, showing greater affinity for zebrafish opioid receptor (ZFOR) 3 (kappa) receptor. ZFOR1 (delta) and ZFOR4 (delta) present higher affinity for zfDYN A than for mDYN A, while the opposing behavior is observed in ZFOR2 (mu). Functional [(35)S]guanosine 5'-[gamma-thio]triphosphate (GTPgammaS) stimulation experiments indicate that these two peptides fully activate the zebrafish opioid receptors, although the mean effective dose (EC(50)) values obtained for ZFOR2 and ZFOR3 receptors are lower than those seen for ZFOR1 and ZFOR4. A comparative study indicates that mammalian and zebrafish opioid receptors might bind their corresponding dynorphin A in a similar fashion, hence suggesting an important role of the opioid system through the vertebrate evolution.

Binding profile of the endogenous novel heptapeptide Met-enkephalin-Gly-tyr in zebrafish and rat brain.

Zebrafish is considered a model organism, not only for the study of the biological functions of vertebrates but also as a tool to analyze the effects of some drugs or toxic agents. Five opioid precursor genes homologous to the mammalian opioid propeptide genes have recently been identified; one of these, the zebrafish proenkephalin, codes a novel heptapeptide, the Met-enkephalin-Gly-Tyr (MEGY). To analyze the pharmacological properties of this novel ligand, we have labeled it with tritium ([(3)H]MEGY). In addition, we have also synthesized two analogs: (d-Ala(2))-MEGY (Y-d-Ala-GFMGY) and (d-Ala(2), Val(5))-MEGY (Y-d-Ala-GFVGY). The binding profile of these three agents has been studied in zebrafish and rat brain membranes. [(3)H]MEGY presents one binding site in zebrafish, as well as in rat brain membranes, although it shows a slight higher affinity in zebrafish brain. The observed saturable binding is displaced by naloxone, thus confirming the opioid nature of the binding sites. Competition binding assays indicate that the methionine residue is essential for high-affinity binding of MEGY and probably of other peptidic agonists in zebrafish, whereas the change of a Gly for a d-Ala does not dramatically affect the ligand affinity. Our results show that the percentage of [(3)H]MEGY displaced by all the ligands studied is higher than 100%, thus inferring that naloxone (used to determine nonspecific binding) does not bind to all the sites labeled by [(3)H]MEGY. Therefore, we can deduct that some of the MEGY binding sites should not be considered classical opioid sites.

Cloning and characterization of a full-length pronociceptin in zebrafish: evidence of the existence of two different nociceptin sequences in the same precursor.

We report the cloning and molecular characterization of a zebrafish pronociceptin gene, which is expressed in brain and intestine. This fish precursor codes for two different nociceptin peptides, one of which presents an opioid sequence in its N-terminus, being more similar to dynorphin A than its mammalian counterparts. Our results represent a new tool to investigate the function of nociceptin and its receptor in relation to pain and drug addiction.

Characterization of zebrafish proenkephalin reveals novel opioid sequences.

Cloning and molecular characterization of an homologous gene to proenkephalin in a teleost, the zebrafish Danio rerio is presented in this paper. The new zebrafish proenkephalin (zfPENK) encodes a 249 amino acid polypeptide that displays an identity of 40% to mammalian PENKs and which contains the consensus sequences for four Met-enkephalins, one Leu-enkephalin and one Met-enkephalin-Gly-Tyr, described for the first time in teleosts. Expression studies indicate that zfPENK is selectively expressed in zebrafish brain. Our findings support the concept that PENK genes might have been highly conserved throughout evolution and that its origins might be placed more than 400 million years ago. Moreover, we present evidence that the heptapeptide Met-enkephalin-Gly-Tyr present in fish might be anterior in evolution to the heptapeptide Met-enkephalin-Arg-Phe present in tetrapods. Also another homologous sequence to proenkephalin in zebrafish genome is presented. This sequence might stand for the third exon of a possible duplicate gene of zfPENK. Our findings not only present new data in relation to the evolution of opioid peptides in vertebrates, but also we present a new heptapeptide with putative differential activity than the other peptides derived from the mammalian proenkephalins. Future research will define the functional role of this new heptapeptide in the mechanism that describes opioid activity.