Day-night specific binding of 2-[125I]iodomelatonin and melatonin content in gill, small intestine and kidney of three fish species.

Some of melatonin's (Mel) well-established physiological effects are mediated via high-affinity cell-membrane receptors belonging to the superfamily of G-protein-coupled receptors. Specific binding of ligand 2-[(125)I]iodomelatonin, using membrane preparations from osmoregulatory tissues of flounder, rainbow trout and sea bream, together with Mel concentrations in the tissues and plasma were studied. The kidney, gill and small intestine samples were collected during the day and at night. The dissociation constants (K (d)) and maximal binding densities (B (max)) were calculated for each tissue at 11:00 and 23:00 h. The binding sites with K (d) values in the tissues in the picomolar range indicated the high affinity. K (d) and B (max) values were tissue- and species-dependent. The GTP analogue [Guanosine 5'-O-(3-thiotriphosphate)] treatment significantly reduced the B (max) value, indicating that the 2-[(125)I]iodomelatonin-binding sites are probably coupled to a G-protein. No daily variations in K (d) and B (max) values were observed. These are the first studies of the presence of 2-[(125)I]iodomelatonin-binding sites in the small intestine, kidney tubule and gill of fish. The data strongly suggest new potential targets for Mel action and the influence of Mel on water/ion balance in fish. The intestine seems to be a site of Mel synthesis and/or an active accumulation of the hormone.

Coexpression of corticotropin-releasing hormone and urotensin i precursor genes in the caudal neurosecretory system of the euryhaline flounder (Platichthys flesus): a possible shared role in peripheral regulation.

CRH and urotensin I (UI) are neuroendocrine peptides that belong to the superfamily of corticotropin-releasing factors. In mammals, these peptides regulate the stress response and other central nervous system functions, whereas in fish an involvement for UI in osmoregulation has also been suggested. We have identified, characterized, and localized the genes encoding these peptides in a unique fish neuroendocrine organ, the caudal neurosecretory system (CNSS). The CRH and UI precursors, isolated from a European flounder CNSS library, consist of 168 and 147 amino acid residues, respectively, with an overall homology of approximately 50%. Both precursors contain a signal peptide, a divergent cryptic region and a 41-amino acid mature peptide with cleavage and amidation sites. Genomic organization showed that whole CRH and UI coding sequences are contained in a single exon. Northern blot analysis and quantitative PCR of a range of tissues confirmed the CNSS as a major site of expression of both CRH and UI and thus serves as a likely source of circulating peptides. In situ hybridization demonstrated that CRH and UI colocalize to the same cells of the CNSS. Our findings suggest that, in euryhaline fish, the CNSS is a major site of production of CRH and probably contributes to the high circulating levels observed in response to specific environmental challenges. Furthermore, the localization of CRH and UI within the same cell population suggests an early, possibly shared role for these peptides in controlling stress-mediated adaptive plasticity.

Isolation, synthesis, and biological activity of flounder [Asn1,Ile5,Thr9] angiotensin I.

A novel angiotensin I (ANG I) has been isolated from incubates of plasma and kidney extracts of the flounder, Platichthys flesus, using ion-exchange, gel-permeation, and reverse-phase high performance liquid chromatography (HPLC). Its sequence was determined as H-Asn-Arg-Val-Tyr-Ile-His-Pro-Phe-Thr-Leu-OH by sequence analysis and mass spectrometry. No vasopressor activity was detected at the elution position of [Asp(1)] ANG I in ion-exchange HPLC. The sequence was confirmed by identity of the elution position with the synthetic peptide in two different HPLC systems. When compared with ANG I isolated from other teleost fish, flounder ANG I uniquely has an isoleucine at position 5 rather than valine. Injection of angiotensin II (ANG II) into chronically cannulated flounder resulted in a dose-dependent pressor response, native [Asn(1),Ile(5)] ANG II, was found to elicit pressor responses comparable with those seen when teleost [Asn(1),Val(5)] ANG II and human [Asp(1),Ile(5)] ANG II were injected into flounder over the dose range 0.02-1.00 nmol/kg(-1). Plasma concentrations of the neurohypophysial peptide AVT were measured in chronically cannulated flounder following the injection of ANG II to examine the effect of ANG II on circulating AVT concentration. The injection of [Asn(1),Ile(5)] ANG II (1 nmolkg(-1)) or [Asp(1),Ile(5)] ANG II (2.5 nmolkg(-1)) resulted in a significant fall in the circulating levels of AVT suggesting that ANG II either directly or indirectly negatively influences AVT secretion.