Cloning and functional pharmacology of two corticotropin-releasing factor receptors from a teleost fish.

Although it is well established that fish possess corticotropin-releasing factor (CRF) and a CRF-like peptide, urotensin I, comparatively little is known about the pharmacology of their cognate receptors. Here we report the isolation and functional expression of two complementary DNAs (cDNAs), from the chum salmon Oncorhynchus keta, which encode orthologues of the mammalian and amphibian CRF type 1 (CRF(1)) and type 2 (CRF(2)) receptors. Radioligand competition binding experiments have revealed that the salmon CRF(1) and CRF(2) receptors bind urotensin I with approximately 8-fold higher affinity than rat/human CRF. These two peptides together with two related CRF-like peptides, namely, sauvagine and urocortin, were also tested in cAMP assays; for cells expressing the salmon CRF(1) receptor, EC(50) values for the stimulation of cAMP production were between 4.5+/-1.8 and 15.3+/-3.1 nM. For the salmon CRF(2) receptor, the corresponding values were: rat/human CRF, 9.4+/-0.4 nM; urotensin I, 21.2+/-2.1 nM; sauvagine, 0.7+/-0.1 nM; and urocortin, 2.2+/-0.7 nM. We have also functionally coupled the O. keta CRF(1) receptor, in Xenopus laevis oocytes, to the endogenous Ca(2+)-activated chloride conductance by co-expression with the G-protein alpha subunit, G(alpha16). The EC(50) value for channel activation by rat/human CRF (11.2+/-2.6 nM) agrees well with that obtained in cAMP assays (15.3+/-3.1 nM). We conclude that although sauvagine is 13- and 30-fold more potent than rat/human CRF and urotensin I, respectively, in activating the salmon CRF(2) receptor, neither receptor appears able to discriminate between the native ligands CRF and urotensin I.

Bovine placental lactogen is a potent stimulator of growth and displays strong binding to hepatic receptor sites of coho salmon.

Juvenile coho salmon were treated with bovine placental lactogen (bPL) and bovine growth hormone (bGH) to examine the growth promoting activities of these proteins in a lower vertebrate. Fish were intraperitoneally injected either with 0.5 or 5.0 micrograms/g bPL or with 5.0 micrograms/g bGH once a week for 5 weeks. After only a single injection and 1 week of growth, the high dose of bPL stimulated a significant increase in weight and length relative to untreated fish or fish treated with a control protein, bovine serum albumin. At the end of the experiment, all hormone-treated groups were significantly larger than controls. Fish treated with 5 micrograms/g bPL gained more than three times as much weight as controls. The 5.0 micrograms/g bGH group grew at the same rate as fish treated with one-tenth this dose of bPL, indicating that bPL is a potent stimulator of growth in this species. Radioreceptor assays performed on coho salmon liver membrane preparations indicate that bPL binds with approximately 430-fold higher affinity than bGH, and some 8000-fold higher affinity than bovine prolactin. The action of bPL relative to the structure and function of salmonid pituitary hormones is discussed.

Seasonal changes in osmoregulation, cortisol, and cortisol receptor activity in the gills of parr/smolt of steelhead trout and steelhead-rainbow trout hybrids, Oncorhynchus mykiss.

Steelhead and hybrid trout (rainbow trout male, steelhead female) presmolts and smolts, Oncorhynchus mykiss, exposed to 30/1000 seawater (SW) for 24 hr manifested progressively lower plasma sodium values from December to May. The increase in plasma sodium after 24 hr in SW diminished significantly after March, with the hybrids (which were heavier in weight than the steelheads) reaching their peak osmoregulatory ability in the middle of April, and the steelheads in late May. In fresh water there was little difference in plasma osmotic concentration but steelhead plasma chloride fell significantly from March 30 levels to May 23 levels, whereas hybrid plasma chloride levels did not change during this time. In steelheads but not hybrids, plasma cortisol correlated negatively with plasma sodium of fish exposed 24 hr in SW. However, in both steelheads and hybrids, plasma sodium values 24 hr after SW exposure had an inverse correlation with cytosol cortisol from the gills. Again, in both steelheads and hybrids, plasma values of fish exposed 24 hr in SW correlated positively with cytosol receptor activity. In hybrids, the nuclear extract receptor activity, Nmax, increased significantly until late March and then dropped significantly thereafter. In steelheads, there were no significant differences but Nmax followed the same trends as occurred in hybrids. Thus seasonal changes in cortisol and cortisol receptors in steelhead occur during smoltification and preparation for marine life and suggest an important role for cortisol. In hybrids there appears to be a reduction in seasonality.

Smoltification induced by a 'skeleton' photoperiod in underyearling coho salmon (Oncorhynchus kisutch).

Underyearling coho salmon fry were subjected to three initial photoperiod treatments (6L∶18D, 10L∶14D, 14L∶10D) for two months and subsequently to three final treatments (16L∶8D, 9L∶6D∶1L∶8D, 10L∶14D) in a factorial design. Growth rates and seawater adaptability were monitored regularly. The groups that were exposed initially to 6L∶18D or 10L∶14D and then to 16L∶8D grew faster and had lower plasma sodium ion levels after seawater challenge tests than any of the other groups. Fish which were initially exposed to 6 L or 10 L daylength and then to a 9L∶6D∶1L∶8D skeleton photoperiod, showed a slightly lower growth rate and seawater adaptability than those given the corresponding complete 16L∶8D photoperiod. However fish maintained on skeleton photoperiods had significantly greater growth rates and seawater adaptability than those kept on the 10L∶14D photoperiod. This indicates that it is not the accumulated number of hours of exposure to light that initiates smolting, but rather the time during the day when light is experienced. Fish exposed initially to 14L∶10D showed little or no response to subsequent changes in photoperiod, suggesting that responsiveness to inductive photoperiods depends on the initial photoperiod treatment.