Rainbow trout androgen receptor-alpha fails to distinguish between any of the natural androgens tested in transactivation assay, not just 11-ketotestosterone and testosterone.

We have recently isolated two androgen receptor cDNA clones from the rainbow trout testis. To investigate the functions of the rainbow trout androgen receptor-alpha (rtAR-alpha), we investigated the ligand binding ability and transcriptional activity of rtAR-alpha. Interestingly, in ligand-competition experiments, testosterone (T) (IC(50) 3 x 10(-9) M) competed with [(3)H]mibolerone binding for rtAR-alpha slightly more potently than the teleost fish-specific natural androgen 11-ketotestosterone (11KT) (IC(50) 8 x 10(-9) M), which is thought to be the functional spermatogenesis inducer. In contrast, T (EC(50) 5 x 10(-9) M) and 11KT (EC(50) 6 x 10(-9) M) showed similar efficiency upon cotransfection into EPC cells with a rtAR-alpha expression vector and an androgen-responsive element-based reporter gene. These results indicated that activation of rtAR-alpha does not distinguish between 11KT and T and suggested that a specific system, which is mediated only by 11KT, may exist in the rainbow trout.

Two distinct isoforms of cDNA encoding rainbow trout androgen receptors.

Androgens play an important role in male sexual differentiation and development. The activity of androgens is mediated by an androgen receptor (AR), which binds to specific DNA recognition sites and regulates transcription. We describe here the isolation of two distinct rainbow trout cDNA clones, designated rtAR-alpha and rtAR-beta, which contain the entire androgen receptor coding region. Comparison of the predicted amino acid sequence of rtAR-alpha to that of rtAR-beta revealed 85% identity. Interestingly, despite this high homology, rtAR-alpha activated transcription of an androgen-responsive reporter gene in co-transfection assays, but rtAR-beta did not. These results suggest that rainbow trout contains two distinct isoforms of androgen receptors whose functions differ. The region of rtAR-beta responsible for its inactivity was mapped to its ligand binding domain by analyzing chimeras of the rtAR-alpha, rtAR-beta, and rtGR-I (glucocorticoid) receptors. Alteration of any one of three out of four segments within this domain restored activity. Extracts made from COS-1 cells transfected with an rtAR-alpha expression plasmid produced a high level of [3H]mibolerone binding, whereas no binding was observed by extracts of cells transfected with an rtAR-beta expression plasmid. These data demonstrate that the lack of transactivation activity of rtAR-beta is due to its inability to bind hormone.

Pharmacological detection of AMPA receptor heterogeneity by use of two allosteric potentiators in rat hippocampal cultures.

1. In order to examine whether a recently developed allosteric potentiator for AMPA receptors, 4-[2-(phenylsulphonylamino)ethylthio]-2,6-difluoro-phenoxyaceta mide (PEPA), can be utilized as an indicator of AMPA receptor heterogeneity, the action of PEPA upon the increase of intracellular free calcium ion concentration ([Ca2+]i) elicited by AMPA was investigated in rat hippocampal cultures, and the action was compared with that of cyclothiazide, a well characterized allosteric modulator of AMPA receptors. 2. PEPA dose-dependently potentiated AMPA-induced increase of [Ca2+]i. In 90% (72 out of 80) of the cells in which cyclothiazide acts, PEPA potentiated the increased [Ca2+]i induced by AMPA with pronounced cell-to-cell variation in rat hippocampal cultures. 3. The ratio of the potentiation by PEPA to the potentiation by cyclothiazide (P/C ratio) also varied with cells between 0 and 2.15. It was found that the cultured hippocampal cells consisted of multiple populations with different P/C ratios. Among them two populations exhibited characteristic P/C ratios; low (0 to 0.15; 27 out of 80 cells, 34%) and high (> or = 2.00; 1 out of 80 cells, 1%) P/C ratios. The P/C ratios of the other populations were between 0.25 and 1.20, and these cells constituted 65% (52 out of 80 cells) of the cells tested. 4. Reverse transcriptase-polymerase chain reaction analysis suggested that GluR2-flip, GluR1-flip, GluR2-flop, and GluR1-flop were abundantly expressed (in this rank order) in the cultures used. 5. In Xenopus oocytes expressing GluR1, GluR3, or these subunits plus GluR2, the potentiation of AMPA response by PEPA and by cyclothiazide varied with subunit and splice-variant combinations, and the P/C ratio was between 0.19 and 2.20. Oocytes with low P/C ratios (0.19 to 0.50) and low sensitivity to PEPA potentiation (1.9 fold to 6.41 fold) were those expressing flip variants predominantly, and oocytes with high P/C ratios (1.8 to 2.2) were those expressing flop variants predominantly. Oocytes with intermediate P/C ratios (0.51 to 1.20) were those expressing various combinations of flip and flop variants, and it was impossible to specify the relative abundance of flip and flop variants in these cells. Therefore, the P/C ratio can be used to infer subunit/splice variant expression only when the ratio is low or high. 6. These results suggest that the potentiation by PEPA alone reveals cell-to-cell heterogeneity of AMPA receptors, but a comparison of the actions of PEPA and cyclothiazide further facilitates the detection of the heterogeneity.

A novel allosteric potentiator of AMPA receptors: 4--2-(phenylsulfonylamino)ethylthio--2,6-difluoro-phenoxyaceta mide.

We report that a novel sulfonylamino compound, 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluoro-phenoxyacetam ide (PEPA), selectively potentiates glutamate receptors of the AMPA subtype. PEPA (1-200 microM) dose dependently potentiated glutamate-evoked currents in Xenopus oocytes expressing AMPA (GluRA-GluRD), but not kainate (GluR6 and GluR6+KA2) or NMDA (zeta1 + epsilon1-epsilon4), receptor subunits. PEPA was effective at micromolar concentrations and, in contrast to the action of cyclothiazide, preferentially modulated AMPA receptor flop isoforms. At 200 microM, PEPA potentiated glutamate responses by 50-fold in oocytes expressing GluRCflop (EC50 approximately 50 microM) versus only threefold for GluRCflip; a similar preference for flop isoforms was observed for other AMPA receptor subunits. Dose-response analysis for GluRCflop revealed that 100 microM PEPA produced a sevenfold increase in AMPA receptor affinity for glutamate. PEPA produced considerably weaker potentiation of kainate-evoked than glutamate-evoked currents, suggesting modulation of the process of receptor desensitization. In human embryonic kidney 293 cells transfected with AMPA receptor subunits, PEPA either abolished or markedly slowed the rate of onset of desensitization and potentiated steady-state equilibrium currents evoked by glutamate with subunit (GluRC >/= GluRD > GluRA) and splice-variant (flop > flip) selectivity similar to that observed in oocytes. Our results show that PEPA is a novel, flop-preferring allosteric modulator of AMPA receptor desensitization at least 100 times more potent than aniracetam.

A cDNA encoding fish fibroblast growth factor-2, which lacks alternative translation initiation.

Here, we describe the isolation of a rainbow trout cDNA clone that contains the entire fibroblast growth factor-2 (FGF-2; basic FGF) coding region. Interestingly, the rainbow trout cDNA contains a translation stop codon just upstream of the primary initiating methionine codon and so cannot give rise to the longer forms of FGF-2 that are produced in mammals by alternative translation initiation at leucines farther upstream. Transfection of human FGF-2 cDNA into fish cells shows that fish cells can initiate protein synthesis at an upstream leucine CUG codon; surprisingly, however, synthesis is initiated only at the most 5' CUG and not at the two subsequent CUG codons or the methionine AUG codon also used in mammalian cells. Like other FGF-2 proteins, bacterially produced rainbow trout FGF-2 protein binds tightly to heparin-Sepharose and also promotes the proliferation of fibroblast cells. However, the protein differs from all others previously identified at amino acids 121-123, which are part of the proposed highly conserved receptor-binding domain. Comparisons of the efficacies of recombinant wild-type and mutant rainbow trout FGF-2 proteins demonstrate that these three amino acids are critical to the activity of FGF-2.

Fish glucocorticoid receptor with splicing variants in the DNA binding domain.

Here we describe the isolation of a rainbow trout cDNA containing an entire GR coding region. Although the encoded protein is highly homologous to other GRs, especially in its DNA binding domain, it contains a nine amino acid insertion between the two zinc fingers. This novel form is found in all rainbow trout tissues examined; however, the testis also contains a splice variant lacking this insert, making it completely continuous to other GRs. In transient transfection assays of cultured cells, the two rainbow trout GR variants activated transcription from the glucocorticoid-responsive mouse mammary tumor virus promoter to comparable levels.

Immunocytochemical localization of the Pit-1 protein in the pituitary of the rainbow trout (Oncorhynchus mykiss).

Pit-1 is a pituitary-specific transcription factor responsible for activating the growth hormone (GH) gene family. The localization of Pit-1 protein in the pituitary of rainbow trout was studied by immunocytochemical analysis. Fish Pit-1 was found to be extensively localized in the nuclei of cells in the proximal pars distalis (PPD), rostral pars distalis (RPD), and pars intermedia (PI). Sagittal sections were immunocytochemically double stained with antibodies against Pit-1, GH, prolactin (PRL), and somatolactin (SL). The results showed that Pit-1 protein was present in the GH-containing cells of the PPD, the PRL-containing cells of the RPD, and the SL-containing cells of the PI.