Development of a Retinoic Acid Receptor-Binding Assay with Rainbow Trout Tissue: Characterization of Retinoic Acid Binding, Receptor Tissue Distribution, and Developmental Changes
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abstract
Retinoic acid (RA) regulates the transcription of various genes required for several essential functions in vertebrates through binding to two classes of nuclear receptors, the retinoic acid receptors (RAR) and retinoid X receptors (RXR). We investigated nuclear RA binding in tissues from rainbow trout using the radiolabeled all-trans and 9-cis isomers of RA. Specific binding (indicative of receptor binding) of both all-trans- and 9-cis-RA was found in all tissues tested, including the adult trout ovary, testis, gill, liver, kidney, blood, white muscle, and heart. The kinetics and absolute amount of RA binding were dependent on both the tissue and the isomer of RA used. All-trans-RA bound with high affinity (K(d) approximately 1.0-3.9 nM), and low capacity (B(max) approximately 75-484 fmol RA/mg protein), while 9-cis-RA bound with lower affinity (K(d) approximately 7-56 nM), but with a greater capacity (B(max) approximately 214-1076 fmol RA/mg protein). The B(max) results were used to estimate RAR and RXR levels and revealed that the gill possesses primarily RARs while the liver possesses primarily RXRs. The RAR-specific competitor TTNPB was able to effectively displace all-trans-[3H]RA in most tissues, and the RXR-specific competitor AGN 194204 was able to effectively displace 9-cis-[3H]RA. However, TTNPB and AGN 194204 could not displace all of the RA in the kidney and testis, suggesting the existence of another nuclear RA binding protein. Binding of all-trans- and 9-cis-RA was also found in developing trout embryos and fry. Kinetic analysis revealed that RAR levels predominated at the eyed-embryo stage, but decreased 87% by the swim-up fry stage, while RXR levels remained relatively constant over the same time period. These findings suggest that RA and its receptors may play a key role in early trout development. This study has provided a simple and rapid radioligand binding assay that can identify RAR and RXRs in trout tissues.
