Demonstration and Characterisation of a Testicular Receptor for 1,25-Dihydroxycholecalciferol in the Rat.

Defective luteinizing-hormone-mediated camp generation in rat testis was previously demonstrated by us in acute and chronic uremia. This defect was abolished by administration of 1,25-dihydroxycholecalciferol (1,25-dihydroxycalciol). In the present study, we furnish evidence for a cytosolic 1,25-dihydroxycaliol receptor in the rat testis. Testes of non-rachitic, rachitic or acutely uremic male Sprague-Dawley rats were homogenized in 0.3 M KCl, 10 mM Tris/HCl, 1.5 mM EDTA, 1 mM dithiothreitol, pH 7.4. Fresh purified cytosol was incubated with 1,25-[³H]-dihydroxycalciol (with or without 200-fold molar excess of unlabeled 1,25-dihydroxycalciol, subjected to 5-20% sucrose density-gradient separation (centrifugation with 255000 x g, 21 h, 4°C) and analysed with the hydroxyapatite assay. The data document the presence of a macromolecule migrated ar 3.5 S and could clearly be distinguished from the 6-S tissue-binding site for 25-hydroxycholecalciferol. The 1,25-dihydroxycalciol receptor was present in non-rarchitic and acutely uremic rats. Scatchard analysis of the receptor demonstrates high affinity and selectivity (1,25-dihydroxycalciol » 25-hydroxycholecalciferol > 1 α-hydroxycholecalciferol > 24(r),25-dihydroxycholecalciferol), low capacity (N_max = 82 fmol/mg protein) and an equilibrium constant of K_d = 2.6 x 10^-10 M. The receptor is protein according to enzyme degradation studies (trypsin, pronase, RNAase, DNAase). Competition studies with and without the alkylating reagent N-ethylmaleimide demonstrate a cysteine residue in or close to the binding site for 1,25-dihydroxycalciol Binding of 1,25-dihydroxycalciol protects the receptor against inactivation with N-ethylmaleimide. [ABSTRACT FROM AUTHOR]