Demonstration and characterisation of a testicular receptor for 1,25-dihydroxycholecalciferol in the rat.

Abstract

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‐dihydroxycalciol 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 255000xg, 21 h, 4°C) and analysed with the hydroxyapatite assay. The data document the presence of a macromolecule in testicular cytosol with the properties of the intestinal 1,25‐dihydroxycalciol receptor. The binding macromolecule migrated at 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‐rachitic, rachitic 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 (Nmax= 82 fmol/mg protein) and an equilibrium constant of Kd= 2.6 × 10−10 M. The receptor is a 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.