Demonstration and characterization of a 1 alpha,25-(OH)2D3 receptor-like macromolecule in cultured rat pituitary cells.
Recent studies have demonstrated the importance of prolactin (PRL) and growth hormone (GH) in the regulation of 25-hydroxycholecalciferol-1 alpha-hydroxylase activity. We have previously shown that 1 alpha,25-dihydroxycholecalciferol (1 alpha,25-(OH)2D3) reduces PRL and GH production by a clonal strain of rat pituitary tumour cells (GH3). The biologically active form of vitamin D3, 1 alpha,25-(OH)2D3, acts via an initial binding to cytoplasmic receptor proteins in target cells, and we demonstrate in this study the presence of specific receptors for 1 alpha,25-(OH)2D3 in the GH3 cells. GH3 cell cytosol was incubated with [3H]1 alpha,25-(OH)2D3 at 0-4 degrees C. Maximal binding was obtained between 2 and 6 h, and Scatchard analysis showed one single class of binding sites with Kd of 0.33 +/- 0.05 nM (mean + SD) and a Bmax of 103 +/- 26 fmol/mg cytosol protein. Competitive binding experiments revealed the following potency order: 1 alpha,25-(OH)2D3 greater than 25-OHD3 greater than 1 alpha-OHD3, 24,25-(OH)2D3. In contrast, corticosterone, testosterone, progesterone and oestradiol showed negligible ability to displace [3H]1 alpha,25-(OH)2D3 from its receptor. Sucrose gradient ultracentrifugation in high salt concentration revealed that GH3 cell cytosol possessed at 3.7S [3H]1 alpha,25-(OH)2D3 receptor protein which was inactivated by heating and protease treatment, but not after incubation with DNase or RNase. The receptor protein aggregated in salt-free sucrose gradients since the 3.7S complex was shifted reversibly to a approximately 6S form. Isoelectric focussing localized most of the [3H]1 alpha,25-(OH)2D3 to a protein peak with an isoelectric point of approximately 6 (pI 5.8-6.2). Since this 1 alpha,25-(OH)2D3 receptor protein has similar properties as the corresponding 1 alpha,25-(OH)2D3 receptors found in normal rat tissues, we suggest that lactotropes and somatotropes represent true target cells for 1 alpha,25-(OH)2D3.
D000818:Animals; D002116:Calcitonin / Q000494:pharmacology; D002117:Calcitriol / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D003600:Cytosol / Q000378:metabolism; D013006:Growth Hormone / Q000096:biosynthesis; D007525:Isoelectric Focusing; D007700:Kinetics; D010281:Parathyroid Hormone / Q000494:pharmacology; D010902:Pituitary Gland / Q000032:analysis*; D010911:Pituitary Neoplasms / Q000032:analysis*; D011388:Prolactin / Q000096:biosynthesis; D051381:Rats; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000032:analysis*; D013997:Time Factors
D010281:Parathyroid Hormone; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D011388:Prolactin; D013006:Growth Hormone; D002116:Calcitonin; D002117:Calcitriol