Characterization of 1,25-dihydroxyvitamin D3 receptors and in vivo targeting of [3H]-1,25(OH)2D3 in the sheep placenta.
We sought to detect the presence of receptors for 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in placental tissues of five late gestational pregnant sheep and to quantitate their biochemical properties and abundance. Cytosol prepared from cotyledonary tissue was found to contain two [3H]-1,25(OH)2D3 binding macromolecules that sedimented at 3.2 S and 4.1 S, respectively, on linear (4-20 per cent) hypertonic sucrose gradients. The 4.1 S component cosedimented with serum that had been prelabelled with [3H]-25-hydroxyvitamin D3 (25-OHD3) and was present in cytosols despite extensive washing of the tissue prior to homogenization. Concurrent incubation of the cytosol with [3H]-1,25(OH)2D3 and a tenfold molar excess of radioinert 25-OHD3 resulted in complete resolution of the 3.2 S macromolecule and disappearance of the 4.1 S binding component. The binding of [3H]-1,25(OH)2D3 to the 3.2 S component was completely abolished by coincubation with a 100-fold molar excess of radioinert 1,25(OH)2D3 and was replaced by a well resolved peak in the 4.1 S region. Scatchard analysis of cytosol binding to [3H]-1,25(OH)2D3 in the presence of a tenfold molar excess of radioinert 25OHD3 revealed a single class of non-interacting saturable binding site in the cotyledon and the endometrium of high affinity and low capacity. The mean +/- s.e. of the dissociation constant of the cotyledonary receptor of 0.21 +/- 0.06 nM was not different from that of 0.16 +/- 0.03 nM for the endometrial receptor. However, the abundance of the cotyledonary receptor was fourfold higher than that in the endometrium (110 +/- 20 versus 28 +/- 7 fmol/mg protein). Since it is not possible to completely separate endometrial tissue from cotyledonary tissue, the low abundance of receptor in endometrial cytosols may merely represent contamination of endometrial tissue with cotyledonary tissue. Further analysis of the [3H]-1,25(OH)2D3 occupied receptor in cotyledonary cytosols showed that it bound to DNA cellulose and was eluted with 0.16 M KCl. This in vitro binding of [3H]-1,25(OH)2D3 to DNA was confirmed in vivo by the finding of preferential nuclear targetting of [3H]-1,25(OH)2D3 (56 per cent of total cellular activity), 4 h after fetal intravenous administration of [3H]-1,25(OH)2D3 to five chronically catheterized fetal sheep. Total placental uptake of [3H]-1,25(OH)2D3 at this time amounted to 3.7 +/- 0.9 per cent of the injected dose. Preliminary analysis of ovine placental cytosols revealed a calcium binding protein of similar molecular weight to that found in the ovine intestine and in the intestine and placenta of rodents.(ABSTRACT TRUNCATED AT 400 WORDS)
Ross
R
R
Department of Paediatrics, University of Cincinnati College of Medicine, OH 45267-0541.
Florer
J
J
Halbert
K
K
McIntyre
L
L
D000818:Animals; D001345:Autoradiography; D002117:Calcitriol / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004717:Endometrium / Q000378:metabolism; D005260:Female; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000378:metabolism*; D064030:S100 Calcium Binding Protein G / Q000378:metabolism*; D012756:Sheep
D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D064030:S100 Calcium Binding Protein G; D002117:Calcitriol