Vitamin D receptor levels and binding are reduced in aged rat intestinal subcellular fractions

Abstract

The hormonal form of vitamin D, 1alpha,25(OH)(2)-vitaminD(3) [1alpha,25(OH)(2)D(3)], stimulates signal transduction pathways in intestinal cells. To gain insight into the relative importance of the vitamin D receptor (VDR) in the rapid hormone responses, the amounts and localization of the VDR were evaluated in young (3 months) and aged (24 months) rat intestinal cells. Immune-fluorescence and Western blot studies showed that VDR levels are diminished in aged enterocytes. Confocal microscopy assays revealed that the VDR and other immune-reactive proteins have mitochondrial, membrane, cytosol and perinuclear localization. Western blot analysis using specific antibodies detected the 60 and 50 kDa bands expected for the VDR in the cytosol and microsomes and, to a lesser extent, in the nucleus and mitochondria. Low molecular weight immune-reactive proteins were also detected in young enterocytes subcellular fractions. Since changes in hormone receptor levels appear to constitute a common manifestation of the ageing process, we also analyzed 1alpha,25(OH)(2)D(3) binding properties and VDR levels in subcellular fractions from young and aged rats. In competition binding assays, employing [(3)H]-1alpha,25(OH)(2)D(3) and 1alpha,25(OH)(2)D(3), we have detected specific binding in all subcellular fractions, with maximum binding in mitochondrial and nuclear fractions. Both, VDR protein levels and 1alpha,25(OH)(2)D(3) binding, were diminished with ageing. Age-related declines in VDR may have important consequences for correct receptor/effector coupling in the duodenal tissues and may explain age-related declines in the hormonal regulation of signal transduction pathways that we previously reported.