No abstract

Although aged rats reportedly have reduced intestinal vitamin D receptor (VDR) concentrations, it is unclear whether an analogous age-related defect occurs in man. Thus, we assessed the interrelationship among serum 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], calcium absorption and intestinal VDR in 44 healthy, ambulatory women, ages 20-87 yr. Fractional calcium absorption was measured after oral administration of 45Ca (20 mg CaCl2 as carrier); serum 1,25-(OH)2D3, by the calf thymus binding assay; and serum intact PTH, by a two-site immunochemiluminometric assay. Vitamin D receptor concentration was measured, by a new immunoradiometric assay, in biopsy specimens taken from the second part of the duodenum during gastroduodenoscopy in 35 of the women. Despite an age-related increase in serum PTH (r = 0.48; P less than 0.001) and in serum 1,25-(OH)2D3 concentration (r = 0.32; P less than 0.05), intestinal VDR concentration decreased with age (r = -0.38; P = 0.03) and fractional calcium absorption did not change with age. Although a contribution of decreased 25-hydroxyvitamin D 1 alpha-hydroxylase activity to the blunting of the increase in serum 1,25-(OH)2D3 concentration late in life is not excluded, the data are far more consistent with impaired intestinal responsiveness to 1,25-(OH)2D3 action. This defect could lead to compensatory increases in PTH secretion and 1,25-(OH)2D3 production which maintain calcium absorption and serum ionic calcium, but at the expense of increased bone loss.

The level of mRNA encoding the 1,25-dihydroxyvitamin D3 receptor in the intestine of vitamin Ddeficient rats given 1,25-dihydroxyvitamin D3 was determined by Northern blot analysis using a 32P-labeled cDNA probe to the 1,25-dihydroxyvitamin D3 receptor. mRNA levels increased 10-fold above deficiency levels at 6 and 12 hr after an intravenous dose of 1,25-dihydroxyvitamin D3, returning to predosing levels at 24 hr. Total receptor protein level determined by an immunoradiometric assay was increased 2-fold at 12 hr. No change in unoccupied receptor levels determined by ligandbinding assay was observed during this period. These results suggest that 1,25-dihydroxyvitamin D3 increases receptor mRNA and total receptor level to maintain constant levels of unoccupied receptor.

The effects of vitamin D status, serum calcium, and serum phosphorus levels on 1,25-dihydroxyvitamin D receptor levels in kidney were investigated. Weanling rats were fed for 4 weeks on a diet with various levels ofcalcium and phosphorus with or without vitamin D. The 1,25-dihydroxyvitamin )3 receptor concentration in kidney was determined by an immunoradiometric assay. In the absence ofvitamin D, total receptor concentration is increased 2-fold by an increase in serum calcium concentration. At normal serum calcium levels, the ministration of vitamin D resulted in a 5-fold increase in receptor concentration. In hypocalcemic anima, however, vitamin D did not change receptor levels. Serum phosphorus levels could not be linked to any changes in 1,25-dihydroxyvitamnin D3 receptor concentration. This study demonstrates that serum calcium levels and vitamin D regulate 1,25-dihydroxyvitamin D3 receptor concentration in vivo in kidney. On the other hand, vitamin D is unable to exert control of receptor levels in kidney under hypocalcemic conditions. 1,25-Dihydroxyvitamin D3 [1,2D3] is the main regulator ofplasma calcium and phosphorus levels (1). Vitamin D3 undergoes a two-step hydroxylation, first in the liver and then in the kidney, to form the active metabolite 1,25-(OH)2D3. The hormone acts through an intracellular receptor protein to modulate specific gene transcription in target tissues (2).One key to a better understanding of the physiological responses to vitamin D is to study regulation of the 1,25-(OH)2D3 receptor (VDR). The importance of receptor regulation is emphasized by the observations that biological response to 1,25-(OH)2D3 is a direct function of receptor number (3) and occupancy (4,5). Previous work in cell culture systems has shown that 1,25-(OH)2D3 up-regulates its own receptor (6). Regulation of the receptor has also been studied in vivo in the rat (7,8). Measured by ligand binding, 1,25-(OH)2D3 was found to have no effect on intestinal receptor levels after 24 hr and caused only a marginal increase after 5 days of treatment. The VDR in kidney, however, was significantly up-regulated at both time points.By using an immunoradiometric assay (IRMA) for total vitamin D3 receptor protein (9), we have shown a 2-fold increase in receptor protein levels in rat intestine in vivo 12 hr after intravenous administration of 1,25-(OH)2D3, although no change was detected by ligand binding (8).In this study we examined the effects of dietary vitamin D, serum calcium, and serum phosphorus on VDR concentration in rat kidney in vivo using an IRMA. We Buffers. The following buffers were used: phosphatebuffered saline (PBS), 1.5 mM KH2PO4/8.1 mM Na2HPO4, pH 8.0/137 mM NaCl/2.7 mM KCI; PBS/Triton, 0.5% Triton X-100/PBS; TE, 50 mM Tris HCl, pH 7.4/1.5 mM EDTA; TED, 5 mM dithiothreitol/TE; TEDK300, 300 mM KCIl/ TED; TEDK300/bovine serum albumin, 0.5% bovine serum albumin/0.02% NaN3/TEDK300; TEDNalS0, 150 mM NaCl/5 mM dithiothreitol'TED; homogenization buffer, 5 mM diisopropyl fluorophosphate/TEDK300. Sample Col...