C.S. Ritter, H.J. Armbrecht, E. Slatopolsky, A.J. Brown

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25-Hydroxyvitamin D3 suppresses PTH synthesis and secretion by bovine parathyroid cells C.S. Ritter, H.J. Armbrecht, E. Slatopolsky, A.J. Brown Kidney International Volume 70, Issue 4, Pages 654-659 (August 2006) DOI: 10.1038/sj.ki.5000394 Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 1 Reverse transcription-PCR of 1αOHase mRNA from cultured bPTC. Total RNA was isolated from confluent cultures of bPTC, and reverse transcription-PCR was performed using primers specific for bovine 1αOHase (CYP27B1) as described in Materials and Methods. Kidney International 2006 70, 654-659DOI: (10.1038/sj.ki.5000394) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 2 1αOHase activity in cultured bPTC. Confluent cultures of bPTC were incubated with the specified concentration of 25(OH)D3 for 24h. Medium was analyzed for 1-hydroxylated metabolites by RIA (IDS Inc., Fountain Hills, AZ, USA). Data are expressed as mean±s.d. (n=3). *P≤0.001 vs substrate control. Kidney International 2006 70, 654-659DOI: (10.1038/sj.ki.5000394) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 3 Suppression of PTH secretion by 25(OH)D3 and 1,25(OH)2D3. Confluent cultures of bPTC were incubated with the specified concentrations of 25(OH)D3 or 1,25(OH)2D3 for 72h with daily media changes. The cells were placed in fresh medium for 3h and PTH in the medium was measured by enzyme-linked immunosorbent assay (Immutopics). Data are from two independent experiments and are expressed as mean±s.d. (n=12). *P<0.05 vs ethanol control; **P<0.01 vs ethanol control. Kidney International 2006 70, 654-659DOI: (10.1038/sj.ki.5000394) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 4 Reduction of PTH mRNA secretion by 25(OH)D3 and 1,25(OH)2D3. Confluent cultures of bPTC were incubated with the specified concentrations of 25(OH)D3 or 1,25(OH)2D3 for 72h with daily media changes. PTH mRNA and 18S rRNA were measured by Northern blot analysis. Data are expressed as mean±s.d. (n=3). *P<0.05 vs ethanol control; **P<0.01 vs ethanol control. Kidney International 2006 70, 654-659DOI: (10.1038/sj.ki.5000394) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 5 Inhibition of parathyroid cell 1αOHase by clotrimazole. Confluent bPTC were incubated with 100nM 25(OH)D3 and increasing concentrations of clotrimazole for 24h. Medium was analyzed for 1-hydroxylated metabolites by RIA. Data are expressed as mean±s.d. (n=3). P<0.001 vs no clotrimazole control. Kidney International 2006 70, 654-659DOI: (10.1038/sj.ki.5000394) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 6 Effect of clotrimazole on suppression of PTH mRNA by 25(OH)D3. Confluent bPTC were incubated with ethanol vehicle or 100nM 25(OH)D3 in the absence or presence of 1μM clotrimazole (clot) for 72h with daily media changes. Total RNA was isolated and analyzed for PTH mRNA and 18S rRNA by Northern blot analysis. Data are from two independent experiments and are expressed as mean±s.d. (n=6 total). *P<0.001 vs no 25(OH)D3 control. Kidney International 2006 70, 654-659DOI: (10.1038/sj.ki.5000394) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 7 Effect of clotrimazole on induction of 24-OHase mRNA. Confluent bPTC were incubated with ethanol vehicle or 100nM 25(OH)D3 in the absence or presence of 1μM clotrimazole (clot) for 72h with daily media changes. Total RNA was isolated and 24-OHase mRNA and 18S rRNA were measured by Northern blot analysis. Data are expressed as mean±s.d. (n=3). *P<0.001 vs no 25(OH)D3 control. Kidney International 2006 70, 654-659DOI: (10.1038/sj.ki.5000394) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 8 Competitive VDR binding of 25(OH)D3 and 1,25(OH)2D3. Confluent bPTC were incubated with [3H]1,25(OH)2D3 (0.5nM) with increasing concentrations of radioinert 1,25(OH)2D3 or 25(OH)D3 for 2h. The cells were chilled, washed rapidly, and sonicated. The sonicates were treated with charcoal/dextran and centrifuged to remove unbound ligand, and the supernatant containing the VDR was counted for tritium (see Materials and Methods for details). Data are expressed as mean±s.d. (n=3). Kidney International 2006 70, 654-659DOI: (10.1038/sj.ki.5000394) Copyright © 2006 International Society of Nephrology Terms and Conditions