Metabolites and analogs of 1α,25-dihydroxyvitamin D₃: evaluation of actions in bone

Analogs of 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] activate both genomic mechanisms via the nuclear vitamin D₃ receptor (nVDR) and nongenomic pathways via the plasma membrane vitamin D₃ receptor (pmVDR). Both of these pathways are normally activated by 1α,25(OH)₂D₃, but as a result of synthesis of numerous analogs of 1α,25(OH)₂D₃ these pathways can be distinguished. We used increasing doses of vitamin D₃ analogs to determine their potencies of action on these two distinct pathways, measuring calcium channel potentiation as an indicator of the nongenomic action and measuring increases in osteocalcin mRNA and protein release and bone resorption as indicators of genomic action. We found that both 25(OH)-16,23E-diene-D₃ (R) and 1α,25(OH)₂-16,23E-diene-D₃ (A) are 10-fold more potent than 1α,25(OH)₂D₃ for activation of the nongenomic pathway because double bonds in the side chain and the D ring increase the affinity for calcium channel potentiation. While the C-1α-hydroxyl group is not necessary for potentiation of calcium channels, methyl groups at this position can alter the affinity for calcium channel potentiation. On the other hand, 1000 fold higher concentrations of nongenomic analogs were needed compared to 1α,25(OH)₂D₃ to increase osteocalcin mRNA or protein release. 1α,25-Dihydroxy-16-ene-23-yne-26,27-hexafluorovitamin D₃, (E) is an agent that is 10 fold more potent than 1α,25(OH)₂D₃ at increasing osteocalcin mRNA and protein release, whereas 1α,25(OH)₂-3-epi-D₃ increases osteocalcin mRNA and protein with a potency over 10 fold lower than 1α,25(OH)₂D₃. These results suggest that double bonds in the side chain and the D ring stabilize action on the nongenomic pathway whereas F₆ on the terminal portion of the side chain increases potency for nVDR. On the other hand, while the C-1α-hydroxyl group is necessary for activation of genomic events via nVDR, the activation of nongenomic events occurs in the absence of this group.