Effect of 1,25(OH)₂D₃ and its analogues on membrane phosphoinositide turnover and [Ca²⁺](i) in Caco-2 cells

Our laboratory has recently reported that 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] rapidly stimulates membrane polyphosphoinositide turnover and increases intracellular calcium concentration ([Ca²⁺](i)) in Caco-2 cells. The role of binding to the vitamin D receptor (VDR) in the regulation of these rapid biochemical events, however, remains unclear. The present studies were, therefore, conducted using analogues of 1,25(OH)₂D₃, which differ markedly in their affinities for the VDR, to assess and compare their effects on [Ca²⁺](i) and on inositol 1,4,5-trisphosphate (IP₃) formation. Competitive binding studies performed with both intact cells and high-salt cytosolic extracts from Caco-2 cells demonstrated that 1,25(OH)₂D₃ and 1,24-(OH)₂-22-ene-24-cyclopropyl-D₃ (BT) have high affinities for the VDR; 25(OH)-16-ene,23-yne-D₃ (AT), however, has a much lower affinity (~1,000- fold less) for the VDR. Despite these large differences in binding affinities for the VDR, AT and BT produced similar concentration-dependent increases in [Ca²⁺](i) and in IP₃ formation while 1,25(OH)₂D₃ was ~10-fold less active. These results indicate that the structural requirements for the rapid action of these secosteroids on signal transduction in Caco-2 cells are different from those for receptor binding and transcriptional regulation.