1,25-Dihydroxyvitamin D₃ inhibits Na⁺-H⁺ exchange by stimulating membrane phosphoinositide turnover and increasing cytosolic calcium in CaCo-2 cells

We have examined the effects of 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] on the phosphoinositol signal transduction pathway in the human colon cancer-derived cell line CaCo-2 and have studied the regulation of intracellular calcium ([Ca²⁺]_i) and pH (pH_i) by this secosteroid. CaCo-2 cells were prelabeled with [³H]myoinositol and treated with 10^-8 M 1,25-(OH)₂D₃ or vehicle for 90 sec. 1,25-(OH)₂D₃ caused a decrease in labeled phosphatidylinositol-4-5-bis-phosphate and an increase in labeled inositol 1,4,5-trisphosphate. Treatment with 10^-8 M 1,25-(OH)₂D₃ for 90 sec also raised the cellular content of diacylglycerol. In a dose-dependent manner, 1,25-(OH)₂D₃ caused the translocation of protein kinase-C activity from the cytosolic to the membrane fraction, which occurred after as little as 15 sec of exposure to the secosteroid, peaked at about 1-5 min, and then returned toward baseline values. In these CaCo-2 cells, baseline [Ca²⁺]_i was 258 ± 2 nM (mean ± SE), as assessed using the fluorescent dye fura-2. After exposure to 10^-8 M 1,25-(OH)₂D₃, [Ca²⁺]_i rapidly increased to 392 ± 14 nM after 100 sec, fell, and then subsequently rose to a plateau of 350 ± 3 nM after 400 sec. In Ca²⁺-free buffer, 1,25-(OH)₂D₃ caused only a transient rise in [Ca²⁺]_i, indicating that 1,25-(OH)₂D₃ stimulated both the release of intracellular calcium stores and calcium influx. 1,25-(OH)₂D₃ caused a dose-dependent decrease in pH_i in CaCo-2 cells, as assessed by the fluorescent dye BCECF, which was not observed in cells suspended in Na⁺-free buffer or pretreated with amiloride, indicating that the secosteroid inhibited Na⁺-H⁺ exchange. No effect of 1,25-(OH)₂D₃ on pH_i was observed in cells in a Ca²⁺-free buffer or pretreated with the phospholipase-C inhibitor U-73,122, which also blocked the rise in [Ca²⁺]_i, or in cells pretreated with the Ca²⁺/calmodulin inhibitor calmidazolium. Taken together, these studies indicate that 1,25-(OH)₂D₃ rapidly stimulates membrane phosphoinositide breakdown in CaCo-2 cells, generating the second messengers inositol 1,4,5-trisphosphate and diacylglycerol, causing translocation of protein kinase-C to the membrane, and increasing [Ca²⁺]_i by both releasing calcium stores and promoting calcium influx. Secondary to the rise in [Ca²⁺]_i, Na⁺-H⁺ exchange is inhibited by a calcium/calmodulin-dependent pathway.