title: 1,25-dihydroxyvitamin D3 inhibits Na(+)-H+ exchange by stimulating membrane phosphoinositide turnover and increasing cytosolic calcium in CaCo-2 cells.
abstract: 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 (pHi) 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 Ca(2+)-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 pHi 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 pHi was observed in cells in a Ca(2+)-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.
authors.affiliation: Department of Medicine, University of Chicago, Illinois 60637.
authors.forename: R K
authors.initials: RK
authors.lastname: Wali
authors.forename: C L
authors.initials: CL
authors.lastname: Baum
authors.forename: M J
authors.initials: MJ
authors.lastname: Bolt
authors.forename: T A
authors.initials: TA
authors.lastname: Brasitus
authors.forename: M D
authors.initials: MD
authors.lastname: Sitrin
mesh_terms: D000584:Amiloride / Q000494:pharmacology; D002117:Calcitriol / Q000494:pharmacology*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors*; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003110:Colonic Neoplasms; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004075:Diglycerides / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007294:Inositol / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008563:Membrane Lipids / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers
chemical_list: D002352:Carrier Proteins; D004075:Diglycerides; D007295:Inositol Phosphates; D008563:Membrane Lipids; D010716:Phosphatidylinositols; D017923:Sodium-Hydrogen Exchangers; D007294:Inositol; D015759:Ionomycin; D000584:Amiloride; D012964:Sodium; D011493:Protein Kinase C; D002117:Calcitriol