title: Lysophosphatidylinositol: a potential mediator of 1,25-dihydroxyvitamin D-induced increments in hepatocyte cytosolic calcium.
abstract: Recent studies in our laboratory demonstrate that 1,25-dihydroxyvitamin D [1,25-(OH)2D] increases hepatocyte cytosolic calcium in the absence of extracellular calcium and activates phospholipase-A-induced deacylation of phosphatidylinositol within 5 min. To determine whether inhibition of phospholipase-A affects D-induced increments in cytosolic calcium and which metabolite of phosphatidylinositol mediates these increments, cytosolic calcium is measured in cultured hepatocytes, loaded with Fura 2AM. Cellular fluorescence is determined at excitation wavelengths of 340 and 380 nm, and cytosolic calcium is calculated. 1,25-(OH)2D treatment for 5 min increases cytosolic calcium levels in the cultured hepatocyte. Inhibition of phospholipase-A with bromophenacylbromide blocks the vitamin D effect on cytosolic calcium, indicating that phospholipase-A activation precedes increments in cytosolic calcium. Neither arachidonic acid nor sodium arachidonate affects cytosolic calcium. In contrast, LPI increases hepatocyte cytosolic calcium in the presence and absence of extracellular calcium. The effect is not blocked by bromophenacylbromide. Neither cell viability nor supernatant fluorescence is altered by LPI, indicating that LPI is increasing fluorescence within the cell. 1,25-(OH)2D treatment for 5 min increases cytosolic pH in cultured hepatocytes. Inhibition of cell alkalinization with amiloride blocks the vitamin D effect on both cytosolic pH and cytosolic calcium, but not the effect of LPI on calcium. NH4Cl increases hepatocyte pH but not cytosolic calcium. The results indicate that phospholipase-A activation is necessary for 1,25-(OH)2D-induced increments in hepatocyte cytosolic calcium and that LPI, a deacylation product of phosphatidylinositol, mediates these increments. Furthermore, inhibition of Na+/H+ exchange, while not blocking the ability of the cell to increase cytosolic calcium in response to LPI, prevents the 1,25-(OH)2D-induced increments in both pH and calcium. The data suggest that the rapid effects of 1,25-(OH)2D on hepatocyte calcium are preceded by cell alkalinization and phospholipase-A activation, and these effects appear to be linked to the Na+/H+ antiport system and not nonspecific cell alkalinization.
authors.affiliation: Department of Orthopedics, University of Massachusetts Medical Center, Worcester 01655.
authors.forename: D T
authors.initials: DT
authors.lastname: Baran
authors.forename: A M
authors.initials: AM
authors.lastname: Kelly
mesh_terms: D000098:Acetophenones / Q000494:pharmacology; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001572:Benzofurans; D002117:Calcitriol / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D005456:Fluorescent Dyes; D016257:Fura-2; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008246:Lysophospholipids / Q000494:pharmacology*; D010741:Phospholipases A / Q000037:antagonists & inhibitors; D051381:Rats; D013050:Spectrometry, Fluorescence
chemical_list: D000098:Acetophenones; D001572:Benzofurans; D005456:Fluorescent Dyes; D008246:Lysophospholipids; C025449:lysophosphatidylinositol; D000584:Amiloride; D010741:Phospholipases A; D002117:Calcitriol; C006821:4-bromophenacyl bromide; D002118:Calcium; D016257:Fura-2