title: Novel role of phospholipase C-delta1: regulation of liver mitochondrial Ca2+ uptake.
abstract: Mitochondrial Ca2+ (mCa2+) handling is an important regulator of liver cell function that controls events ranging from cellular respiration and signal transduction to apoptosis. Cytosolic Ca2+ enters mitochondria through the ruthenium red-sensitive mCa2+ uniporter, but the mechanisms governing uniporter activity are unknown. Activation of many Ca2+ channels in the cell membrane requires PLC. This activation commonly occurs through phosphitidylinositol-4,5-biphosphate (PIP2) hydrolysis and the production of the second messengers inositol 1,4,5-trisphosphate [I(1,4,5)P3] and 1,2-diacylglycerol (DAG). PIP2 was recently identified in mitochondria. We hypothesized that PLC exists in liver mitochondria and regulates mCa2+ uptake through the uniporter. Western blot analysis with anti-PLC antibodies demonstrated the presence of PLC-delta1 in pure preparations of mitochondrial membranes isolated from rat liver. In addition, the selective PLC inhibitor U-73122 dose-dependently blocked mCa2+ uptake when whole mitochondria were incubated at 37 degrees C with 45Ca2+. Increasing extra mCa2+ concentration significantly stimulated mCa2+ uptake, and U-73122 inhibited this effect. Spermine, a uniporter agonist, significantly increased mCa2+ uptake, whereas U-73122 dose-dependently blocked this effect. The inactive analog of U-73122, U-73343, did not affect mCa2+ uptake in any experimental condition. Membrane-permeable I(1,4,5)P3 receptor antagonists 2-aminoethoxydiphenylborate and xestospongin C also inhibited mCa2+ uptake. Although extra mitochondrial I(1,4,5)P3 had no effect on mCa2+ uptake, membrane-permeable DAG analogs 1-oleoyl-2-acetyl-sn-glycerol and DAG-lactone, which inhibit PLC activity, dose-dependently inhibited mCa2+ uptake. These data indicate that PLC-delta1 exists in liver mitochondria and is involved in regulating mCa2+ uptake through the uniporter.
authors.affiliation: Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee 37232-4753, USA.
authors.forename: Clayton D
authors.initials: CD
authors.lastname: Knox
authors.forename: Andrey E
authors.initials: AE
authors.lastname: Belous
authors.forename: Janene M
authors.initials: JM
authors.lastname: Pierce
authors.forename: Aya
authors.initials: A
authors.lastname: Wakata
authors.forename: Ian B
authors.initials: IB
authors.lastname: Nicoud
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Anderson
authors.forename: C Wright
authors.initials: CW
authors.lastname: Pinson
authors.forename: Ravi S
authors.initials: RS
authors.lastname: Chari
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002120:Calcium Channel Agonists / Q000494:pharmacology; D015220:Calcium Channels; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D004963:Estrenes / Q000494:pharmacology; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000502:physiology*; D008297:Male; D008856:Microscopy, Fluorescence; D008930:Mitochondria, Liver / Q000201:enzymology* / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D054803:Phospholipase C delta; D011760:Pyrrolidinones / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013096:Spermine / Q000037:antagonists & inhibitors / Q000494:pharmacology; D013268:Stimulation, Chemical; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000502:physiology*
chemical_list: D002120:Calcium Channel Agonists; D015220:Calcium Channels; D002135:Calcium-Binding Proteins; D004963:Estrenes; D005456:Fluorescent Dyes; D007527:Isoenzymes; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; C034703:mitochondrial calcium uniporter; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C085029:adenophostin A; D013096:Spermine; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; D054803:Phospholipase C delta; C518026:Plcd1 protein, rat; D000241:Adenosine; D002118:Calcium