Novel role of phospholipase C-δ1:: regulation of liver mitochondrial Ca2+ uptake

Mitochondrial Ca2+ (mCa(2+)) 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 mCa(2+) 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)P-3] and 1,2-diacylglycerol (DAG). PIP2 was recently identified in mitochondria. We hypothesized that PLC exists in liver mitochondria and regulates mCa(2+) 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 mCa(2+) uptake when whole mitochondria were incubated at 37degreesC with Ca-45(2+). Increasing extra mCa(2+) concentration significantly stimulated mCa(2+) uptake, and U-73122 inhibited this effect. Spermine, a uniporter agonist, significantly increased mCa(2+) uptake, whereas U-73122 dose-dependently blocked this effect. The inactive analog of U-73122, U-73343, did not affect mCa(2+) uptake in any experimental condition. Membrane-permeable I(1,4,5)P3 receptor antagonists 2-aminoethoxydiphenylborate and xestospongin C also inhibited mCa(2+) uptake. Although extra mitochondrial I(1,4,5)P3 had no effect on mCa(2+) uptake, membrane-permeable DAG analogs 1-oleoyl-2-acetyl-sn-glycerol and DAG-lactone, which inhibit PLC activity, dose-dependently inhibited mCa(2+) uptake. These data indicate that PLC-delta1 exists in liver mitochondria and is involved in regulating mCa(2+) uptake through the uniporter.