Ca2+ handling by Hdh -HET and Hdh -KO cells. Fluorimetric measurements of cytoplasmic Ca2+ using Fura-2 (A-D) or FuraFF (E and F) in permeabilized Hdh-HET and Hdh-KO MEF cells were conducted as described in Materials and Methods. A. Steady state [Ca2+]c values and the size of the intracellular calcium pools in HET1, HET5, KO12 and KO27 cells. Prestimulation steady state [Ca2+]c obtained in presence of ATP 2 mM, creatine phosphate 5 mM, creatine phosphokinase 5 units/ml and succinate 2 mM. To obtain the size of InsP3, ER and total ionomycin-sensitive pools, 7.5 μM InsP3, 2 μM thapsigargin (Tg) and 10 μM ionomycin were added respectively. Mean ± SE of at least five independent experiments with multiple parallels (HET1: n = 12; HET5; n = 12; KO12, n = 13; KO27, n = 10). B. Analog traces showing the [Ca2+]c responses to suboptimal (250 nM) and optimal InsP3 (7.5 μM) and Tg for HET1, HET5, KO12 and KO27 cells. Changes were normalized to the size of the ER Ca2+ pool. C. Suboptimal InsP3 (250 nM)-induced [Ca2+]c increase normalized to total InsP3-sensitive (7.5 μM) pool. D. Maximal InsP3 (7.5 μM)-induced [Ca2+]c increase normalized to total Tg-sensitive (2 μM) ER pool. E. passive Ca2+ buffering assessed as the [Ca2+]c increase evoked by the addition of 10 μM CaCl2 in Tg and Ruthenium Red (3 μM)- pretreated MEF cells. F. Mitochondrial Ca2+ uptake caused by the addition of 40 μM CaCl2. Rate of uptake was calculated for initial 30 seconds after the challenge during which the rate was linear. Correction was made for the Ruthenium Red insensitive component. Data values show mean ± standard error of at least three independent experiments with multiple parallels. * indicates statistically significantly different p values (p < 0.03).