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ADP and CCCP ‐induced increases in mitochondrial free Ca 2+ : greater contribution of matrix Ca 2+ buffering by ATP/ADP
Author(s) -
Haumann Johan,
Patel Runa,
Aldakkak Mohammed,
Rhodes Samhita S,
Vinnakota Kalyan C,
Dash Ranjan K,
Camara Amadou KS,
Beard Daniel A,
Stowe David F
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.756.6
Subject(s) - chemistry , bioenergetics , ruthenium red , biophysics , membrane potential , mitochondrial matrix , calcium , mitochondrion , biochemistry , cytosol , biology , enzyme , organic chemistry
Mitochondrial free calcium (mfCa 2+ ) is a key regulator of bioenergetics, but mfCa 2+ is also affected by matrix Ca 2+ buffering capacity. We tested how changes in matrix pH (pH m ) and ATP/ADP affect mfCa 2+ . We measured mfCa 2+ (indo‐1), pH m (BCECF), membrane potential (ΔΨ m )(rhodamine‐123) and redox state (NADH) using spectrofluorometry in guinea pig heart mitochondria suspended in Ca 2+ free buffer with 2.5 mM EGTA and 0.5 mM pyruvic acid. ADP (250 μM) and CaCl 2 (free [Ca 2+ ] of 25–400 nM) were added to infer the dynamics. Ruthenium red (RR) was given to block Ca 2+ uniport. Carbonyl cyanide chlorophenylhydrazone (CCCP) was added to abolish the H + gradient and lower pH m . Fluorescence signals for mfCa 2+ were corrected for NADH in energized mitochondria and for any dye‐induced spectral shifts. We found that mfCa 2+ increased more with added ADP than CCCP. RR blocked increases in mfCa 2+ with added buffer CaCl 2 , but did not affect increases due to ADP and CCCP. CCCP alone caused a marked lowering of pH m and abolished ΔΨ m , while ADP decreased pH m and ΔΨ m to a lesser extent. pH m , mfCa 2+ , and ΔΨ m reverted to baseline as ADP was converted to ATP. We conclude the increase in mfCa 2+ after adding ADP is due more to decreased buffering of ADP vs. ATP than to matrix acidification by CCCP. Changes in protein buffering and ion exchange activity may also modulate mfCa 2+ during alterations in mitochondrial activity.