title: Dynamic buffering of mitochondrial Ca2+ during Ca2+ uptake and Na+-induced Ca2+ release.
abstract: In cardiac mitochondria, matrix free Ca(2+) ([Ca(2+)]m) is primarily regulated by Ca(2+) uptake and release via the Ca(2+) uniporter (CU) and Na(+)/Ca(2+) exchanger (NCE) as well as by Ca(2+) buffering. Although experimental and computational studies on the CU and NCE dynamics exist, it is not well understood how matrix Ca(2+) buffering affects these dynamics under various Ca(2+) uptake and release conditions, and whether this influences the stoichiometry of the NCE. To elucidate the role of matrix Ca(2+) buffering on the uptake and release of Ca(2+), we monitored Ca(2+) dynamics in isolated mitochondria by measuring both the extra-matrix free [Ca(2+)] ([Ca(2+)]e) and [Ca(2+)]m. A detailed protocol was developed and freshly isolated mitochondria from guinea pig hearts were exposed to five different [CaCl2] followed by ruthenium red and six different [NaCl]. By using the fluorescent probe indo-1, [Ca(2+)]e and [Ca(2+)]m were spectrofluorometrically quantified, and the stoichiometry of the NCE was determined. In addition, we measured NADH, membrane potential, matrix volume and matrix pH to monitor Ca(2+)-induced changes in mitochondrial bioenergetics. Our [Ca(2+)]e and [Ca(2+)]m measurements demonstrate that Ca(2+) uptake and release do not show reciprocal Ca(2+) dynamics in the extra-matrix and matrix compartments. This salient finding is likely caused by a dynamic Ca(2+) buffering system in the matrix compartment. The Na(+)- induced Ca(2+) release demonstrates an electrogenic exchange via the NCE by excluding an electroneutral exchange. Mitochondrial bioenergetics were only transiently affected by Ca(2+) uptake in the presence of large amounts of CaCl2, but not by Na(+)- induced Ca(2+) release.
authors.affiliation: Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: Christoph A
authors.initials: CA
authors.lastname: Blomeyer
authors.forename: Jason N
authors.initials: JN
authors.lastname: Bazil
authors.forename: David F
authors.initials: DF
authors.lastname: Stowe
authors.forename: Ranjan K
authors.initials: RK
authors.lastname: Pradhan
authors.forename: Ranjan K
authors.initials: RK
authors.lastname: Dash
authors.forename: Amadou K S
authors.initials: AK
authors.lastname: Camara
mesh_terms: D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D006168:Guinea Pigs; D008929:Mitochondria, Heart / Q000737:chemistry / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000378:metabolism*; D012430:Ruthenium Red / Q000737:chemistry; D012964:Sodium / Q000737:chemistry / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger / Q000737:chemistry / Q000378:metabolism*
chemical_list: D024101:Mitochondrial Proteins; D019831:Sodium-Calcium Exchanger; D012430:Ruthenium Red; D012964:Sodium; D002118:Calcium