title: Enhanced charge-independent mitochondrial free Ca(2+) and attenuated ADP-induced NADH oxidation by isoflurane: Implications for cardioprotection.
abstract: Modulation of mitochondrial free Ca(2+) ([Ca(2+)](m)) is implicated as one of the possible upstream factors that initiates anesthetic-mediated cardioprotection against ischemia-reperfusion (IR) injury. To unravel possible mechanisms by which volatile anesthetics modulate [Ca(2+)](m) and mitochondrial bioenergetics, with implications for cardioprotection, experiments were conducted to spectrofluorometrically measure concentration-dependent effects of isoflurane (0.5, 1, 1.5, 2mM) on the magnitudes and time-courses of [Ca(2+)](m) and mitochondrial redox state (NADH), membrane potential (ΔΨ(m)), respiration, and matrix volume. Isolated mitochondria from rat hearts were energized with 10mM Na(+)- or K(+)-pyruvate/malate (NaPM or KPM) or Na(+)-succinate (NaSuc) followed by additions of isoflurane, 0.5mM CaCl(2) (≈200nM free Ca(2+) with 1mM EGTA buffer), and 250μM ADP. Isoflurane stepwise: (a) increased [Ca(2+)](m) in state 2 with NaPM, but not with KPM substrate, despite an isoflurane-induced slight fall in ΔΨ(m) and a mild matrix expansion, and (b) decreased NADH oxidation, respiration, ΔΨ(m), and matrix volume in state 3, while prolonging the duration of state 3 NADH oxidation, respiration, ΔΨ(m), and matrix contraction with PM substrates. These findings suggest that isoflurane's effects are mediated in part at the mitochondrial level: (1) to enhance the net rate of state 2 Ca(2+) uptake by inhibiting the Na(+)/Ca(2+) exchanger (NCE), independent of changes in ΔΨ(m) and matrix volume, and (2) to decrease the rates of state 3 electron transfer and ADP phosphorylation by inhibiting complex I. These direct effects of isoflurane to increase [Ca(2+)](m), while depressing NCE activity and oxidative phosphorylation, could underlie the mechanisms by which isoflurane provides cardioprotection against IR injury at the mitochondrial level.
authors.affiliation: Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, WI, USA.
authors.forename: Bhawana
authors.initials: B
authors.lastname: Agarwal
authors.forename: Amadou K S
authors.initials: AK
authors.lastname: Camara
authors.forename: David F
authors.initials: DF
authors.lastname: Stowe
authors.forename: Zeljko J
authors.initials: ZJ
authors.lastname: Bosnjak
authors.forename: Ranjan K
authors.initials: RK
authors.lastname: Dash
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004734:Energy Metabolism / Q000187:drug effects; D007530:Isoflurane / Q000494:pharmacology*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008929:Mitochondria, Heart / Q000187:drug effects* / Q000378:metabolism; D015428:Myocardial Reperfusion Injury / Q000517:prevention & control*; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D051381:Rats; D017208:Rats, Wistar
chemical_list: D009243:NAD; D000244:Adenosine Diphosphate; D007530:Isoflurane; D002118:Calcium