Cytosolic and mitochondrial [Ca2+] in whole hearts using indo-1 acetoxymethyl ester: Effects of high extracellular Ca2+

Joop H.M. Schreur, Vincent M. Figueredo, Masami Miyamae, David M. Shames, Anthony J. Baker, S. Albert Camacho

Research output: Contribution to journalArticlepeer-review

47 Scopus citations

Abstract

Assessment of free cytosolic [Ca2+] ([Ca2+]c) using the acetoxymethyl ester (AM) form of indo-1 may be compromised by loading of indo-1 into noncytosolic compartments, primarily mitochondria. To determine the fraction of noncytosolic fluorescence in whole hearts loaded with indo-1 AM, Mn2+ was used to quench cytosolic fluorescence. Residual (i.e., noncytosolic) fluorescence was subtracted from the total fluorescence before calculating [Ca2+]c. Noncytosolic fluorescence was used to estimate mitochondrial [Ca2+]. In hearts paced at 5 Hz (N = 17), noncytosolic fluorescence was 0.61 ± 0.06 and 0.56 ± 0.07 of total fluorescence at λ385 and λ456, respectively. After taking into account noncytosolic fluorescence, systolic and diastolic [Ca2+]c was 673 ± 72 and 132 ± 9 nM, respectively. noncytosolic [Ca2+] was 183 ± 36 nM and increased to 272 ± 12 when extracellular Ca2+ was increased from 2 to 6 mM. This increase in noncytosolic [Ca2+] was inhibited by ruthenium red, a blocker of Ca2+ uptake by mitochondria. We conclude that cytosolic and mitochondrial [Ca2+] can be determined in whole hearts loaded with indo-1 AM by using Mn2+ to quench cytosolic fluorescence.

Original languageEnglish (US)
Pages (from-to)2571-2580
Number of pages10
JournalBiophysical Journal
Volume70
Issue number6
DOIs
StatePublished - Jun 1996
Externally publishedYes

ASJC Scopus subject areas

  • Biophysics

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