Cytosolic and mitochondrial [Ca2+] in whole hearts using indo-1 acetoxymethyl ester: effects of high extracellular Ca2+.
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 lambda 385 and lambda 456, 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.
Schreur
J H
JH
Department of Medicine, Cardiology, San Francisco General Hospital, California 94110, USA.
Figueredo
V M
VM
Miyamae
M
M
Shames
D M
DM
Baker
A J
AJ
Camacho
S A
SA
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002118:Calcium / Q000378:metabolism*; D002614:Chelating Agents; D003600:Cytosol / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D007211:Indoles; D008297:Male; D051379:Mice; D008929:Mitochondria, Heart / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D002614:Chelating Agents; D005456:Fluorescent Dyes; D007211:Indoles; C059587:Indo-1 pentaacetoxymethyl ester; D002118:Calcium