Investigation of factors affecting fluorometric quantitation of cytosolic [Ca2+] in perfused hearts.
The goal of these studies was to examine the effects of several factors that may artifactually influence quantitation of cytosolic [Ca2+], [Ca2+]c, while using the fluorescent calcium indicator Indo-1. The following factors were investigated: 1) a possible fluorescence contribution from unhydrolized Indo-1/AM (by Mn2+ quenching), 2) Ca2+ buffering by Indo-1 (by varying [Indo-1]), 3) endothelial and mitochondrial Indo-1 loading (by bradykinin stimulation and calculations), and 4) effects of changing tissue fluorescence (predominantly NAD(P)H) on calculated [Ca2+]c during hypoxia (by a new method which allowed simultaneous determination of [Ca2+]c and changes in [NAD(P)H]). No significant contribution of Indo-1/AM was found. With increasing [Indo-1], calculated systolic [Ca2+]c fell significantly. Indo-1 incorporation (< 18%) into endothelial cells, caused a slight underestimation of systolic [Ca2+]c, while mitochondrial Indo-1 loading may cause overestimation of [Ca2+]c. With increased tissue fluorescence, during hypoxia, systolic [Ca2+]c may be underestimated by approximately 27% (for Indo-1 loading factors three to five times original tissue fluorescence). These studies suggest conditions in which experimental artifacts could be minimized to allow reliable quantitation of [Ca2+]c in intact perfused hearts using Indo-1 fluorometry. The major problem of obtaining reliable results depended on the ability to correct for changing NAD(P)H fluorescence while keeping [Indo-1] low.
Brandes
R
R
Cardiovascular Research Institute, University of California, San Francisco.
Figueredo
V M
VM
Camacho
S A
SA
Baker
A J
AJ
Weiner
M W
MW
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002021:Buffers; D002118:Calcium / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism; D005456:Fluorescent Dyes; D000860:Hypoxia / Q000378:metabolism; D066298:In Vitro Techniques; D007211:Indoles; D008297:Male; D008929:Mitochondria, Heart / Q000378:metabolism; D008955:Models, Cardiovascular; D009206:Myocardium / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010477:Perfusion; D051381:Rats; D017207:Rats, Sprague-Dawley; D013050:Spectrometry, Fluorescence
D002021:Buffers; D005456:Fluorescent Dyes; D007211:Indoles; D009243:NAD; C059587:Indo-1 pentaacetoxymethyl ester; D009249:NADP; C048960:indo-1; D002118:Calcium