Investigation of factors affecting fluorometric quantitation of cytosolic [Ca2+] in perfused hearts.

Abstract

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.

• Authors: R Brandes, V M Figueredo, S A Camacho, A J Baker, M W Weiner
• Journal: Biophysical journal (Biophys J) Vol. 65 Issue 5 Pg. 1983-93 (Nov 1993) ISSN: 0006-3495 [Print] United States
• PMID: 8298028 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S., Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Buffers
• Fluorescent Dyes
• Indoles
• NAD
• Indo-1 pentaacetoxymethyl ester
• NADP
• indo-1
• Calcium

Topics

• Animals
• Biophysical Phenomena
• Biophysics
• Buffers
• Calcium (metabolism)
• Cytosol (metabolism)
• Endothelium, Vascular (metabolism)
• Fluorescent Dyes
• Hypoxia (metabolism)
• In Vitro Techniques
• Indoles
• Male
• Mitochondria, Heart (metabolism)
• Models, Cardiovascular
• Myocardium (metabolism)
• NAD (metabolism)
• NADP (metabolism)
• Perfusion
• Rats
• Rats, Sprague-Dawley
• Spectrometry, Fluorescence