Quantitative assessment of [Ca2+]i levels in rat skeletal muscle in vivo.
Intracellular free Ca2+ concentration ([Ca2+]i) plays an essential role in physiological regulatory processes and common pathological conditions. Better understanding of these phenomena is still hampered by problems encountered in the quantitative assessment of [Ca2+]i changes, especially in blood-perfused organs. This study demonstrates that the ratiometric fluorescence technique can be adapted for quantitative in vivo [Ca2+]i determinations. The rat spinotrapezius muscle was topically loaded with indo 1-AM and imaged by a cooled digital camera. Ratio images were calculated in small regions (100 micrometers x 100 micrometers) practically devoid of large vessels in the resting state, after 30 min of ischemia, 20 min of reperfusion, or ionomycin or manganate treatments. When we assumed an average [Ca2+]i of 100 nM in the resting blood-perfused muscle, ischemia increased [Ca2+]i to approximately 200 nM. During reperfusion [Ca2+]i decreased to approximately 140 nM. Ionomycin induced an increase in [Ca2+]i to well above 750 nM. Manganate reduced Ca2+-dependent fluorescence to virtually zero. Our main conclusion is that changes in [Ca2+]i can be monitored and quantitatively determined in vivo.
Tóth
A
A
Second Department of Physiology, Semmelweis Medical University, H-1082 Budapest, Hungary.
Ivanics
T
T
Ruttner
Z
Z
Slaaf
D W
DW
Reneman
R S
RS
Ligeti
L
L
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D007091:Image Processing, Computer-Assisted; D008297:Male; D008856:Microscopy, Fluorescence / Q000379:methods; D018482:Muscle, Skeletal / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D002118:Calcium