AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 275: H1652-H1662, 1998;
0363-6135/98 $5.00
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Vol. 275, Issue 5, H1652-H1662, November 1998

Quantitative assessment of [Ca2+]i levels in rat skeletal muscle in vivo

András Tóth1, Tamás Ivanics1, Zoltán Ruttner1, Dick W. Slaaf2, Robert S. Reneman3, and László Ligeti1

1 Second Department of Physiology, Semmelweis Medical University, H-1082 Budapest, Hungary; and Departments of 2 Biophysics and 3 Physiology, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands

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 µm × 100 µm) 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 ~200 nM. During reperfusion [Ca2+]i decreased to ~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.

intravital microscopy; indo 1-acetoxymethyl ester ratiometric method; ischemia-reperfusion; ionomycin


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