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Division of Physiology, Department of Medicine, University of California, San Diego, La Jolla, California 92093-0623
The purpose of this research was to
develop a technique for rapid measurement of O2 uptake
(
O2) kinetics in single isolated skeletal muscle cells. Previous attempts to measure single cell O2 have utilized polarographic-style
electrodes, thereby mandating large fluid volumes and relatively poor
sensitivity. Thus our laboratory has developed an ~100-µl,
well-stirred chamber for the measurement of
O2 in isolated Xenopus laevis
myocytes using a phosphorescence quenching technique [Ringer solution
with 0.05 mM Pd-meso-tetra(4-carboxyphenyl)porphine] to
monitor the fall in extracellular PO2 (which is
proportional to cellular O2 within the
sealed chamber). O2 in single living
myocytes dissected from Xenopus lumbrical muscles was
measured from rest across a bout of repetitive tetanic contractions
(0.33 Hz) and in response to a ramp protocol utilizing an increasing
contraction frequency. In response to the square-wave contraction bout,
the increase in O2 to steady state (SS)
was 16.7 ± 1.3 ml · 100 g
1 · min1 (range
13.0-21.9 ml · 100 g1 · min1;
n = 6). The rise in O2
at contractions onset (n = 6) was fit with a time delay
(2.1 ± 1.2 s, range 0.0-7.7 s) plus monoexponential rise to SS (time constant = 9.4 ± 1.5 s, range
5.2-14.9 s). Furthermore, in two additional myocytes,
O2 increased progressively as
contraction frequency increased (ramp protocol). This technique for
measuring O2 in isolated, single
skeletal myocytes represents a novel and powerful investigative tool
for gaining mechanistic insight into mitochondrial function and
O2 dynamics without potential complications of the circulation and other myocytes.
phosphorescence quenching; Xenopus laevis; skeletal muscle; oxygen uptake kinetics
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