Assessment of O2 uptake dynamics in isolated single skeletal myocytes.
The purpose of this research was to develop a technique for rapid measurement of O(2) uptake (Vo(2)) kinetics in single isolated skeletal muscle cells. Previous attempts to measure single cell Vo(2) have utilized polarographic-style electrodes, thereby mandating large fluid volumes and relatively poor sensitivity. Thus our laboratory has developed an approximately 100-microl, well-stirred chamber for the measurement of Vo(2) 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 Po(2) (which is proportional to cellular Vo(2) within the sealed chamber). Vo(2) 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 Vo(2) to steady state (SS) was 16.7 +/- 1.3 ml x 100 g(-1) x min(-1) (range 13.0-21.9 ml x 100 g(-1) x min(-1); n = 6). The rise in Vo(2) 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, Vo(2) increased progressively as contraction frequency increased (ramp protocol). This technique for measuring Vo(2) in isolated, single skeletal myocytes represents a novel and powerful investigative tool for gaining mechanistic insight into mitochondrial function and Vo(2) dynamics without potential complications of the circulation and other myocytes.
Kindig
Casey A
CA
Division of Physiology, Department of Medicine, University of California, San Diego, La Jolla 92093-0623, USA. ckindig@ucsd.edu
Kelley
Kevin M
KM
Howlett
Richard A
RA
Stary
Creed M
CM
Hogan
Michael C
MC
D000818:Animals; D002469:Cell Separation; D004558:Electric Stimulation; D005260:Female; D006019:Glycolysis; D007700:Kinetics; D008163:Luminescent Measurements; D032342:Muscle Cells / Q000378:metabolism* / Q000502:physiology; D009119:Muscle Contraction / Q000502:physiology; D018482:Muscle, Skeletal / Q000378:metabolism* / Q000502:physiology; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D014982:Xenopus laevis