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Division of Physiology, Department of Medicine, University of California, San Diego, La Jolla, California 92093-0623
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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THE STUDY OF
O2 uptake (O2) at the
transition to a higher metabolic rate offers valuable insight into
mechanisms regulating metabolic control during muscle
contractions. In this regard, O2
kinetics measured at the mouth (pulmonary) have very similar characteristics to that seen across skeletal muscle (1,
10). However, Whipp et al. (28) speculate that
metabolic heterogeneities associated with skeletal muscle fiber
type and fiber-type recruitment make metabolic control inferences based
on pulmonary O2 kinetic profiles
difficult. In addition to fiber-type issues, concerns within intact,
exercising muscle, including convective O2 delivery, O2 delivery-to-O2 matching,
and O2 diffusion, further compound this issue
(7). At the other end of the spectrum, although metabolic
control has been studied extensively in isolated mitochondria, these
preparations reveal that mitochondria do not necessarily perform the
same in culture as in vivo (e.g., Refs. 24,
29). Thus the ability to study
O2 dynamics in single, intact myocytes would represent a powerful tool that would either eliminate or allow
control of the aforementioned potentially confounding variables.
With the use of polarographic-style O2
electrodes, O2 has been measured
previously in both isolated muscles (e.g., Refs. 6,
14) and myocytes (e.g., Refs. 5,
25). However, these investigations were hindered by
inherent complications associated with the O2 electrode,
including 1) the necessity for relatively large chamber
fluid volumes, resulting in a low signal-to-noise ratio; 2)
intrinsic O2 consumption; and 3) inertia (slow
response times). An optical method for extremely rapid assessment of
PO2 using O2-dependent quenching of
phosphorescence has been pioneered by Wilson (for review, Ref.
30). This technique has been adapted for measurement of
both in vitro and in vivo PO2 within
biological systems (3, 4, 11, 23, 31). This present
investigation was undertaken to develop a method using phosphorescence
quenching techniques (to avoid complications associated with
polarographic style O2 electrodes) for rapid assessment of
O2 in isolated single living skeletal
myocytes with high signal-to-noise resolution across the
rest-to-contractions transition.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Adult female African clawed frogs (Xenopus laevis) were doubly pithed and decapitated. Isolated single skeletal myocytes with tendon intact were microdissected from the lumbrical muscles (II-IV). Myocyte fiber type was assessed during dissection according to twitch characteristics (i.e., both the speed of contraction/relaxation rate as well as fatigue rate in response to ramp protocol) and appearance under dark-field illumination (both size and opacity of individual fibers) (26). All procedures were approved by the University of California-San Diego Animal Care and Use Committee and conform to National Institutes of Health guidelines.
Principle of O2-dependent phosphorescence quenching.
The O2-dependent quenching of phosphorescence is an
optical method for measuring PO2
(31) that can be described quantitatively by the
Stern-Volmer equation where
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(1) |
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(2) |
o and are the phosphorescence lifetimes
at anoxia and a given PO2 and
kq, the quenching constant (in Torr), is a second-order rate constant that is related to the frequency of collisions between O2 and the excited triplet state of the
porphyrin and the probability of energy transfer when collisions
occur. The constants for the compound used,
Pd-meso-tetra(4-carboxyphenyl)porphine bound to albumin in
solution have been well characterized (17). Pd-based
O2 probes have very little pH or temperature dependence within the physiological range (30).
The chamber was calibrated with PO2 values
ranging from 0 Torr (Ringer solution bubbled with 100% N2)
to 159 Torr (room air) at 20°C. The measured phosphorescence
lifetime decay in the anoxic environment and the relationship between
measured PO2 and the corresponding
phosphorescence lifetime as PO2 increased were
used to calculate the phosphorescence quenching parameters. From this calibration, kq was set at 180 Torr/s and
o at 601 µs.
The phosphorescence quenching of the porphyrin probe was measured
through a system consisting of a flash lamp (Oxygen Enterprises, Philadelphia, PA), a 425-nm band-pass excitation filter, a 630-nm cut-on emission filter, and a photomultiplier tube for collection of
the phosphorescence. To calculate phosphorescence lifetimes from the
porphyrin probe, the phosphorescence-decay curves from a series of five
flashes (15 Hz) were averaged, and a monoexponential function was fit
to the subsequent best-fit decay curve (Medical Systems Analysis
software, Greenvale, NY). Measurements were taken every 2 s.
Measurement of O2.
Myocyte O2 was measured from the change
in PO2 in the Ringer solution (with 0.05 mM
Pd-porphyrin probe bound to bovine serum albumin) within a sealed
chamber (described below) on the basis of the supposition that the fall
in Ringer solution PO2 is due solely to
O2 uptake from the myocyte. Thus
O2 can be calculated from the fall in
PO2 where O2 solubility in
physiological Ringer solution is 0.03015 ml O2 /ml at
20°C and 760 Torr (2). The chamber (100 µl volume) was
constructed of 0.5-cm Lucite on four sides, whereas the top and bottom
are composed of glass. The top is removable and sealed with silicon
gel. The chamber has dual infusion-withdrawal ports with a platinum
post on one end. The platinum post serves as the site for a stainless
steel stirrer (dielectrically coated and driven by a rotating magnet
located outside the chamber) and connection site for the platinum clip fastened to the myocyte tendon. The other end of the chamber has a
0.5-mm (ID) port (sealed from the atmosphere with silicon gel) through
which a thread attached to the contralateral tendon passes. The thread
is fastened to an external, adjustable force transducer (model 400A,
Aurora Scientific, Aurora, Ontario, Canada), which is coupled to a data
acquisition system (AcqKnowledge, Biopac Systems, Santa Barbara, CA)
for subsequent analysis.
Experimental protocol.
After microdissection, the isolated myocyte was placed in the chamber.
All air bubbles were removed, the chamber was sealed as described
above, and optimum muscle length was set on the basis of maximal
tetanic force output. Fibers were stimulated electrically to induce
direct tetanic muscle contractions (50 impulses/s of 2-ms duration,
200-ms train duration, 8-10 V). Isolated myocytes were then
subjected to a contraction protocol consisting of either 1)
3 min at 0.33 Hz (n = 6) or 2) incremental
ramp protocol (2-min intervals) from 0.1, 0.2, 0.25, 0.33, 0.5, and 1.0 Hz or until developed force dropped to ~60% of peak values
(n = 2). This latter protocol resulted in an estimated
peak O2. After the contraction protocol,
the fibers were mounted at a constant muscle length, and fiber width
(widest and narrowest at 2 locations) and length measurements were
taken in duplicate. Volume (V) was calculated assuming an ellipse as
V = 
· r(1) · r(2) · length,
where r is the cell radius and 1 and 2 are narrowest and
widest, respectively (5), and was converted to a mass
(1.10 g/cm3).
Kinetic modeling.
For kinetic analysis of O2 dynamics from
rest to steady-state contractions, Kaliedagraph data-analysis software
(Synergy Software, Reading, PA) was used. A monoexponential model was
used as follows
![<A><AC>V</AC><AC>˙</AC></A><SC>o</SC><SUB>2</SUB>(<IT>t</IT>)<IT>=</IT><A><AC>V</AC><AC>˙</AC></A><SC>o</SC><SUB>2</SUB>(b)+<IT>A</IT> · [1 − <IT>e</IT><SUP>−(t−TD)/&tgr;</SUP>]](/content/vol94/issue1/fulltext/353/img003.gif)
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(3) |
is the time constant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Metabolic and force output responses to a typical
"square-wave" contraction trial in one representative fiber are
shown in Fig. 1. For single myocytes
(n = 6) subjected to 3 min of tetanic contractions at
0.33-Hz frequency, the O2 A
was 16.7 ± 1.3 ml · 100 g1 · min1 (range
13.0-21.9 ml · 100 g1 · min1), whereas
the TD was 2.1 ± 1.2 (range 0.0-7.7 s) and was 9.4 ± 1.5 s (range 5.2-14.9 s) as determined by Eq. 3. For all cells, force production did not drop below 75% of peak
(initial) tension.
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Figure 2 demonstrates the responses of a
fast-twitch (more glycolytic) and slow-twitch (more oxidative) muscle
cell type to a "ramp" protocol of increasing contraction frequency.
The fall in extracellular PO2 was significantly
greater per a given contraction frequency in the glycolytic myocyte
(Fig. 2A) due, in part, to a threefold greater mass
(slow-twitch = 34 vs. fast-twitch = 104 µg). The more
oxidative myocyte was capable of sustaining force production at a level
above 60% of initial values significantly longer than the more
glycolytic myocyte. However, absolute force production was greater for
the glycolytic fiber, thereby mandating a higher
O2 at a given contractile frequency (at
0.1 and 0.2 Hz) compared with the oxidative fiber (Fig. 2B).
In general, and as expected, O2
increased as contraction frequency increased. Furthermore, peak
O2 was greater in the oxidative (31.9 ml · 100 g1 · min1) compared
with the more glycolytic (26.7 ml · 100 g1 · min1) cell
(Fig. 2B).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Phosphorescence-quenching techniques were utilized in the present
study to develop a novel procedure for measuring steady-state O2 during contractions and
O2 dynamics across the
rest-to-contractions transition in single isolated X. laevis
myocytes. To our knowledge, this is the first investigation to quantify
O2 on-kinetics in a single muscle cell.
Agreement with previous findings.
Muscle O2 has been measured in a number
of species, including humans, dogs, rats, and frogs. Maximal values
obtained either volitionally [e.g., humans, ~60
ml · 100 g1 · min1
(22)] or induced electrically [e.g., rat, ~9
ml · 100 g1 · min1
(20); dog, ~27 ml · 100 g1 · min1
(13)] vary on the basis of species. Furthermore, with the
use of a polarographic O2 electrode, values for isolated
X. laevis iliofibularis myoyctes reached ~19.5
ml · 100 g1 · min1
(25). With use of the technique described herein, maximal
O2 values for the two myocytes in which
this was measured ranged from ~25 to 32 ml · 100 g1 · min1 (Fig. 2),
which fall within the range for that reported previously.
O2 kinetics in single
myocytes. However, muscle O2 on-kinetics
have been reported in intact muscle groups such as human quadriceps
[e.g., half-time of the response (t1/2) = 28 s (10); t1/2 = 25 s (1)] and canine gastrocnemius-plantaris muscle
[t1/2 = 16.5 s (21);
t1/2 = ~20 s (9)].
One question, which has remained elusive, is whether an actual TD in
oxidative phosphorylation exists at the rest-to-work transition. When
Bangsbo et al. (1) corrected for blood transit time from
femoral vein to sampling port, the TD before an increase in
O2 occurred between 2 and 6 s from
exercise onset. Data presented in this study suggest that the
readjustment of oxidative metabolism to a higher metabolic demand can
begin quite rapidly and, in some cases, almost immediately.
After the TD, the increase in muscle O2
has been well characterized by a monoexponential rise to steady-state
levels (e.g., Refs. 9, 21). The value of for the myocytes studied in the investigation was ~9 s. This value is
comparatively fast to that shown for human and dog muscle, yet it is
similar to the pulmonary O2 primary
component time constant at the transition to a higher running speed in
the horse (16). It seems plausible that much of the
variability seen both within and across species regarding
O2 kinetics and amplitude can be
explained by fiber type (14) and/or oxidative capacity
(25). However, to date, that issue has yet to be resolved.
As discussed below, this single myocyte preparation, coupled with the
facility to discriminate fiber type and assess oxidative capacity,
offers a strong tool to elucidate the contribution of fiber type per se
(vs. oxidative capacity) in determining not only the maximal aerobic
capacity of muscle but also the rate at which aerobic metabolism
adjusts to step increases in metabolic demand.
As discussed above, the time between metabolic stimulus and a
discernable increase in oxidative metabolism (i.e., TD) is <6 s in
human quadriceps (1) and was less in most of the myocytes studied in this investigation. Hogan (12) demonstrated
that, at contraction onset, the time before a fall in single frog
myocyte intracellular PO2 was ~13 s during
the first contraction bout and speeded significantly to ~5 s with the
second stimulation. The measurements of
O2 in the present study show somewhat
faster kinetics, as calculated in Eq. 3, than that seen in
the fall in intracellular PO2 (11,
12). Although it is uncertain at present as to why this
discrepancy exists, it may be that if the O2 diffusing capacity of the myocyte exceeds the metabolic rate of the cell, an
O2 gradient will only exist temporally; i.e., there may be measurable flux of O2 without a large
PO2 gradient. Consequently, the TD before the
fall in intracellular PO2 (12)
does not necessarily equate with the onset of aerobic metabolism but
rather the point at which the rate of aerobic metabolism becomes
greater than the O2 diffusive capacity of the cell.
Moreover, if the mitochondria are located primarily in the periphery of
the cell, then a loss of O2 may occur from the
extracellular fluid before mean intracellular PO2 is altered significantly.
Methodological limitations.
A major concern in the present preparation was adequate mixing within
the chamber. However, in the present study, acute manipulations in
PO2 within the chamber, either by exposing the
chamber to the atmosphere or infusing Ringer solution at a different
PO2, were detected within 1 s.
Furthermore, the minimal TD in the O2
increase at contraction onset further suggests that the unstirred layer surrounding the myocyte is likely too thin to contribute significantly to the O2 diffusion barrier present under these
experimental conditions. A second concern is that O2
conductance is inherently different in this preparation than from seen
in an intact muscle preparation where O2 flux takes place
primarily between the discrete capillary-to-myocyte interface
(19). However, as it has traditionally remained
technically formidable to not only quantify the myocyte-to-capillary
surface area ratio in whole muscle but also to determine the
PO2 within the capillary network, the ability
to carefully set and control a homogeneous O2 environment
surrounding the single myocyte in the present preparation eliminates
the need for these determinations. A third concern is that
PO2 within the chamber falls (i.e., becoming more hypoxic), due toO2, over the
duration of the contraction protocol given the finite O2
volume within the sealed chamber. However, over the arguably
most critical time period (first 30-60 s) of contraction onset,
chamber PO2 drops 1-5 Torr, which would not be expected to alter significantly the initial metabolic response.
Advantages and applications of this technique.
Previous attempts to measure O2
within single muscle cells (e.g., Refs. 5,
25) or isolated muscle fiber bundles (e.g., Refs.
6, 15) have invariably used a polarographic
style O2 electrode, which necessitates larger fluid volumes
and introduces noise in the electrode signal as a result of the voltage
pulse necessary to stimulate the fiber (18). Given the
stability of the Pd-porphyrin O2 probe, its innocuous
nature when in contact with biological tissue and the rapidity with
which it can be sampled (for review, see Ref. 30), the
phosphorescence-quenching technique offers a viable solution for
measuring PO2 in small volumes. Indeed, phosphorescence quenching is currently used to measure frog single myocyte intracellular PO2 (11) as
well as rat microvascular PO2 (3).
O2 on-kinetic response is
hindered by heterogeneity associated with fiber-type composition as
well as convective and conductive O2 delivery
(27). The technique described herein is particularly
advantageous in that O2 dynamics can be
studied independent of fiber type, muscle recruitment, blood flow
patterns, and/or humoral variables. Furthermore, the mechanistic basis
for the O2 slow component that occurs at
work performed above the lactate threshold is likely confounded by or
associated with muscle recruitment (8). Thus this
single-myocyte preparation offers an excellent tool to assess whether
the additional O2 cost arises within the individual
myocytes or is associated with some alteration in fiber recruitment.
Furthermore, this technique can be developed for measurement of economy
and efficiency of different fiber types at different time points during
contractions, including during fatiguing and hypoxic
conditions. One final and particularly powerful application of
this methodology is that intracellular biochemical responses assessed
via fluorescence microscopy and O2 can
be measured simultaneously.
These results demonstrate that O2 can be
quantified in isolated single skeletal myocytes with the use of a
phosphorescence-quenching-based technique.
O2 values in the Xenopus
lumbrical myocytes, both amplitudes and kinetics, are consistent with
that seen in skeletal muscle of other species as well as in other
muscles of the frog. This technique can be utilized to study
single myocyte O2 independent of
confounding variables associated with whole muscle preparations and
thus represents a powerful tool in understanding the readjustment of
oxidative phosphorylation to an increased metabolic demand.
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ACKNOWLEDGEMENTS |
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This work was supported, in part, by National Institute of Arthritis and Muscloskeletal and Skin Diseases (NIAMSD) Grant AR-40155 (to M. C. Hogan), NIAMSD Grant 1 F32 AR-48461 (to C. A. Kindig), and National Institute on Aging Grant 1 F32 AG-20014 (to K. M. Kelley). R. A. Howlett is a Parker B. Francis fellow.
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FOOTNOTES |
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Address for reprint requests and other correspondence: C. A. Kindig, Div. of Physiology, Dept. of Medicine, Univ. of California, San Diego, 9500 Gilman Dr., MC0623A, La Jolla, CA 92093-0623 (E-mail: ckindig{at}ucsd.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
October 11, 2002;10.1152/japplphysiol.00559.2002
Received 26 June 2002; accepted in final form 28 September 2002.
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|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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 C. A. Kindig, R. A. Howlett, and M. C. Hogan Effect of contractile duration on intracellular PO2 kinetics in Xenopus single skeletal myocytes J Appl Physiol, May 1, 2005; 98(5): 1639 - 1645. [Abstract] [Full Text] [PDF]
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L. S. Ziemer, W. M. F. Lee, S. A. Vinogradov, C. Sehgal, and D. F. Wilson Oxygen distribution in murine tumors: characterization using oxygen-dependent quenching of phosphorescence J Appl Physiol, April 1, 2005; 98(4): 1503 - 1510. [Abstract] [Full Text] [PDF]
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 P. McDonough, B. J Behnke, D. J Padilla, T. I Musch, and D. C Poole Control of microvascular oxygen pressures in rat muscles comprised of different fibre types J. Physiol., March 15, 2005; 563(3): 903 - 913. [Abstract] [Full Text] [PDF]
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 Y. Chung, P. A. Mole, N. Sailasuta, T. K. Tran, R. Hurd, and T. Jue Control of respiration and bioenergetics during muscle contraction Am J Physiol Cell Physiol, March 1, 2005; 288(3): C730 - C738. [Abstract] [Full Text] [PDF]
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C. A. Kindig, C. M. Stary, and M. C. Hogan Effect of dissociating cytosolic calcium and metabolic rate on intracellular PO2 kinetics in single frog myocytes J. Physiol., January 15, 2005; 562(2): 527 - 534. [Abstract] [Full Text] [PDF]
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 S. Koga, D. C. Poole, T. Shiojiri, N. Kondo, Y. Fukuba, A. Miura, and T. J. Barstow Comparison of oxygen uptake kinetics during knee extension and cycle exercise Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2005; 288(1): R212 - R220. [Abstract] [Full Text] [PDF]
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D. P Wilkerson, I. T Campbell, and A. M Jones Influence of nitric oxide synthase inhibition on pulmonary O2 uptake kinetics during supra-maximal exercise in humans J. Physiol., December 1, 2004; 561(2): 623 - 635. [Abstract] [Full Text] [PDF]
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E. G. Mik, T. G. van Leeuwen, N. J. Raat, and C. Ince Quantitative determination of localized tissue oxygen concentration in vivo by two-photon excitation phosphorescence lifetime measurements J Appl Physiol, November 1, 2004; 97(5): 1962 - 1969. [Abstract] [Full Text] [PDF]
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 A. Lo, A. J. Fuglevand, and T. W. Secomb Theoretical simulation of K+-based mechanisms for regulation of capillary perfusion in skeletal muscle Am J Physiol Heart Circ Physiol, August 1, 2004; 287(2): H833 - H840. [Abstract] [Full Text] [PDF]
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A. Lo, A. J. Fuglevand, and T. W. Secomb Oxygen delivery to skeletal muscle fibers: effects of microvascular unit structure and control mechanisms Am J Physiol Heart Circ Physiol, August 7, 2003; 285(3): H955 - H963. [Abstract] [Full Text] [PDF]
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C. A. Kindig, R. A. Howlett, and M. C. Hogan Effect of extracellular PO2 on the fall in intracellular PO2 in contracting single myocytes J Appl Physiol, May 1, 2003; 94(5): 1964 - 1970. [Abstract] [Full Text] [PDF]
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