(Circulation. 1998;98:1990-1992.)
© 1998 American Heart Association, Inc.
Brief Rapid Communication |
Systemic Hypoxia Elevates Skeletal Muscle Interstitial Adenosine Levels in Humans
From the Section of Cardiology, The Milton S. Hershey Medical Center, Pennsylvania State University, Hershey.
Correspondence to D.A. MacLean, Section of Cardiology, Department of Medicine, The Milton S. Hershey Medical Center, Pennsylvania State University, PO Box 850, Hershey, PA 17033. E-mail dmaclean{at}med.hmc.psghs.edu
Abstract
Background—Adenosine is a potent vasodilator that has been shown to increase in cardiac tissue in response to hypoxia. However, peripheral vasodilatation also occurs during hypoxia, and the vasoactive substance(s) responsible for skeletal muscle vasodilation have not yet been completely identified. Therefore, the purpose of this study was to measure and quantify skeletal muscle interstitial adenosine during acute systemic hypoxia.
Methods and Results—Skeletal muscle interstitial adenosine concentrations were determined by the microdialysis technique, in which 4 semipermeable microdialysis probes were inserted into the vastus lateralis muscle of 6 healthy male subjects and perfused at a rate of 5 µL/min with Ringer's solution. Sixty minutes after the insertion of the microdialysis probes, systemic hypoxia was induced for 30 minutes by having the subjects breathe a mixture of 10.5% O2 in N2. Arterial oxygen saturation (fingertip oximeter) was lowered (P<0.05) from 96±0.7% to 74.9±1.4%, and forearm blood flow was increased 28%. During normoxia, the interstitial adenosine concentration was 0.44±0.08 µmol/L, and it was increased to 1.03±0.15 (P<0.05) and 0.85±0.09 (P<0.05) after 15 and 30 minutes of hypoxia, respectively.
Conclusions—These data are consistent with the concept that during acute systemic hypoxia, interstitial adenosine plays a key role in stimulating peripheral vasodilation.
Key Words: dialysis • blood flow • oxygen
Systemic hypoxia is commonly seen in a variety of disease states, including heart failure, shock, pulmonary embolism, and lung disease. To compensate for the decreased O2 delivery to tissues, a number of mechanisms are activated, resulting in an increase in ventilation, heart rate, cardiac output, and peripheral (muscle) blood flow.1 2 3 Although a number of different compounds have been suggested to be responsible for the hypoxia-induced increase in blood flow, the most widely recognized and investigated has been adenosine. Much of the previous work has used various heart models,4 5 6 and most of the evidence for a role of adenosine came from studies showing that hypoxia-induced hyperemia was attenuated by adenosine deaminase,7 adenosine receptor blockade,4 or the inhibition of adenosine production.8
In contrast, the effects of hypoxia on skeletal muscle blood flow have been investigated to a much lesser extent. However, it has been shown that during systemic hypoxia, the release of adenosine from canine skeletal muscle was increased.9 Furthermore, it has been demonstrated in rodent muscle that adenosine largely mediates the vasodilation associated with systemic hypoxia10 11 and that this vasodilation was dependent on nitric oxide synthesis.10 However, whether these same substances or mechanisms are responsible for the hypoxia-induced vasodilatation in human skeletal muscle is unclear. Furthermore, human skeletal muscle interstitial adenosine levels have never been measured during hypoxia, and these data would greatly advance our understanding of the regulation of peripheral blood flow. Therefore, the purpose of the present study was to measure and quantify interstitial adenosine levels during acute systemic hypoxia in humans.
Methods
Subjects
The experimental protocol was approved by the institutional
clinical investigation committee, and written informed consent was
obtained. Six healthy male subjects with a mean age of 32 years (range,
26 to 40 years) participated in this study. None of the subjects were
taking any medication, and all refrained from the ingestion of
caffeine-containing beverages for 24 hours before the studies.
Microdialysis
The fibers used to construct the microdialysis probes were
obtained from an artificial dialysis kidney (GFE 18) that had a
molecular weight cutoff of 3000 Da (ID, 0.20 mm; OD, 0.22
mm). Each end of a single fiber was inserted 
1 cm into a hollow
nylon tube (ID, 0.50 mm; OD, 0.63 mm) and glued. The actual
probe length (distance between the 2 nylon tubes) was 4 cm. Four
microdialysis probes were inserted into the vastus lateralis muscle
(50% type I and 50% type II fibers) of 1 leg, with the skin and
subcutaneous tissue at the microdialysis probe entrance and exit sites
anesthetized with a local injection of lidocaine HCl. The
probes were then inserted into the muscle via a 14-gauge cannula in a
direction parallel to muscle fiber orientation. After insertion, the
microdialysis probes were attached to a perfusion pump (CMA model 102)
and perfused at a rate of 5 µL/min with a Ringer's solution
containing 0.5 mmol/L lactate and 3.0 mmol/L
glucose.
Materials
The subjects reported to the laboratory after an overnight fast,
and after microdialysis probe insertion, they rested supine for 60
minutes before the experimental protocol was initiated. A sealed face
mask with separate valves for inspired and expired gases was positioned
over the subject's mouth and nose for the delivery of a hypoxic gas
mixture and the monitoring of minute ventilation
(
E) and end-tidal
CO2. A 15-minute baseline collection period was
conducted while the subjects breathed room air (normoxia), during which
heart rate (ECG), blood pressure (Finapres),
E (pneumotachometer), end-tidal
CO2 (RGM, Ohmeda), and arterial
oxygen saturation (O2 Sat, fingertip oximeter)
were monitored every minute and collected on a Gould recorder.
Meanwhile, microdialysis dialysate was collected over the entire
15-minute period. Systemic hypoxia was induced by having the
subjects breathe a mixture of 10.5% O2 in
N2 for 30 minutes. All
cardiovascular and ventilatory variables were
collected every minute, whereas dialysate was collected in two
15-minute blocks (0 to 15 and 16 to 30 minutes). In 1 subject, because
of technical problems, hypoxia gas delivery was suspended after
only 15 minutes. In 3 subjects, venous occlusion plethysmography was
used to measure forearm blood flow (FBF). With this technique, forearm
circumference is measured in a mercury-in-Silastic strain-gauge
plethysmograph while a cuff is inflated to suprasystolic
pressure to exclude blood flow to the hand,12 and
blood flow is expressed as mL ·
min-1 · 100 mL-1.
It should be noted that all cardiopulmonary data were averaged
over the 15-minute collection periods to match the time frame used for
dialysate collection.
Analyses
Dialysate adenosine (40 µL) concentrations were
determined by the method of Tullson et al13 and
high-performance liquid chromatography (HPLC).
To determine the exchange fraction (called "probe recovery") of
adenosine over the microdialysis membrane, a small amount of
[2-3H]adenosine (<0.1 µCi/mL) was
included in the perfusate as the internal reference marker, as
previously described.14 Five microliters of
perfusate and collected dialysate was added to 3 mL of
scintillation fluid and counted in a liquid scintillation counter for
the determination of adenosine specific activity. Probe
recovery was then calculated on the basis of the relative loss of
[2-3H]adenosine
(perfusatedpm-dialysatedpm/perfusatedpm),
and interstitial adenosine was calculated by
dividing dialysate minus perfusate adenosine by probe
recovery. It should be noted that despite the addition of a small
amount of adenosine
([2-3H]adenosine, nmol/L range) to the
perfusate, on analysis by HPLC, no adenosine
was detected in any of the perfusate samples.
Statistics
The differences between normoxia and hypoxia were
analyzed with a paired Student's t test.
Significance was accepted at P<0.05, and all values are
expressed as mean±SEM.
Results
The cardiovascular and ventilatory data are
presented in the
Table
. There were
17% and 12% decreases (P<0.05) in
O2 Sat and end-tidal CO2,
respectively, during hypoxia compared with normoxia. In
contrast, hypoxia resulted in 23% and 41% increases
(P<0.05) in heart rate and E,
respectively, compared with normoxia. In the 3 subjects in whom FBF was
measured, hypoxia resulted in a 1.3-mL ·
min-1 · 100 mL-1
increase (28%) in skeletal muscle blood flow. There were no
significant shifts in mean arterial pressure throughout the
experiment.
|
Microdialysis probe recovery remained unchanged from normoxia
(51.7±2.2%) to hypoxia (0 to 15 minutes, 50.3±2.1%; 16 to
30 minutes, 49.3±2.2%). Interstitial adenosine
levels more than doubled (P<0.05) during the first 15
minutes of hypoxia (Figure) and
remained elevated (P<0.05) throughout the remainder of the
experiment. It appears from the data that there was a small decrease in
the interstitial adenosine levels during the final
15 minutes (16 to 30 minutes) of hypoxia. However, this
decrease is primarily due to a reduction in sample size (n=6 to n=5) as
a result of having to suspend hypoxic gas delivery in 1 subject during
the final 15 minutes of the experiment.
|
Discussion
The major finding of this study was that during acute systemic
hypoxia (75% O2 Sat),
interstitial adenosine levels were more than
doubled, and these observations represent the first time that
skeletal muscle interstitial adenosine has been
measured under such conditions. It is well documented that during
systemic hypoxia, peripheral blood flow is
increased.1 3 In the present study, FBF was
determined in 3 subjects to confirm this finding. It was observed that
skeletal muscle blood flow was increased 1.3 mL ·
min-1 · 100 mL-1,
whereas in a study by Leuenberger et al3 using
exactly the same protocol, FBF was increased 1.2 mL ·
min-1 · 100 mL-1.
These findings suggest that skeletal muscle blood flow was increased in
the present study during hypoxia and that the increase was
similar to that previous observed.
There are only a handful of studies in which interstitial adenosine levels have been measured, especially during hypoxia, and even these have yielded conflicting results. For example, both an increase8 and no change15 in interstitial adenosine levels have been observed in cardiac tissue in response to systemic hypoxia. Meanwhile, the only study in which skeletal muscle interstitial adenosine levels were determined was performed by Hellsten et al16 and illustrated that during 1-legged knee extensor exercise in humans, interstitial adenosine levels were increased as a function of exercise intensity and blood flow. These data provide new information regarding the direction and magnitude of change of interstitial adenosine in the face of increased metabolic demand and blood flow. In the present study, the resting interstitial adenosine concentration was 0.44±0.08 µmol/L, which is slightly higher than that seen by Hellsten et al16 for human skeletal muscle but very close to those values observed by Wang et al8 for cardiac tissue.
In the present study, an acute bout of systemic hypoxia resulted in a doubling of the interstitial adenosine concentrations. These findings suggest that in human skeletal muscle, adenosine plays a very important role in the regulation of local blood flow. Similar findings have been reported for rodent muscle11 and canine muscle9; it should be noted, however, that the level of hypoxia used in the present study is mild compared with those used in various animal models. It has been suggested, in animal preparations, that other compounds interact with adenosine to mediate the vasodilation associated with hypoxia. For example, it has been demonstrated that adenosine stimulates ATP-sensitive K+ channels on skeletal muscle to open, and the resulting release of K+ acts as a vasodilator.11 More recently, it has been shown that vasodilation in rats induced by adenosine during systemic hypoxia was dependent on nitric oxide synthesis.17 However, it is unclear whether a similar interaction between adenosine and nitric oxide exists in human skeletal muscle. These findings illustrate that the mechanisms associated with hypoxia-induced vasodilation are complex and that although adenosine is a key mediator, other compounds may interact with adenosine and also contribute to increased peripheral blood flow.
The measurement of interstitial compounds provides information not readily obtained from other conventional methods, such as arteriovenous differences and muscle biopsies. Detection of changes in interstitial concentrations is important because the interstitium is the site at which many of the metabolites are either produced or exert their effects. Therefore, interstitial determinations provide valuable information regarding not only the direction and magnitude of change in key metabolites but also their role in regulatory mechanisms. Furthermore, from the above discussion, it will be important in the future to measure all the putative regulators when investigating the mechanisms associated with hypoxia-induced vasodilation. In conclusion, the present study demonstrates that skeletal muscle interstitial adenosine is increased in response to acute systemic hypoxia. Furthermore, the microdialysis technique allows the measurement and quantification of compounds in the interstitial space and, when applied to future studies, will lead to valuable new insights into the mechanisms associated with hypoxia-induced vasodilation.
Acknowledgments
This work was supported by NIH grants R01-AG-12227 and HL-02654 and an NIH-sponsored General Clinical Research Center with Division of Research Resources grant M01-RR10732. The authors wish to thank K. Gray and M. Herr for their excellent technical support.
Received June 15, 1998; revision received September 16, 1998; accepted September 22, 1998.
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