Erythrocyte and The Regulation

Erythrocyte and the Regulation of Human Skeletal Muscle Blood Flow and Oxygen
Delivery: Role of Circulating ATP

Jos Gonzlez-Alonso, David B. Olsen and Bengt Saltin
Circ Res. 2002;91:1046-1055; originally published online October 31, 2002;
doi: 10.1161/01.RES.0000044939.73286.E2
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Erythrocyte and the Regulation of Human Skeletal Muscle

Blood Flow and Oxygen Delivery
Role of Circulating ATP
Jos Gonzlez-Alonso, David B. Olsen, Bengt Saltin
AbstractBlood flow to contracting skeletal muscle is tightly coupled to the oxygenation state of hemoglobin. To
investigate if ATP could be a signal by which the erythrocyte contributes to the regulation of skeletal muscle blood flow
and oxygen (O2) delivery, we measured circulating ATP in 8 young subjects during incremental one-legged
knee-extensor exercise under conditions of normoxia, hypoxia, hyperoxia, and COnormoxia, which produced
reciprocal alterations in arterial O2 content and thigh blood flow (TBF), but equal thigh O2 delivery and thigh O2 uptake.
With increasing exercise intensity, TBF, thigh vascular conductance (TVC), and femoral venous plasma [ATP]
augmented significantly (P0.05) in all conditions. However, with hypoxia, TBF, TVC, and femoral venous plasma
[ATP] were (P0.05) or tended (P0.14) to be elevated compared with normoxia, whereas with hyperoxia they tended
to be reduced. In COnormoxia, where femoral venous O2Hb and (O2CO)Hb were augmented compared with
hypoxia despite equal arterial deoxygenation, TBF and TVC were elevated, whereas venous [ATP] was markedly
reduced. At peak exercise, venous [ATP] in exercising and nonexercising limbs was tightly correlated to alterations in
venous (O2CO)Hb (r20.93 to 0.96; P0.01). Intrafemoral artery infusion of ATP at rest in normoxia (n5) evoked
similar increases in TBF and TVC than those observed during exercise. Our results in humans support the hypothesis
that the erythrocyte functions as an O2 sensor, contributing to the regulation of skeletal muscle blood flow and O2
delivery, by releasing ATP depending on the number of unoccupied O2 binding sites in the hemoglobin molecule. (Circ
Res. 2002;91:1046-1055.)
Key Words: skeletal muscle blood flow erythrocytes oxygen sensor oxygen delivery
he signals regulating the rapid adjustments of skeletal

muscle blood flow to exercise and altered inspiratory O2
fraction have been the focus of more than a century of
research. Recently, the idea has been advanced that optimal
local circulatory adjustments to alterations in blood oxygenation and exercise require the existence of extracellular and
cellular O2 sensors coupled to signal-transduction systems,
which are capable of evoking the appropriate vascular responses in contracting and resting muscle.19 The extensive
available evidence demonstrating that alterations in circulating O2 are inversely related to changes in exercising skeletal
muscle blood flow provides indirect support for this notion.10 20 To assess the primary extracellular site of O2
sensing, this laboratorys initial approach was to elucidate
whether alterations in skeletal muscle blood flow to exercise
in humans were associated with either the amount of O2
dissolved in circulating plasma or the amount of O2 bound to
hemoglobin. 16,19 Using normoxia, hypoxia, anemia,
anemiahypoxia, COnormoxia, and COhyperoxia as interventions, we found compelling evidence indicating that
elevations in exercising muscle blood flow and vascular
conductance are independent of pronounced alterations in

PaO2 (40 to 540 mm Hg) but are closely linked to the
reductions in arterial oxyhemoglobin.16,19 Our findings suggest that the main vascular O2 sensor locus is located in the
erythrocyte itself, rather than in the PO2 sensitive regions of
the endothelium or vascular smooth muscle.
The red blood cell itself might be involved in the regulation
of local blood flow and O2 delivery by signaling O2 availability in the erythrocyte. This theory is consistent with the
findings from two independent groups of researchers who
have demonstrated that red blood cells release ATP3 and
NO4,5 in response to a fall in hemoglobin O2 saturation. The
release of NO from the S-nitrosohemoglobin molecule with
the lowering in oxyhemoglobin is thought to induce the
diffusion of the NO group to the vascular endothelium where
it stimulates vessel relaxation.4 5 ATP in turn can induce
vasodilatation by binding to P2y-purinergic receptors located
on the vascular endothelial cells to release NO- and/or
endothelium-derived hyperpolarization factors, which diffuse
to the vascular smooth muscle and result in vasodilatation.3
The vasodilator potency of ATP is clearly supported by
Original received August 9, 2002; revision received October 21, 2002; accepted October 23, 2002.
From The Copenhagen Muscle Research Centre, Rigshospitalet, University of Copenhagen, Denmark.
Correspondence to Jos Gonzlez-Alonso, The Copenhagen Muscle Research Centre, Rigshospitalet, Section 7652, Blegdamsvej 9, DK-2100
Copenhagen , Denmark. E-mail jga@cmrc.dk
2002 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000044939.73286.E2
1046
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by guest on March 3, 2014
Gonzlez-Alonso et al
Figure 1. Blood flow, blood pressure, vascular conductance,

and oxygen parameters measured at rest, during incremental
knee-extensor exercise and after 10 minutes of recovery with
exposure to normoxia, hypoxia, hyperoxia, and COnormoxia.
*Values during hypoxia and COnormoxia are significantly different from normoxia (P0.05).
reports where ATP was infused in isolated arterioles2124 and

human forearm.25,26 Although in vitro data demonstrate that
ATP is released from red blood cells with exposure to
hypoxia in the presence of hypercapnia,27 hypoxia alone,3 and
mechanical deformation,28 the physiological significance of
this mechanism has never been studied in intact humans.
Accordingly, the aim of this study was to test the hypothesis that the erythrocyte contributes to the local regulation of
skeletal muscle blood flow and O2 delivery by releasing ATP
in direct proportion to the number of unoccupied O2 binding
sites in the hemoglobin molecule. To accomplish this aim, we
combined the use of different inspiratory O2 fractions and CO
inhalation with the one-legged knee-extensor exercise model
to precisely manipulate the amount of O2 and CO bound of
hemoglobin in arterial and femoral venous blood during a
wide range of muscle O2 demands.
Materials and Methods

Thirteen healthy recreationally active subjects (12 males and 1
female) participated in two studies. They possessed a mean (SD)
age of 243 years, body weight of 759 kg, and height of 1837
cm. The subjects were fully informed of any risks and discomforts
associated with the experiments before giving their informed written
consent to participate. The studies conformed to the code of Ethics of
the World Medical Association (Declaration of Helsinki) and were
approved by the Ethics Committee of Copenhagen and Frederiksberg
communities.
In the first study, 8 of the subjects completed 3 to 4 four-minute
knee-extensor exercise bouts at 271, 461, 642, and 852%
(SEM) of their normoxic peak power output (785 W; 60 rpm)
Control of Oxygen Delivery by Circulating ATP
1047
under the following conditions: (1) normoxia (inspiratory oxygen

fraction, FIO2 21%); (2) systemic hypoxia (FIO2 10%); (3)
systemic hyperoxia (FIO2 100%); and (4) carbon monoxide (CO)
breathing combined with normoxia (211% circulating carboxyhemoglobin fraction (FCOHb); FIO2 21%). Each experimental condition was separated by 1 hour of rest. The normoxic, hypoxic, and
hyperoxic trials were counterbalanced across subjects, whereas the
COnormoxia trial was performed last, due to the relatively long
elimination period required to return to baseline COHb levels.
The subjects reported to the laboratory at 8 AM, after the ingestion
of a normal breakfast. On arrival, they rested in a supine position.
Catheters were placed under local anesthesia into the femoral artery
and vein of the exercising thigh, and the femoral vein or forearm vein
of nonexercising limbs using the Seldinger technique. The femoral
artery and veins catheters were positioned 1 to 2 cm proximal or
distal from the inguinal ligament. A thermistor to measure venous
blood temperature was inserted through the femoral venous catheter
orientated in the anterograde direction for the determination of
femoral venous blood flow. Electrodes to measure 3-lead ECG were
also attached. After 10 to 15 minutes of supine rest on the
knee-extensor ergometer while exposed to either normal, low, or
very high inspiratory O2 fraction, thigh hemodynamics and blood
samples were obtained for the later determination of baseline values.
The subjects then started to exercise in a given condition, followed
by 10 minutes of recovery. Before and during exercise and during
recovery in the CO trial, the subjects breathed in a closed-circuit
system previously described.19 CO was administered into the closedcircuit system using calibrated plastic syringes (total CO administered 32536 mL). During exercise, heart rate and arterial blood
pressure were recorded continuously. Thigh blood flow was measured after 60 and 150 seconds of exercise. Arterial and venous
blood samples (1 to 3 mL) were withdrawn simultaneously before
blood flow measurements.
The other five subjects participated in a second investigation
aimed to determine whether intraarterial infusion of ATP in the
femoral artery at rest (in the supine position) would increase skeletal
muscle blood flow and vascular conductance to the same extent than
exercise under normoxic conditions. ATP (Sigma A7699) dissolved
in isotonic saline (1 mg/mL) was infused into the femoral artery
during stepwise 4 minutes infusion periods with 10 minutes washout
intervals between doses. The ATP infusion rates given were 200,
400, 1000, 3000, 8000, 16 000, and 25 000 nmol/min. Furthermore,
a bolus injection (1 mL) was used to determine the rapidity and
duration of response to a 2000 nmol/mL dose.
In the first study, femoral venous blood flow (ie, thigh blood flow,
TBF, largely indicative of quadriceps blood flow during kneeextensor exercise) was determined by the constant infusion thermodilution technique.29 TBF was calculated using a heat balance
equation. During the ATP infusion study, TBF was measured in the
femoral artery using an ultrasound Doppler.30 Femoral artery blood
flow (FaBFVmean A 6104, L/min) was calculated by multiplying the weighted mean blood velocity (Vmean, m/s) by the crosssectional area of the femoral artery (A).30 To avoid contribution of
blood from the lower leg in either study, an occlusion cuff placed just
below the knee was inflated to 260 mm Hg. TBF values in both
studies represent the average of two measurements made from 2 to
3 minutes of exercise and 1 to 2 minutes after the start of ATP
infusion. Heart rate was obtained from the continuously recorded
ECG signal. Arterial blood pressure was continuously monitored by
a pressure transducer (Pressure Monitoring Kit, Baxter) inserted in
the femoral artery at the level of inguinal region.
[ATP] in plasma and whole blood samples were determined using
the luciferin-luciferase technique.31 Blood samples (2.7 mL) for
determination of either plasma or whole blood [ATP] were obtained
using vacuum syringes containing 4.3 mg EDTA (S-monovette, 2.7
mL KE; Sarstedt). For plasma [ATP] analysis, blood samples in the
syringes were centrifugated immediately for 30 seconds at 14 000
rpm (4C; Sigma 1 to 15 K, Osterode am Harz). Plasma was then
pipetted into prechilled tubes and frozen down in dry ice. The
duration of the whole procedure from blood withdrawal to plasma
separation was 90 seconds. For whole blood [ATP] analysis, 25 L
1048
Circulation Research
November 29, 2002
Results
Skeletal Muscle Hemodynamics and Circulating
ATP Under Normoxic Conditions
Figure 2. Plasma ATP responses at rest, during incremental

knee-extensor exercise and after 10 minutes of recovery with
*Significantly different from resting values (P0.05).
of whole blood was pipetted into a tube containing 2.5 mL of 2.5%

trichloroacetic acid (TCA). To measure whole blood [ATP] in the
same range as plasma samples, 2.5 L of the TCA-blood mixture
was then pipetted into another tube containing 2.5 mL Tris-EDTA
and frozen down in dry-ice. All samples were stored at 40C until
assayed. [ATP] was measured using a commercially available ATP
Kit (ATP Kit SL 144 to 041; BioTherma AB) using the internal ATP
standard procedure. Samples were measured in duplicates. The
coefficient of variation of 10 repeated resting plasma samples was
11%.
Hematocrit was measured in triplicate after microcentrifugation
and corrected for trapped plasma (0.98). Hemoglobin concentration,
carboxyhemoglobin fraction, and blood O2 saturation were determined spectrophotometrically (OSM-3 Hemoximeter, Radiometer).
PO2, PCO2, and pH were determined with Astrup technique (ABL5,
Radiometer) and corrected for measured femoral venous blood
temperature.
Heart rate and mean arterial pressure were calculated as the
average response over the period in which TBF was measured. Thigh
vascular conductance was calculated as the quotient between TBF
and mean arterial pressure. Blood O2 content (ctO2) was calculated as
(1.39[Hbfunctional]O2 saturation)(0.003PO2), where Hbfunctional was calculated as [Hbtotal](100COHb/100). FO2Hb
was estimated as the fraction oxygenated hemoglobin in relation to
total Hb whereas F(O2CO)Hb was fraction of oxygenated and
O2 )
carboxylated Hb in relation to total Hb. Thigh O2 uptake (thigh V
was calculated by multiplying the TBF by the difference in contents
in O2 between the femoral artery and vein (a-vO2diff). Leg O2
delivery was calculated by multiplying TBF by arterial ctO2 (ctaO2).
The ratio between a-vO2diff and ctaO2 was calculated as an index of
O2 extraction.
Statistical Analysis
A two-way repeated measures analysis of variance (ANOVA) was
performed to test significance between and within treatments. After
a significant F test, pair-wise differences were identified using
Tukeys honestly significant difference (HSD) post hoc procedure.
When appropriate, significant differences were also identified using
Students paired t tests. The significance level was set at P0.05.
Data are presented as meanSEM.
TBF, mean arterial pressure, and TVC increased progressively during incremental exercise under normoxic conditions
and subsequently returned to values that were not significantly higher than rest after 10 minutes of recovery (Figure
1). With an unchanged ctaO2 during incremental exercise, the
gradual elevation in O2 delivery to the thigh was the sole
result of the increase in TBF. Femoral arterial and venous
plasma [ATP] did not change in the transitions from rest to 20
W or the transition from rest to passive exercise (n4), which
augmented blood flow by 0.90.1 L/min. However, they
increased progressively during incremental exercise, being
significantly higher at 67 W compared with rest
([ATP]a997143 versus 654110 nmol/L, respectively;
[ATP]v 1835410 versus 64282 nmol/L, respectively; both
P0.05), and remained elevated after 10 minutes of recovery
(1500 nmol/L). Thigh arterial and venous plasma ATP,
which accounts for the changes in thigh plasma flow, increased progressively from 11922 to 63501567 nmol/min
(P0.05) but declined to 1100 (355) nmol/min after 10
minutes of recovery (Figure 2). In the nonexercising limbs,
venous plasma [ATP] also increased from 42823 nmol/L at
rest to 929171 nmol/L at 67 W (P0.05) and remained at
912212 nmol/L after 10 minutes of recovery.
Skeletal Muscle Hemodynamics and Circulating

ATP When Exposed to Systemic Hypoxia, Systemic
Hyperoxia, and CO in Combination With Normoxia
The exposure to systemic hypoxia and COnormoxia
equally reduced ctaO2 by 22% compared with normoxia,
whereas the exposure to systemic hyperoxia increased ctaO2
by 9% above normoxic levels. No significant changes in
ctaO2 were observed during incremental exercise in any of the
experimental conditions (Table). Despite the similar decline
in arterial ctO 2 , O 2 Hb, and FO 2 Hb in hypoxia and
COnormoxia, arterial O2 saturation and F(O2CO)Hb in
COnormoxia were similar to normoxia, but they were
significantly reduced in hypoxia (99 versus 77%, respectively). In COnormoxia, thigh O2 extraction was significantly reduced compared with the other trials (eg, 20 W: 49%
versus 54% to 596%, respectively; P0.05), reflecting a
high ctvO2 similar to that in normoxia. Concomitant to
changes in ctaO2 and ctvO2, TBF and TVC were elevated with
hypoxia (11% to 14%) and COnormoxia (20% to 25%), but
were reduced with hyperoxia (8% to 11%; Figure 1). The
compensatory adjustments in TBF and a-vO2diff with hypoxia, hyperoxia, and COnormoxia afforded similar thigh O2
O2 among the 4 experimental conditions
delivery and V
(Figure 1).
At rest, femoral arterial and venous plasma [ATP] with
normoxia, hypoxia, hyperoxia, and COnormoxia ranged
from 495 to 838 nmol/L (PNS). During incremental exercise, plasma [ATP] and thigh plasma ATP increased significantly with increasing exercise intensity in all conditions (all
P0.001; Figures 2 and 3). Moreover, venous plasma [ATP]
at a given work rate tended (P0.14) to be higher with
1049
Femoral Blood Variables at Rest During Incremental Knee-Extensor Exercise and After 10 Minutes of Recovery When Exposed to
Normoxia, Hypoxia, Hyperoxia, and CO Inhalation Combined With Normoxia
Normoxia
Power Output, W
Hypoxia
Rest
21 W
35 W
50 W
67 W
Recovery
Rest
21 W
35 W
50 W
Recovery
8.30.2
8.40.2
8.30.2
8.30.2
8.50.2
8.20.2
8.30.2
8.40.2
8.50.2
8.60.2*
8.30.2
8.30.2
8.40.2
8.30.2
8.40.2
8.60.2*
8.10.2
8.30.2
8.40.2
8.50.2
8.60.2*
8.20.2
41.11.3
41.21.3
41.30.9
41.60.9*
42.30.9*
40.70.9
41.41.1
41.60.9
41.90.9*
42.80.8*
41.71.2
41.31.0
41.61.0
41.61.0
41.51.0
42.31.0*
40.71.0
41.31.0
41.70.8
42.20.9*
42.70.7*
41.10.8
1.80.1
1.80.1
1.70.1
1.70.1
1.70.1
1.60.1
1.70.1
1.70.1
1.60.1
1.70.1
1.60.1
1.50.1
1.20.2
1.10.2
1.00.2
1.00.2
1.60.2
1.60.1
1.20.1
1.00.1
1.10.1
1.40.1
1805
1814
1815
1815
1854
1794
1527
1446
1394*
1434
1324*
1259
707*
667*
606*
546*
1366
1106
525*
435*
96.80.3
96.80.2
97.00.2
97.00.2
97.00.2
96.90.2
81.42.7
66.93.7
37.03.2*
35.12.9*
31.82.7*
27.91*
74.72.8
59.32.7
98.60.2
98.50.1
98.70.2
98.70.1
98.70.2
98.50.1
68.43.7
38.23.3*
36.33.0*
32.82.8*
28.92.5*
1012
1012
1062
1072*
373
231*
[Hb], mmol/L
Hct, %
COHb, %
O2Hb, mL/L
375*
934
76.72.5* 73.41.5*
74.11.5*
71.30.9*
27.62.6*
22.82.5*
19.12.4*
51.22.5*
83.12.7
78.32.4* 75.01.5*
75.81.5*
72.90.9*
76.32.9
60.92.7
28.82.6*
23.82.5*
20.22.4*
52.62.6*
1092*
1042*
474
402
371
361
360
221*
444*
302
191*
171*
161*
271
FO2Hb, %
F(O2CO)Hb, %
PO2, mm Hg
231*
231*
ctO2, mL/L
a
1835
1824
1537
1466
1404*
1269
1844
707*
1845
677*
1855
616*
1884
556*
1376*
1116
525*
445*
98.60.2
98.50.1
98.70.2
98.70.1
98.70.2
98.50.1
82.82.8
68.03.8
37.53.3*
35.62.9*
32.12.7*
28.22.5*
75.92.9
60.32.7
391
401
391
391
391
371
461
551*
591*
631*
702*
442
1444
1334*
375*
944*
78.02.5* 74.61.5*
75.41.6*
72.50.9*
27.92.6*
23.02.5*
19.32.4*
51.92.6
361
341
331
311*
321*
421
471*
481*
511*
361*
sO2, %
PCO2, mm Hg
pH
a
7.410.01 7.400.01 7.400.01 7.390.01 7.370.01* 7.370.01* 7.430.01 7.440.01 7.450.01 7.440.01 7.440.01
7.370.01 7.330.01* 7.300.01* 7.270.01* 7.210.01* 7.340.01 7.400.01
7.370.01 7.360.01* 7.310.01* 7.410.01
Hyperoxia
Power Output, W
CONormoxia
Rest
21 W
35 W
50 W
67 W
Recovery
Rest
21 W
35 W
50 W
Recovery
8.30.3
8.30.3
8.40.3
8.30.3
8.40.3
8.20.3
7.90.2
8.00.2
8.00.2
8.20.2*
7.80.2
8.20.3
8.30.3
8.30.3
8.30.3
8.50.2*
8.00.3
7.80.3
8.00.2
8.10.2*
8.10.2*
7.80.2
41.11.3
41.21.3
41.31.3
41.31.3
41.91.2*
41.01.2
39.61.1
39.71.1
39.91.0
40.51.1*
39.01.0
41.11.0
41.21.2
41.31.1
41.51.2
42.21.3*
40.21.5*
39.31.2
40.01.0
40.01.0
40.21.0*
39.00.9
1.80.1
1.70.1
1.60.1
1.50.1
1.50.1
1.50.1
19.40.4# 20.80.4# 21.70.9*# 21.90.1*# 21.10.7*#
1.70.1
1.30.2
1.20.2
1.10.1
1.10.2
1.60.1
19.30.3# 20.80.4# 21.70.8*# 21.70.8*# 21.10.7*#
1826
1826
1846
1846
1867
1816
1424
1404
1395
1434
1374
1476
1588
1218
623*
573*
524*
1025
Hb, mmol/L
Hct, %
COHb, %
O2Hb, mL/L
796*
747*
685*
655*
1050
November 29, 2002
Continued
Normoxia
Power Output, W
Rest
21 W
Hypoxia
35 W
50 W
67 W
Recovery
Rest
21 W
35 W
50 W
Recovery
FO2Hb, %
a
98.20.1 98.10.2
98.40.1
98.50.1
98.50.1
98.40.1 80.00.3 78.10.7
77.60.9
77.60.7
78.40.7
78.83.3 42.92.4*
39.93.1*
36.72.3*
34.22.3* 88.02.4 56.74.0 34.01.1*
30.91.3*
28.71.5*
58.82.2*
99.30.3#
99.50.2#
99.40.1#
F(O2CO)Hb, %
a
v
100.00.0 99.80.2
100.00.0
100.00.0
100.00.0
99.90.1 99.40.1# 98.90.6#
80.53.3 44.22.4*
41.13.2*
37.82.4*
35.32.3* 89.72.4 76.13.9# 54.71.1*# 52.61.1*# 50.41.2*# 79.92.4#
60915 58212*
57413* 56912* 1103#
PO2, mm Hg
a
59111*
58710*
495
261*
251*
251*
251*
2007
2006
2026
2026
2037
1496
806*
757*
695*
665*
11314#
11610#
1186#
1166#
323
181*
171*
171*
332
1986
1454
1444
1435
1465
1415
1608*
1228
633*
573*
534*
1035*
99.00.1 99.20.2# 98.60.7#
99.10.3#
99.40.2#
99.30.1#
34.62.3* 89.52.4 70.34.8# 42.91.4#
39.51.4#
36.61.7#
74.53.0#
656*
ctO2, mL/L
sO2, %
a
v
100.00.0 99.80.2
80.23.4 43.52.4*
100.00.0
100.00.0
40.43.2*
37.12.4*
100.00.0
PCO2, mm Hg
a
361
381
381
381
372
351
391#
381#
381#
341*#
371*#
462
562*
602*
622*
702*
432
442
502*
523*
563*#
411*#
7.410.01
7.400.01
pH
a
7.430.01 7.410.01
7.370.01 7.320.01* 7.300.01* 7.280.01* 7.220.01* 7.350.01 7.370.01# 7.330.01*# 7.300.01*# 7.260.01*# 7.340.01#
7.390.01 7.390.01 7.390.01# 7.400.01# 7.390.01# 7.400.01# 7.370.01#
Values are meanSE for 8 subjects. a indicates arterial; v, femoral venous; Hct, hematocrit; Hb, hemoglobin concentration; COHb, carboxyhemoglobin; O2Hb,
oxyhemoglobin; FO2Hb, fraction oxygenated hemoglobin in relation to total Hb; F(O2CO)Hb, fraction of oxygenated and carboxylated Hb in relation to total Hb; ctO2,
total oxygen content of blood; sO2, functional oxygen saturation, which expresses the percentage of oxygenated Hb in relation to the amount of Hb capable of carrying
oxygen.
*Significantly different from rest, P0.05. Significantly different from normoxia, P0.05. #Significantly different from hypoxia, P0.05.
hypoxia compared with the other conditions (Figure 2). The

increases in venous plasma [ATP] with incremental exercise
in all conditions were inversely related to declines in femoral
venous FO2Hb and O2 saturation (Figure 3). Plasma [ATP] in
exercising limb at rest, during peak exercise, and after 10
minutes of recovery tended to be higher than that in the
nonexercising limbs (P0.068; main effect; Figure 4). During peak exercise, venous plasma [ATP] in the exercising and
nonexercising limbs was closely related to alterations in
venous F(O2CO)Hb (r20.93 and r20.96, respectively;
P0.01; Figure 5). Red blood cell [ATP] in the femoral
artery and femoral vein did not change significantly with
either increasing exercise intensity or altered blood oxygenation [1.7 to 1.8 (0.1) mmol/L]. In all experimental conditions, arterial and femoral venous creatine kinase concentrations were not different, and remained unaltered with
increasing exercise intensity and during recovery from exercise [100 (13) U/L].
Skeletal Muscle Hemodynamics With Intrafemoral

Artery ATP Infusion
Intraarterial infusion of ATP in resting normoxic conditions
resulted in significant increases in TBF, TVC, and heart rate,
without altering mean arterial pressure or blood flow in the
control thigh (Figure 6). The constant infusion of ATP did not
produce a significant increase in plasma [ATP] after 2
minutes of the start of infusion. However, when accounting
for the increase in thigh plasma flow, thigh venous plasma
ATP increased from 0.40.1 to 4.61.7 mol/min
(P0.05). The increase in TBF paralleled a proportional drop
in thigh O2 extraction (from 271% to 21%; P0.05),
O2 in the face of a 10-fold
allowing the maintenance of thigh V
increase in O2 delivery. The bolus infusion of 2000 nmol/mL
of ATP, which augmented arterial plasma [ATP] from
130445 to 2652101 nmol/L, increased TBF in less than
10 seconds reaching a value of 2.30.1 L/min after 30
seconds (baseline TBF 0.40.1 L/min).
Discussion
There are several novel findings in this study that, on one
hand, indicate a tight coupling between alterations in circulating plasma ATP and changes in the oxygenation and
carboxylation state of hemoglobin and, on the other hand,
demonstrate the physiological relevance of such variations in
circulating ATP: (1) the progressive increases in femoral
venous plasma [ATP] during incremental exercise with normoxia, hypoxia, hyperoxia, and COnormoxia closely mirrored the declines in femoral venous O2Hb; (2) differences in
1051
Figure 3. Relationship between femoral venous plasma [ATP] vs

femoral venous O2Hb fraction, (O2CO)Hb fraction, O2 saturation, and PO2 during incremental knee-extensor exercise with
venous plasma [ATP] among conditions were strongly related

to variations in venous F(O2CO)Hb (r20.93); (3) at peak
exercise, venous plasma [ATP] in the nonexercising limbs
tended to be lower than in the exercising limbs, being
strongly correlated to venous F(O2CO)Hb (r20.96); (4)
COnormoxia, which produced the same decline in ctaO2
than hypoxia but increased femoral venous O2Hb and
(O2CO)Hb, resulted in an abrupt lowering in plasma [ATP];
and (5) intrafemoral artery infusion of ATP at rest in
normoxia evoked similar increases in TBF and TVC than
those observed during dynamic exercise. Together, our results support the hypothesis that the erythrocyte functions as
an O2 sensor, contributing to the regulation of local skeletal
muscle blood flow and O2 delivery by releasing ATP depending on the number of unoccupied O2 binding sites in the
hemoglobin molecule.
Exercising Skeletal Muscle Blood Flow Responds

to Changes in the Affinity and Amount of O2
Bound to Hemoglobin, Rather Than to Changes in
the Amount of O2 Dissolved in Blood
The present elevations in exercising TBF and TVC with
hypoxia and COnormoxia, and reductions with hyperoxia,
are generally consistent with previous reports producing large
changes in ctaO2.1120 The main difference compared with our
recent studies was that with greater herein COHb levels in the
COnormoxia trial (22% versus 18%) thigh O2 delivery did
not increase significantly, and that with lower herein FIO2
levels in the hypoxia trial (10% versus 12%) thigh O2 delivery
Figure 4. Plasma [ATP] at rest, at peak exercise and after 10

minutes of recovery with exposure to normoxia, hypoxia, hyperoxia, and COnormoxia. Note that venous [ATP] in the exercising and nonexercising limbs increased (P0.05) or tended to
increase during exercise and recovery compared with rest
(P0.066 and P0.089).
tended to decline somewhat at high work rates.19,20 In every

O2 was remarkably similar among
study, however, thigh V
conditions owing to compensatory adjustments in TBF and
a-vO2diff. These hemodynamic data illustrate the ability of
the circulatory system in adjusting skeletal muscle blood flow
so that total O2 flux from muscle capillaries to mitochondria
is maintained in conditions of vastly different O2 gradients.
Such precise circulatory regulation is thought to be controlled
by extracellular and cellular O2 sensors, which are capable of
changing vascular responses in contracting and resting muscle in accordance with O2 availability.19
The present results shed more light into the quest for the
primary extracellular site of O2 sensing. Collectively, our
present and previous reports using normoxia, hypoxia, anemia, anemiahypoxia, COnormoxia, and COhyperoxia
as interventions13,14,16,19 provide compelling evidence that
elevations in TBF and TVC during steady state exercise are
independent of pronounced alterations in PaO2 (40 to
540 mm Hg), but are closely linked to reductions in arterial
O2Hb and altered venous O2Hb. New evidence from the
comparison of present and previous results also argues
against the idea that skeletal muscle hemodynamics carefully
senses changes in O2 dissolved in arterial blood. In the present
study, we observed that hyperoxia that elevated PaO2 to
1052
November 29, 2002
Figure 5. Relationship between femoral venous plasma [ATP] vs

femoral venous O2Hb fraction, (O2CO)Hb fraction, O2 saturation, and PO2 in the exercising and nonexercising limbs during
peak knee-extensor exercise with exposed to normoxia, hypoxia, hyperoxia, and COnormoxia.
580 mm Hg, lowered TBF by 0.4 L/min (9%) compared with normoxia. This contrasts sharply to our previous
observation that COhyperoxia increased TBF by 1.0
L/min (33%) compared with normoxia despite the similarly
elevated PaO2.19 Therefore, it seems that the main vascular O2
sensor locus lies in the erythrocyte itself, rather than in the
PO2-sensitive regions of the endothelium or vascular smooth
muscle. It is worth noting that intracellular O2 markers such
as PO2 and MbO2 saturation cannot explain the profound
increases in TBF with CO inhalation because quadriceps
muscle PO2 and MbO2 saturation have been shown to be
similar in normoxia, COnormoxia, and COhyperoxia.20 A
central question then relates to how the red blood cell signals
the vascular endothelium to increase or decrease skeletal
muscle blood flow in direct proportion to O2Hb. According to
the model proposed by Ellsworth et al,3 the erythrocyte would
release ATP in proportion to the offloading of O2 from
hemoglobin. Free plasma ATP would in turn bind to purinergic receptors (P2y) in the vascular endothelium, resulting in a
vasodilatory response mediated by NO- and/or endotheliumderived hyperpolarization factor. Hence, treatments that modify the amount of O2 bound to Hb, such as those used in this
study, would be hypothesized to alter plasma [ATP] and
muscle blood flow.
Changes in Circulating Plasma ATP Are Closely

Coupled to Changes in Oxygenation and
Carboxylation State of Hemoglobin
Venous plasma ATP has previously been shown to increase
significantly during forearm exercise compared with rest in
Figure 6. Blood flow, mean arterial pressure, vascular conductance, and plasma [ATP] during progressive intraarterial infusion
of ATP in the femoral artery. *Significantly higher than resting
values (P0.05).
humans.32,33 A major novel finding in this study was that the

magnitude of increase in femoral venous plasma [ATP]
during incremental knee-extensor exercise varied profoundly
with alterations in blood oxygenation, being more tightly
coupled to alterations in O2Hb and (O2CO)Hb fractions
than PO2. This conclusion is based on three observations: (1)
the greater venous plasma [ATP] at a given work rate with
hypoxia was associated with lower venous O2Hb, whereas the
opposite occurred with COnormoxia, which resulted in
higher venous O2Hb than hypoxia (Figure 3); (2) the drop in
femoral PvO2 cannot explain the higher venous plasma [ATP]
in hypoxia compared with COnormoxia as PvO2 values
were essentially the same in both trials; and (3) the greater
plasma [ATP] in the exercising compared with nonexercising
limbs was intimately related to the lower (O2CO)Hb fraction, but weakly related to PvO2 (Figure 5). Thus, the present
alterations in plasma [ATP] with vast variations in blood
oxygenation and metabolic O2 demands are intimately linked
to the offloading of O2 from Hb as well as the binding of CO
to Hb, but are poorly related to changes in circulating free O2.
The question then arises as to whether the release of ATP
from the red blood cell could account for the present changes
in plasma ATP during exercise. Theoretically, the release of
very small amounts of the ATP contained in red blood cells
can cause large increases in plasma ATP because the [ATP]
in red blood cells is almost 3000-fold higher than in plasma
(1.8 mmol/L versus 0.5 to 2 mol/L, respectively). Several in
vitro studies have clearly documented an enhanced ATP
release from red blood cells with exposure to hypoxia in the
presence of hypercapnia,27 hypoxia alone,3 and mechanical
deformation.28 Furthermore, a recent in vitro study has
elegantly shown that CO, which displaces O2 from the heme
subunits of the Hb molecule and increases the affinity of the
remaining subunits for O2,34 drastically inhibits ATP release
from red blood cells.8 In congruence with this in vitro finding,
the present correlation at peak exercise is stronger between
plasma [ATP] and F(O2CO)Hb (r20.93 to 0.96) than
plasma [ATP] and O2 saturation (r20.68 to 0.86) or plasma
[ATP] and FO2Hb (r20.03 to 0.45) (Figure 5). This suggests
that not only the lower O2 offloading, but also the persistent
binding of CO to Hb explains the lower venous plasma ATP
in COnormoxia compared with the other trials. In this
context, the erythrocyte might be seen as the major source
accounting for the observed differences in plasma ATP.
However, there are other potential sources of plasma
ATP. Although the effects of hypoxia, hyperoxia, and
COnormoxia on interstitial ATP are unknown, studies
with microdialysis in human skeletal muscle in normoxia
have shown that adenosine, AMP, ADP, and ATP increase
in the muscle interstitium in proportion to the intensity of
contraction.35 The augmented interstitial [ATP], which
possibly reflects increases in sympathetic activity and/or
intracellular ATP turnover, could be another potential
source for plasma ATP during exercise. It has long been
known that sympathetic nerves corelease ATP with noradrenaline depending on the frequency of stimulation.21 In
this light, results from a parallel study showed that hypoxia
and COnormoxia caused remarkably similar significant
increases in muscle sympathetic nerve activity (MSNA)
during dynamic handgrip exercise.36 A similar increase in
plasma ATP would be expected in COnormoxia and
hypoxia if sympathetic nerves were major contributors to
plasma ATP. However, the observations that venous
plasma [ATP] was lower with COnormoxia despite
MSNA was equally high in COnormoxia and hypoxia in
our parallel study, suggest that sympathetic nerves were
not the main source of plasma ATP.
Muscle cells do not appear to serve as a source for
extracellular ATP as intracellular ATP content decreases
with incremental exercise in all the present conditions.20
Flow- or shear stressinduced increase in ATP from
endothelial cells cannot explain our results either, because
COnormoxia resulted in lower [ATP] than hypoxia,
despite the somewhat higher TBF than in hypoxia. Lastly,
the strikingly constant creatine kinase concentration
throughout the entire protocol clearly argues against a
noticeable leakage of ATP from interstitium. Together
results from the present and parallel studies seem to
indicate that the erythrocyte is the source accounting for
the majority of the changes in plasma ATP.
1053
Similar Increase in Skeletal Muscle

Hemodynamics With Intraarterial Infusion of ATP
Than During Incremental Exercise
Regardless of the mechanism or the source, the present study
demonstrates that elevations in human skeletal muscle blood
flow during incremental exercise are, in part, related to
progressive increases in plasma ATP. A crucial question is
whether the observed increases in plasma ATP during incremental exercise (0.5 to 2 mol/L or 0.1 to 8 mol/min)
would be sufficient to increase skeletal muscle blood flow,
independently of many other vasoactive compounds that also
rise during exercise. Underpinning the physiological relevance of circulating ATP, we found that the infusion of ATP
in the femoral artery at rest in normoxia evoked a dosedependent increase in TBF and TVC comparable to that
observed during exercise. This finding agrees with the wellcharacterized vasodilator effect of ATP infusion in the human
forearm25,26 and isolated arterioles23,24 and extends previous
results by directly comparing the hemodynamic effects of
intraarterial infusion of ATP to those associated with endogenous ATP during exercise. The present observation that
neither mean blood pressure nor blood flow in the control
thigh were altered suggest that the infused ATP primarily
acted locally.
Notably, the constant infusion of ATP up to 25 mol/min
in the femoral artery did not result in a corresponding
elevation in femoral venous plasma [ATP] (Figure 6). There
are several reasons for this finding: (1) ATP rapidly binds to
surface receptors21,22; (2) ATP degrades to other nucleotides
by the action of actonucleotidases21,22; and (3) changes in
plasma flow should be taken into account to quantify the
true increase in plasma ATP during incremental exercise.
The effect of rapid decreases in thigh plasma flow is clearly
demonstrated during recovery from exercise, where plasma
[ATP] remains high even after 10 minutes, but the rate of
thigh ATP outflow declines sharply. Furthermore, plasma
[ATP] decreases somewhat when TBF increases with passive
exercise or with adenosine infusion. Therefore, these data
demonstrate that ATP induces a potent dose-dependent vasodilator response in resting human muscle that is comparable
to that observed during incremental exercise.
In conclusion, this study provides evidence in resting and
exercising human limbs that variations in plasma [ATP] are
intimately coupled to the offloading of O2 from the hemoglobin molecule to the muscle cells, the alterations in the affinity
of O2 for Hb and the binding of CO to Hb. Moreover, our
finding that infusion of ATP at rest caused similar increases
in skeletal muscle blood flow than incremental exercise
demonstrate the physiological significance of the presently
observed elevations in plasma ATP. Taken collectively, the
herein results support the theory that the erythrocyte functions as an O2 sensor (see Figure 7), contributing to the
regulation of local skeletal muscle blood flow and O2 delivery
by releasing ATP depending on the number of unoccupied O2
binding sites in the hemoglobin molecule. Our results together with those by McMahon et al,37 which indicate a
varied release of NO/S-nitrosothiol from human red blood
cells as a function of O2Hb, clearly underpin the pivotal role
of the erythrocyte in the control of vascular tone.
1054
November 29, 2002
Figure 7. Model of erythrocyte oxygen

sensing under the conditions of normoxia, hypoxia, hyperoxia, and
COnormoxia during knee-extensor
exercise at 50 W. Unloading of O2 from
the Hb is coupled to the release of ATP,
which in turn can induce vasodilation by
binding to purinergic receptors (P2y) in
the vascular endothelium, resulting in a
vasodilatory response mediated by NOand/or endothelium-derived hyperpolarization factor (EDHF). For example, the
greater venous deoxygenation in hypoxia
is hypothesized to result in a greater
release in ATP, whereas the opposite
would occur in COnormoxia because
of the diminished deoxygenation and
increased number of bound Hb binding
sites. F(O2CO)Hb is the fraction of oxygenated and carboxylated Hb in relation
to total Hb.
Acknowledgments
This study was supported by a grant from The Danish National
Research Foundation (504-14). Special thanks are given to Dr
Arne Lundin from BioThema AB, Sweden, for his insightful
advice in the optimization of the ATP analysis. The excellent
technical assistance of Mads Dalsgaard, Carsten Nielsen, Karin
Hansen, Birgitte Jessen, Heidi Hansen, and Kristina Mller is
acknowledged.
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Correction
In an article by Gonzlez-Alonso et al (Circ Res. 2002;91:1046 1055), Erythrocyte and the
Regulation of Human Skeletal Muscle Blood Flow and Oxygen Delivery: Role of Circulating
ATP, an error appeared in the table. The column headings appearing on page 1050 in the
continuation of the table are incorrectly listed as Normoxia and Hypoxia. The correct column
headings are Hyperoxia and CONormoxia, respectively.
(Circ Res. 2003;92:e61.)

2003 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000067533.37506.16

Erythrocyte and The Regulation

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Erythrocyte and the Regulation of Human Skeletal Muscle Blood Flow and Oxygen

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