1001

J Physiol 556.3 (2004) pp 10011011

Different vasodilator responses of human arms and legs

Sean C. Newcomer1 , Urs A. Leuenberger2 , Cynthia S. Hogeman2 , Brian D. Handly2 and David N. Proctor1
1

Department of Kinesiology The Pennsylvania State University, University Park 16802-6900 and 2 Division of Cardiology, Department of Medicine, The
Pennsylvania State University, College of Medicine, Hershey, PA 17033-0850, USA

Forearm vascular responses to intra-arterial infusions of endothelium-dependent and independent vasodilators have been thoroughly characterized in humans. While the forearm
is a well-established experimental model for studying human vascular function, it is of limited
consequence to systemic cardiovascular control owing to its small muscle mass and blood flow
requirements. In the present study we determined whether these responses could be generalized
to the leg. Based upon blood pressure differences between the leg and arm during upright posture, we hypothesized that the responsiveness to endothelium-dependent vasodilators would
be greater in the forearm than the leg. Brachial and femoral artery blood flow (Q, ultrasound
Doppler) at rest and during intra-arterial infusions of endothelium-dependent (acetylcholine
and substance P) and -independent (sodium nitroprusside) vasodilators were measured in
eight healthy men (2227 years old). Resting blood flows in the forearm before infusion of
acetylcholine, substance P or sodium nitroprusside were 25 4, 30 7 and 29 5 ml min1 ,
respectively, and in the leg were 370 32, 409 62 and 330 30 ml min1 , respectively. At
the highest infusion rate of acetylcholine (16 g (100 ml tissue)1 min1 ) there was a greater
(P < 0.05) increase in Q to the forearm (1864 476%) than to the leg (569 86%). Similarly,
at the highest infusion rate of substance P (125 pg (100 ml tissue)1 min1 ) there was a greater
(P < 0.05) increase in Q to the forearm (911 286%) than to the leg (243 58%). The
responses to sodium nitroprusside (1 g (100 ml tissue)1 min1 ) were also greater (P < 0.05)
in the forearm (925 164%) than in the leg (326 65%). These data indicate that vascular
responses to both endothelium-dependent and -independent vasodilator agents are blunted
in the leg compared to the forearm.
(Received 15 December 2003; accepted after revision 24 February 2004; first published online 27 February 2004)
Corresponding author S. Newcomer: Pennsylvania State University, College of Medicine, Hershey Medical Center
H0477, 500 University Drive, Hershey, PA 17033, USA. Email: snewcomer@psu.edu

The endothelium plays a pivotal role in the regulation

of vascular tone, angiogenesis and thrombus formation
(Cooke, 1992). The responses of the forearm vasculature to
intra-arterial infusions of endothelium-dependent vasodilators, such as acetylcholine, substance P and bradykinin,
have been thoroughly characterized in healthy humans
(Taddei et al. 1997a,b; DeSouza et al. 2000, 2002).
Unfortunately, it is not known whether these findings
can be generalized to the vasculature of the legs. While
the forearm is a well-established experimental model
for studying human vascular function, it is of limited
consequence to systemic cardiovascular control due to its
small muscle mass and blood flow requirements.
Upright posture and bipedal locomotion create a
haemodynamic challenge for humans. The hydrostatic
pressure gradient created by the earths gravitational
force exposes the vasculature of the lower extremities


C The Physiological Society 2004

to pressures approximately 65 mmHg greater than those

experienced by the upper extremities (Rowell, 1993;
Malhotra et al. 2002). The impact of this elevated blood
pressure on endothelial vasodilator function in the legs
is still unknown. However, based on research using
coarctation of the aorta to induce hypertension in animals
it seems plausible that endothelial vasodilator function
in the leg is decreased relative to the forearm. This
model of hypertension has parallels to the upright human
condition because it exposes the vasculature proximal to
the coarctation to elevated blood pressures while the blood
pressure distal to the coarctation remains intact. Research
using this model of hypertension suggests that elevated
blood pressure induces vessel remodelling by altering
both endothelial (Hollander et al. 1976; Owens & Reidy,
1985; Ueno et al. 2000) and vascular smooth muscle cells
(Berry & Greenwald, 1976; Bevan, 1976; Bevan et al. 1976;

DOI: 10.1113/jphysiol.2003.059717

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S. C. Newcomer and others

Hollander et al. 1976; Owens & Reidy, 1985; Ueno et al.

2000). Specifically, arterial segments harvested proximal to
the coarctation exhibit increased wall thickness (Berry &
Greenwald, 1976; Hollander et al. 1976; Ueno et al. 2000)
due to proliferation of smooth muscle (Bevan, 1976; Bevan
et al. 1976; Owens & Reidy, 1985; Ueno et al. 2000). This
proliferation of vascular smooth muscle is a consequence
of reductions in nitric oxide synthase (Ueno et al. 2000)
and presumably the bioavailability of nitric oxide. These
findings are supported by experimental studies which
reported impaired endothelium-dependent relaxation in
the vasculature proximal to the obstruction in animals subjected to aortic coarctation (Lockette et al. 1986; Miller
et al. 1987; Lai et al. 1989; Bell & Bohr, 1991; Bell, 1993).
Similar findings have also been reported in the forearm
vasculature of humans following the successful repair of
aortic coarctation (Gardiner et al. 1994).
Evidence for decreased endothelial vasodilator function
in the leg is also indirectly supported by literature
describing the genesis of peripheral arterial disease.
It is well established that peripheral arterial disease
develops first in the vasculature of the lower extremities
before progressing to the vasculature of the upper
extremities (Moore, 2002). These observations may
be indicative of greater endothelial dysfunction and
accelerated atherogenesis in the legs.
The purpose of the present investigation was to
determine whether endothelial vasodilator function is
different in the upper and lower extremities of humans.
Based upon limb differences in blood pressure during
upright posture, we hypothesized that the endothelium
of the lower extremities is less responsive to intra-arterial
infusions of endothelial agonists, acetylcholine and substance P, than the upper extremities.
Methods
Subjects

Studies were performed on eight healthy young men

(age 2227 years). All subjects were free of hypertension
(blood pressure < 140/90 mmHg), hypercholesterolaemia
(fasting cholesterol < 200 mg dl1 ), diabetes (fasting
glucose <120 mg dl1 ) and cardiovascular disease (12 lead
electrocardiogram at rest and during exercise). All subjects had a negative smoking history and were currently
not taking medication. Subjects were excluded from the
study if they had a body mass index (BMI) 30 kg m2
and/or a V O2 ,max > 70th percentile (46.8 ml kg1 min1 ) of
age group norms (American College of Sports Medicine,
2002). Before participation, each subject was verbally
informed of the potential risks and discomforts associated

J Physiol 556.3

with the study and signed a written informed consent form

approved by the Institutional Review Board of the Milton
S. Hershey Medical Center.
Experimental design

This study was separated into three visits (screening,

study 1 and study 2). The purpose of the screening visit
was to ensure that subjects were healthy but sedentary.
During this visit subjects gave a blood sample for analysis
(complete blood count with differential, lipid profile,
electrolytes, uric acid, glucose, creatinine and blood urea
nitrogen) and performed a graded exercise test on a cycle
ergometer (Monark 829E; Monark Exercise AB, Varberg,
Sweden). Metabolic measurements (Sensormedics Vmax
229; Sensormedics, Yorba Linda, CA, USA), in addition
to heart rate, rhythm (Sensormedics) and blood pressure
were obtained during rest and graded exercise.
The purpose of study 1 was to measure limb volume
and blood flow in the non-dominant forearm and leg.
Limb volume was measured in the forearm and leg using
water displacement. Measurements of limb blood flow
were made using Doppler ultrasound (ATL 5000. 1215
MHz non-invasive transducer) at rest and after 10 min of
ischaemia (i.e. peak reactive hyperaemia) in the forearm
and leg.
The purpose of study 2 was to measure the vascular
responses of the forearm and leg to endotheliumdependent and -independent vasodilators. Vascular
responsiveness was determined by measuring blood flow
in the forearm and leg during rest, vehicle infusion (saline),
and during increasing doses of acetylcholine, substance P
or sodium nitroprusside.
Measurements
Limb volume. Forearm volume was measured from the
head of the ulna to the olecranon of the non-dominant
arm using water displacement. Leg volume was measured
from the medial malleolus to approximately 68 cm below
the femoral bifurcation. This location was determined by
taking two-thirds the distance from the proximal patella
to the anterior superior iliac spine.
Blood flow and vascular conductance. Brachial and

femoral arterial blood flows were measured in the

experimental arm and leg with high resolution Doppler
ultrasound (ATL 5000; Philips Medical Systems, Bothwell,
WA, USA). A 125 MHz transducer was positioned
approximately 12 cm proximal to the tip of the intraarterial infusion catheter. During study 1 the transducer position over the brachial artery was moved
proximally by approximately 10 cm to accommodate the

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Limb differences in vasodilator responses

J Physiol 556.3

position of the occlusion cuff. Mean blood velocity was

measured at an insonation angle of 60 . The sample
volume was maximized in an attempt to minimize overestimation of mean blood velocity (Radegran, 1997;
Lott et al. 2001, 2002). Arterial diameter was measured
using a longitudinal view of the artery. Measurement of
arterial diameter was performed at the end of diastole
(determined by ECG) by measuring the distance between
near and far wall intimamedia interface. Blood flow was
calculated as:
Q = r 2 MBV 60
where Q is blood flow (ml min1 ), r is radius (cm) and
MBV is mean blood velocity (cm s1 ). Arterial diameter
and MBV were collected and analysed over a 30 s period
immediately following cuff deflation for measurements of
peak reactive hyperaemia and during the last minute of
drug infusion.
Brachial and femoral vascular conductance were
calculated during rest, saline infusion and drug infusions
as:
Conductance = Q/MAP
where Q is blood flow (ml min1 ) and MAP is mean
arterial pressure (mmHg). Mean arterial pressure was
continuously measured intra-arterially between minutes
three and five of saline or drug infusion.
The percentage increase in blood flow during both
peak reactive hyperaemia and intra-arterial infusion interventions was calculated as:
((Q intervention Q baseline )/(Q baseline )) 100
where Qintervention is either the limb blood flow response to
10 min of ischaemia or drug infusion and Qbaseline is the
blood flow before intervention. The same calculation was
used to assess percentage increase in conductance.
calibrated
mercury-in-Silastic strain gauge was used to express
blood flow measurements as millilitres per 100 millilitres
of tissue per minute (DE Hokanson, Inc., Bellevue, WA,
USA) (Proctor et al. 1996; Groothuis et al. 2003). Briefly,
the subjects lower leg and forearm were positioned
above heart level and a mercury-in-SilasticTM strain
gauge was placed around the widest portion of the limb
segment. To exclude blood flow to the hand and foot
a Hokanson TMC-7 cuff was placed around the wrist
and ankle and inflated to 200 mmHg. During blood flow
measurements a Hokanson SC-10 cuff positioned above
the elbow or knee was inflated to 50 mmHg. Four blood
Venous

occlusion

plethysmography. A

TM


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1003

flow measurements separated by 20 s were taken during

minutes three and five of all drug infusions.

Interventions
Peak reactive hyperaemia (study 1). Blood flow responses
to 10 min of ischaemia were measured in the brachial
and femoral arteries. Briefly, cuffs were positioned around
the wrist and above the elbow of the non-dominant arm
and inflated to 200 mmHg. After 10 min, the upper cuff
was rapidly deflated and brachial artery blood flow was
measured for 30 s using Doppler ultrasound. Peak limb
blood flow elicited by 10 min of arterial occlusion has
been reported to occur within 1015 s (Wascher et al.
1998; Pawelczyk & Levine, 2002). Similar methodology
was used to obtain peak blood flows in the non-dominant
leg. Cuffs were placed around the ankle and upper thigh.
The position of the quadriceps cuff was immediately distal
to the femoral bifurcation. Both cuffs were inflated for
10 min to 200 mmHg. Peak blood flow was measured in
the femoral artery upon deflation of the quadriceps cuff.
The arm and leg were positioned approximately 20 cm
above heart level during the trials.

Intra-arterial infusions (study 2). Each subject reported

at 07.30 to the Milton S. Hershey Medical Centers
General Clinical Research Center in a postabsorptive state
and abstained from caffeine. Upon arrival subjects were
directed to a temperature-controlled room and placed in
a supine position. Under aseptic conditions, two polyethylene catheters (22 and 20 gauge, Arrow International,
Inc., Reading, PA, USA) were inserted proximally into
the brachial (2 cm proximal to antecubital crease) and
common femoral (1 cm proximal to bifurcation) artery
of the non-dominant limb under local anaesthesia (1%
lignocaine). Catheters were used for both intra-arterial
drug infusions and measurements of brachial artery
blood pressure. To ensure accurate arterial blood pressure measurements the pressure transducer (Abbott)
was positioned at heart level and calibrated (OHMEDA,
XCaliber) to 250 mmHg. Blood flow in the control
limbs was measured via strain-gauge venous occlusion
plethysmography and arterial pressure was also monitored
for systemic drug effects.
Separate intra-arterial infusions of acetylcholine and
substance P were used to assess endothelium-dependent
vasodilatation. Acetylcholine was infused at 1, 4 and
16 g (100 ml limb tissue)1 min1 and substance P
at 8, 31 and 125 pg (100 ml limb tissue)1 min1 .

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S. C. Newcomer and others

Table 1. Subject characteristics

Variable

Mean S.E.M.

Age (years)
Height (cm)
Weight (kg)
BMI (kg m2 )
Systolic BP (mmHg)
Diastolic BP (mmHg)
Total cholesterol (mg dl1 )
V O2 peak (ml kg1 min1 )
Forearm volume (ml)
Leg volume (ml)

24 1
181 2
82 2
25 1
113 4
74 4
146 8
34 2
1232 25
8903 288

Sodium nitroprusside was infused intra-arterially to

assess endothelium-independent vasodilatation. Sodium
nitroprusside was infused at rates of 0.063, 0.25 and 1 g
(100 ml limb tissue)1 min1 . These drug infusion rates
were chosen based on pilot studies that revealed marked
increases in limb blood flow without significant systemic
effects.
Each dose of endothelium-dependent and -independent
vasodilators was infused for 5 min. A 2 min washout period
was allowed between drug infusions. The sequence of
drugs was randomized to avoid any ordering effect.
Drug infusion rates were manipulated by adjusting the
speed of infusion of a Harvard Apparatus syringe pump
to the predetermined infusion rates. A vehicle (saline) was
infused at the highest infusion rate of the doseresponse
curve to determine what effect infusion rate had on blood
flow measurements.
Acetylcholine (Novartis Ophthalamics, Duluth,
GA, USA) substance P (Clinalfla AG, Laufelfingen,
Switzerland) and sodium nitroprusside (Abbott
Laboratories, Chicago, IL, USA) were diluted in saline
to the desired concentration before drug infusion. Both

J Physiol 556.3

substance P and sodium nitroprusside were protected

from light by wrapping aluminium and wire insulation
around the syringe and connective tubing.
Statistical analysis

Repeated-measures two-way ANOVA models were applied

to compare limb differences in response to intra-arterial
infusions of acetycholine, substance P and sodium
nitroprusside. For multiple comparisons of simple effects
at the different drug infusion rates a Bonferroni correction
was made. Repeated-measures one-way ANOVA and
Dunnetts test were applied to compare variables to baseline. Statistical significance was set at P < 0.05. All data are
presented as means s.e.m.
Results
Subject characteristics

The subject characteristics are (age, height, weight, BMI,

blood pressure, cholestrol, VO2 peak , limb volume) reported
in Table 1.
Blood flow at rest

During study 1, baseline blood flow was significantly

greater in the leg than in the arm (393 78 versus
47 11 ml min1 , P < 0.05). Baseline blood flows during
study 2 are reported in Tables 2 and 3.
Peak reactive hyperaemia

Peak blood flow in response to 10 min of arterial occlusion

was significantly greater in the leg than in the forearm
(2575 192 versus 546 49 ml min1 , P < 0.05). In
contrast, 10 min of arterial occlusion caused a significantly
greater relative increase in blood flow above baseline
in the forearm compared to the leg (1345 241 versus
720 169%, P < 0.05; Fig. 1)
Acetylcholine infusions

Figure 1. Peak reactive hyperaemia blood flow in forearm (
)

and leg ()
Values are means S.E.M. and are expressed as a percentage above
baseline. Significant difference between forearm and leg (P < 0.05).

Responses to acetylcholine infusions in the forearm and leg

are reported in Tables 2 and 3, respectively. During forearm
infusions brachial artery diameter was significantly greater
(P < 0.05) than baseline at the infusion rate of 16 g
(100 ml tissue)1 min1 . Forearm blood flow and vascular
conductance were significantly (P < 0.05) higher than
baseline during infusions in the forearm at 4 and
16 g (100 ml tissue)1 min1 . Mean arterial pressure was
significantly (P < 0.05) elevated above baseline during
infusions in the forearm of saline and at 16 g (100 ml
tissue)1 min1 acetylcholine. Blood flow to the control

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J Physiol 556.3

Limb differences in vasodilator responses

forearm was significantly decreased below baseline at

acetylcholine infusion rates of 4 and 16 g (100 ml
tissue)1 min1 . Heart rate was not significantly changed
during infusions of acetylcholine in the forearm.
Leg blood flow, vascular conductance and heart rate
were significantly (P < 0.05) increased above baseline
during all three infusion rates of acetylcholine in the leg.
However, femoral artery diameter, mean arterial pressure
and control leg blood flow were not significantly (P < 0.05)
altered during infusions of acetylcholine in the leg.
Acetylcholine at 16 g (100 ml tissue)1 min1 caused
a significantly greater relative increase in blood flow to the
forearm than to the leg (1864 476 versus 569 86%,
P < 0.05; Fig. 2). Forearm vascular conductance was
increased greater than in the leg at 16 g (100 ml
tissue)1 min1 (1760 421 versus 571 81%, P < 0.05;
Fig. 2).
Substance P infusions

1005

At infusion rates of 0.25 and 1 g (100 ml tissue)1 min1 ,

sodium nitroprusside (P < 0.05) increased forearm blood
flow and vascular conductance above baseline. Brachial
artery diameter was significantly (P < 0.05) increased
above baseline at the highest infusion rate of sodium
nitroprusside. However, infusions of sodium nitroprusside
in the forearm caused no significant (P < 0.05) change
in mean arterial pressure, heart rate or blood flow in the
control forearm.
Leg blood flow was significantly (P < 0.05) increased
above baseline during all three infusion rates of sodium
nitroprusside. However, leg vascular conductance was
significantly (P < 0.05) increased above baseline at
0.25 and 1 g (100 ml tissue)1 min1 . Heart rate was
significantly (P < 0.05) increased above baseline at 1 g
(100 ml tissue)1 min1 . In addition, blood flow to the
control leg and mean arterial pressure were significantly
(P < 0.05) decreased below baseline at 0.25 and 1 g
(100 ml tissue)1 min1 , respectively. Infusions of sodium

Responses to substance P infusions in the forearm and

leg are reported in Tables 2 and 3, respectively. At infusion
rates of 31 and 125 pg (100 ml tissue)1 min1 , substance P
significantly (P < 0.05) increased forearm blood flow and
vascular conductance above baseline. Heart rate and blood
flow to the control forearm were significantly (P < 0.05)
increased above baseline at the highest infusion rates of
substance P. Infusions of substance P in the forearm caused
no significant (P < 0.05) change in either brachial artery
diameter or mean arterial pressure.
Leg blood flow and vascular conductance were
significantly (P < 0.05) increased above baseline during
all three infusion rates of substance P. Heart rate and
blood flow to the control leg were significantly (P < 0.05)
increased above baseline at the highest infusion rate of substance P. However, mean arterial pressure was significantly
(P < 0.05) decreased below baseline at the highest infusion
rate of substance P. Infusions of substance P in the leg
caused no significant (P < 0.05) change in femoral artery
diameter.
Substance P at 125 pg (100 ml tissue)1 min1 caused
a significantly greater relative increase in blood flow to
the forearm than to the leg (911 286 versus 243 58%,
P < 0.05; Fig. 3). Forearm vascular conductance was
increased greater than in the leg at 125 pg (100 ml
tissue)1 min1 (890 289 versus 291 66%, P < 0.05;
Fig. 3).
Sodium nitroprusside infusions

Responses to sodium nitroprusside infusions in the

forearm and leg are reported in Tables 2 and 3, respectively.

C The Physiological Society 2004

Figure 2. Blood flow and conductance responses to

acetycholine in forearm () and leg ( )
Values are means S.E.M. and are expressed as a percentage above
baseline. Significant difference between forearm and leg (P < 0.05).

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J Physiol 556.3

Table 2. Haemodynamic responses in forearm to vasodilators

Drug

Dose

Acetylcholine

Base
Veh
1
4
16
Base
Veh
8
31
125
Base
Veh
0.063
0.25
1

Substance P

Nitroprusside

BAD
(cm)
0.45 0.01
0.45 0.01
0.44 0.01
0.46 0.01
0.48 0.01
0.45 0.02
0.44 0.02
0.44 0.02
0.46 0.01
0.46 0.01
0.44 0.01
0.44 0.01
0.45 0.02
0.45 0.01
0.48 0.01

FBF (infusion)
MAP
(mmHg)
(ml min1 )
25 4
30 4
78 16
221 42
394 47
30 7
31 5
75 17
172 29
225 29
29 5
30 5
48 5
132 9
243 15

84 4
87 4
84 4
85 5
88 5
81 3
84 4
81 3
80 4
84 3
87 4
88 4
85 4
86 4
85 4

FVC
(ml min1 mmHg1 )
0.30 0.05
0.36 0.06
0.90 0.16
2.54 0.42
4.48 0.52
0.36 0.07
0.37 0.05
0.95 0.22
2.16 0.33
2.76 0.45
0.33 0.06
0.35 0.06
0.57 0.08
1.54 0.08
2.89 0.20

HR
(b.p.m.)

FBF (control)
(ml (100 ml)1 min1 )

61 3
61 2
60 3
61 3
61 3
57 3
58 3
58 3
59 3
62 3
60 3
60 3
60 3
61 3
64 3

1.7 0.2
1.7 0.2
1.5 0.2
1.3 0.1
1.3 0.1
1.0 0.1
1.0 0.2
1.1 0.1
1.2 0.1
1.5 0.1
1.3 0.1
1.2 0.1
1.1 0.1
1.1 0.1
1.4 0.2

BAD, brachial artery diameter; FBF (infusion), forearm blood flow in infused limb; MAP, mean arterial pressure; FVC,
forearm vascular conductance; HR, heart rate; FBF (control), forearm blood flow in control limb. Values are means S.E.M.
Significantly different from baseline (P < 0.05).

Table 3. Haemodynamic responses in leg to vasodilators

Drug

Dose

Acetylcholine

Base
Veh
1
4
16
Base
Veh
8
31
125
Base
Veh
0.063
0.25
1

Substance P

Nitroprusside

FAD
(cm)
0.96 0.03
0.96 0.04
0.98 0.04
0.99 0.04
0.99 0.04
0.96 0.04
0.95 0.04
0.95 0.04
0.96 0.04
0.94 0.03
0.96 0.04
0.97 0.04
0.98 0.04
0.98 0.04
1.00 0.03

LBF (infusion)
MAP
(ml min1 )
(mmHg)

LVC
(ml min1 mmHg1 )

HR
(b.p.m.)

LBF (control)
(ml (100 ml)1 min1 )

370 32
409 41
940 114
1836 239
2355 249
409 62
399 74
761 84
1052 112
1230 51
330 30
318 44
641 88
1025 116
1301 140

4.47 0.54
4.89 0.75
10.75 1.29
20.88 2.49
27.94 2.59
4.86 0.65
4.84 0.94
9.28 1.13
13.43 1.85
17.09 1.59
3.79 0.32
3.41 0.46
7.24 1.09
12.34 2.00
16.61 2.06

56 3
57 3
62 3
63 3
69 4
56 3
57 3
57 3
61 3
74 4
58 3
58 3
58 3
61 3
74 3

1.3 0.1
1.4 0.1
1.4 0.2
1.3 0.1
1.4 0.1
1.5 0.2
1.7 0.2
1.3 0.2
1.4 0.2
1.9 0.4
1.3 0.1
1.4 0.2
1.2 0.2
1.1 0.2
1.1 0.1

88 5
88 4
86 5
87 5
83 7
84 4
84 4
83 4
80 4
74 4
88 4
88 4
88 4
84 5
76 5

FAD, femoral artery diameter; LBF (infusion), leg blood flow in infused limb; MAP, mean arterial pressure; LVC, leg vascular
conductance; HR, heart rate; LBF (control), leg blood flow in control limb. Values are meas S.E.M. Significantly different
from baseline (P < 0.05).

nitroprusside in the leg caused no significant (P < 0.05)

change in femoral artery diameter.
Sodium nitroprusside at 1 g (100 ml tissue)1 min1
caused a significantly greater relative increase in blood flow
to the forearm than to the leg (925 164 versus 326 65%,
P < 0.05; Fig. 4). Forearm vascular conductance was
increased greater than in the leg at 1 g (100 ml
tissue)1 min1 (948 173 versus 363 79%, P < 0.05;
Fig. 4).

Discussion
Previous studies have characterized endotheliumdependent and -independent vasodilatation in either the
forearm or the leg. Unique to this investigation is the fact
that we were able to characterize endothelium-dependent
and -independent vasodilatation in both the forearm and
the leg of our subjects on the same day. Our findings
demonstrate that the leg exhibits smaller relative increases
in blood flow and vascular conductance than the forearm

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J Physiol 556.3

Limb differences in vasodilator responses

to both pharmacological and physiological vasodilator

stimuli. Specifically, the leg exhibits smaller relative
increases in blood flow and vascular conductance than the
forearm to intra-arterial infusions of the endotheliumdependent vasodilators acetylcholine and substance P,
and the endothelium-independent vasodilator sodium
nitroprusside. In addition, the vasculature of the leg
exhibits smaller relative increases in blood flow and
vascular conductance than the forearm in response to
10 min of ischaemia. To our knowledge, this is the first
study to characterize limb-specific vascular responses to
endothelium-dependent or -independent vasodilators in
humans.

1007

The human forearm has become the preferred model for

investigation of vascular function because intra-arterial
infusions of vasoactive compounds can be limited to

the forearm and systemic effects can be avoided. This

model is often used to assess endothelial and vascular
smooth muscle function in the forearm of healthy subjects and patients with disease. It is important to note
that our brachial artery blood flow responses to intraarterial infusions of acetylcholine, substance P and sodium
nitroprusside are similar to those previously reported in
the forearm of healthy subjects (Taddei et al. 1997a,b;
DeSouza et al. 2000, 2002). However, direct comparisons of
these data are limited owing to methodological variations
in blood flow measurement techniques and drug doses
between studies.
While the forearm is a well-established experimental
model for studying human vascular function, it may
be of limited consequence to systemic cardiovascular
control because of its small muscle mass and blood
flow requirements. However, the leg requires a greater
percentage of cardiac output than the forearm owing
to the fact that it is approximately 10 times the size

Figure 3. Blood flow and conductance responses to substance P

in forearm () and leg ( )
Values are means S.E.M. and are expressed as a percentage above
baseline. Significant difference between forearm and leg (P < 0.05).

Figure 4. Blood flow and conductance responses to sodium

nitroprusside in forearm () and leg ( )
Values are means S.E.M. and are expressed as a percentage above
baseline. Significant difference between forearm and leg (P < 0.05).

Endothelium-dependent and independent

vasodilatation in previous studies


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S. C. Newcomer and others

of the forearm. Relatively few studies have used the leg

as a model for investigating vascular function because
of the complexity of femoral artery catheterization and
the likelihood of systemic drug effects. However, the
vasculature of the leg is the preferred model when
examining skeletal muscle blood flow during exercise.
Fortunately, a few studies investigating exercise hyperaemia have examined the effects of infused endotheliumdependent and -independent vasodilators into the femoral
artery during rest. Our absolute femoral artery blood flow
responses to intra-arterial infusions of acetylcholine and
sodium nitroprusside are similar to those reported in
this literature (Radegran & Saltin, 1999; Kingwell et al.
2003). Once again, direct comparisons of these data are
limited owing to methodological variations in blood flow
measurement techniques and drug doses between studies.
The similarities between our blood flow responses
to both endothelium-dependent and -independent vasodilators and those reported elsewhere support our findings
that the leg exhibits smaller relative increases in blood flow
and vascular conductance than the forearm.
Mechanisms

A number of studies in the literature suggest that the

human forearm and leg differ in their blood flow response
to a variety of stimuli (Imadojemu et al. 2001; Pawelczyk
& Levine, 2002). In addition, studies performed in miniature swine have demonstrated heterogeneous vascular
responses to endothelium-dependent vasodilators in the
brachial and femoral arteries (Laughlin et al. 1998). These
findings of heterogeneous vascular responses in both
humans and animals suggest phenotypic differences in
vascular endothelium and smooth muscle throughout the
vascular tree. Moreover, it is widely accepted that both
endothelial and vascular smooth muscle cell function
are not ubiquitous throughout the vasculature (Hill
et al. 2001). These differences in function have been
attributed to variations in a number of factors, such as
receptor expression and activation, structural proteins
and proteins associated with signal transduction processes
(Hill et al. 2001). However, it is unclear whether these
regional differences in endothelial and vascular smooth
muscle cell function are predetermined during embryonic
development or governed by the microenvironment in
which they reside (Aird, 2003).
In support of the latter of these two hypotheses, research
using coarctation of the aorta to induce forelimb hypertension in animals has demonstrated attenuated vascular
responses to endothelium-dependent vasodilators in
vascular rings proximal to the obstruction (Lockette

J Physiol 556.3

et al. 1986; Miller et al. 1987; Lai et al. 1989; Bell &
Bohr, 1991; Bell, 1993). This model of hypertension
has parallels to the upright human condition because it
creates blood pressure differences in the forelimb and
hindlimb of approximately 5565 mmHg (Bell & Bohr,
1991; Bell, 1993). These blood pressure differences are
similar to those reported between the arms and legs of
upright humans (Rowell, 1993; Malhotra et al. 2002).
It is not surprising then that our findings of smaller
relative increases in blood flow and vascular conductance
in the leg in response to endothelium-dependent vasodilators are in agreement with data collected in the aortic
coarctation model. Taken together, these findings suggest
that increased blood pressure in the leg during upright
posture may be one potential mechanism underlying
reductions in endothelial vasodilator function. However,
it is important to acknowledge that other factors, such as
fluid shear stress, may also play a pivotal role in endothelial
cell phenotype (Topper & Gimbrone, 1999).
Our blood flow and vascular conductance responses
to sodium nitroprusside also may suggest phenotypic
differences in vascular smooth muscle function. As
previously mentioned, it is well established that the
vasculature is made up of a mosaic of phenotypically
different vascular smooth muscle cells (Archer, 1996; Hill
et al. 2001). It is thought that the interaction of the
vascular smooth muscle cells with their environment may
contribute to their heterogeneity (Daemen & De Mey,
1995; Hill et al. 2001). In support of this, functional
changes of vascular smooth muscle have been reported
in animals (Lockette et al. 1986; Otsuka et al. 1988; Lai
et al. 1989) and humans (Gardiner et al. 1994) exposed
to elevated blood pressures through aortic coarctation.
Specifically, vessels exposed to elevated blood pressures
have been reported to have a decreased sensitivity (Lockette
et al. 1986; Otsuka et al. 1988; Lai et al. 1989) and maximal
responsiveness (Gardiner et al. 1994) to a variety of
nitrovasodilators. Our responses to sodium nitroprusside
in the leg are in agreement with these reported changes.
These findings indicate that differences in blood pressure during upright posture may potentially bring about
phenotypic changes to the vascular smooth muscle,
which in turn may alter their response to endotheliumderived relaxing factors. However, it is important to note
that phenotypic differences in vascular smooth muscle
function and morphology have been reported within
branches of the same vascular bed, which are exposed to
equal blood pressures (Daemen & De Mey, 1995; Hill et al.
2001). This suggests that blood pressure is just one of many
factors which may contribute to vascular smooth muscle
heterogeneity.

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J Physiol 556.3

Limb differences in vasodilator responses

Part of the differences we observed in forearm and

leg responses to vasodilators may be attributed to a
ceiling effect if resting blood flow represents a higher
percentage of maximal blood flow in the leg than the
arm. Our data suggest that resting blood flow, when
expressed as a percentage of peak reactive hyperaemia, was
approximately 8% greater in leg than forearm. However,
this calculation relies on the assumption that reactive
hyperaemia elicited by 10 min of ischaemia in the arm or
leg produces a maximal blood flow response. Although no
previous studies have addressed this assumption, many
of our subjects blood flow responses to intra-arterial
infusions of acetylcholine exceeded their responses to
10 min of ischaemia. Therefore, although a ceiling effect is
possible, defining the maximal blood flow is problematic.
Limitations

All infusion rates were normalized to limb volume to

ensure equal concentrations in the vasculature of the
forearm and leg. However, an important assumption of the
present study is that the percentages of skin, fat and muscle
tissue in the forearm and leg are relatively equal. Based
upon surface-to-volume ratio calculations the forearm is
comprised of a larger percentage of skin tissue than the
leg. These differences in tissue composition may have
played a potential role in our observed forearm and leg
responses to endothelium-dependent and -independent
vasodilators. However, based upon research describing
relatively equal distribution of lean and fat tissue in the
arms and legs of young men when measured by dualenergy X-ray absorptiometry, we believe this is unlikely
(Nindl et al. 2002). Another important assumption when
comparing the responsiveness of two distinct vascular beds
to intra-arterial infusions of endothelium-dependent and
-independent vasodilators is that both the microvascular
and receptor (muscarinic and neurokinin) density are
similar. Unfortunately, there is no literature that we know
of that compares these variables in the forearm and leg.
We chose to express our data as a percentage increase in
blood flow above baseline based upon literature suggesting
that it is the preferred method of expression when
interventions cause vasodilatation or vasoconstriction
and baseline blood flow values are statistically different
(Thomas et al. 1994; Buckwalter & Clifford, 2001;
Tschakovsky et al. 2002; Rosenmeier et al. 2003). A
potential problem of expressing our data in this manner is
that any errors in our baseline measurements will directly
affect the magnitude of responses observed. However, we
are confident that this did not occur based upon our
ability to consistently reproduce our baseline blood flows

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1009

between interventions (Tables 2 and 3). In addition, our

baseline blood flows are consistent with those previously
reported in the brachial and femoral arteries (Radegran,
1997; Dinenno et al. 1999; Tschakovsky et al. 2002).
When intra-arterial infusions are used to elicit regional
vascular responses of the forearm and leg, the baroreflexmediated increases in muscle sympathetic nerve activity
are minimized (Pawelczyk & Levine, 2002). However, we
still found evidence of small systemic effects, as mean
arterial pressure was reduced at the highest infusion rates of
substance P and sodium nitroprusside in the leg (Table 3).
These data suggest that a baroreflex-mediated increase
in muscle sympathetic nerve activity may have opposed
vasodilatation in the leg during the highest infusion rates
of substance P and sodium nitroprusside. However, we
believe that it is highly unlikely that baroreflex-mediated
increases in muscle sympathetic nerve activity can account
for the marked limb differences we observed in blood flow
and vascular conductance. This is based on the findings
that blood flow in the leg opposite the drug infusion was
not reduced during infusions of acetylcholine and substance P.
Possible clinical significance

Recently, Malhotra et al. (2002) suggested that the higher

susceptibility of the lower extremities to plaque formation
when compared to the upper extremities was a direct
function of blood pressure differences between these
limbs during upright posture (Malhotra et al. 2002).
This hypothesis is based upon the findings that the
extent and severity of atherosclerosis is directly associated
with the degree of hypertension (Glagov et al. 1961).
The effects of hypertension on endothelial cell integrity
and function have long been hypothesized to play a
pivotal role in atherogenesis (Moore, 2002). Specifically,
decreased nitric oxide availability due to attenuated endothelial cell function has been linked to smooth muscle
cell proliferation, which is a crucial component in the
formation of atherosclerosis. Our data are consistent
with the possibility that stimulated nitric oxide release
through intra-arterial infusions of acetylcholine and
substance P is reduced in the vasculature of the leg
compared to the forearm. These findings support the
hypothesis of Malhotra et al. (2002) by suggesting that
differences in limb blood pressure during upright posture may negatively impact the endothelium of the lower
extremities, rendering them less effective at protecting
the vasculature from plaque formation. However, it is
important to note that the carotid arteries of upright
humans are exposed to lower blood pressures than the arms

1010

S. C. Newcomer and others

and yet appear to be particularly susceptible to peripheral

arterial disease (Moore, 2002). Based on these findings it
is clear that other factors, such as turbulent blood flow
and wall shear stress, may also play a significant role in the
genesis of peripheral arterial disease between limbs.

Conclusion

In conclusion, our findings suggest that the leg exhibits

smaller relative increases in blood flow and vascular
conductance than the forearm in response to intra-arterial
infusions of endothelium-dependent and -independent
vasodilators. We hypothesize that these findings could be
a consequence of elevated blood pressure in the leg during
upright posture, which leads to phenotypic alterations of
both the endothelial and vascular smooth muscle cells. Our
results also suggest that future studies investigating peripheral vascular function in humans should take functional
vascular heterogeneity into account.
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Acknowledgements
The authors would like to thank Kristen S. Gray, Michael D.
Herr and Shelly A. Silber for excellent technical support. The
authors would also like to thank Chester A. Ray and Kevin D.
Monahan for their critical review of this manuscript. This study
was supported by RO1 AG 18246 (D. N. Proctor), RO1 HL 68699
(U. A. Leuenberger), T32 AG 00048 (S. C. Newcomer) and M01
RR 10732 (General Clinical Research Center).