Therole of A2A adenosine receptor in
4670
Journal of Physiology (1996), 492.2, pp.495-503
495
The role of the A2A adenosine receptor subtype in functional
hyperaemia in the hindlimb of anaesthetized cats
S. M. Poucher
Cardiovascular and Metabolism Department, Zeneca Pharmaceuticals, Mereside,
A lderley Park, Macclesfield, Cheshire SK1O 4TG, UK
1.
2.
3.
4.
5.
The present study was designed to investigate the contribution of the A2A adenosine
receptor subtype in the functional hyperaemia response during muscle contraction.
In cats anaesthetized with sodium pentobarbitone and breathing spontaneously following
tracheotomy, the left sciatic and femoral nerves were electrically stimulated at 3 Hz for
20 min to induce muscle contraction, and hindlimb blood flow was measured with a flow
probe. The contribution of the A2A adenosine receptor subtype was assessed using
ZM 241385, a potent and selective A2A adenosine receptor antagonist.
In a control group, the muscle isometric tension measured in the extensor digitorum
longus-tibialis anterior muscle group was 6-64 + 0X66 kg (100 g muscle mass)-1 and
hindlimb vascular conductance was 0X22 + 0 03 ml mmHg-' (kg body mass)-' at 20 min of
contraction. Administration of vehicle did not affect these parameters upon a second
contraction period: 6 31 + 0 61 kg (100 g muscle mass)-1 and 0 23 + 0 03 ml mmHg-1 (kg
body mass)-1, respectively. Total hindlimb conductance during contraction was unaffected
(5 5 + 3 7 % decrease).
ZM 241385 (1 0 mg kg-') did not alter the amount of force produced by the muscle at
20 min of contraction. Hindlimb conductance response was reduced by 27 1 + 4 8%
following the A2A selective adenosine receptor antagonist, similar to that observed with the
non-selective antagonist 8-phenyltheophylline.
These results show that adenosine acting at the A2A subtype receptor can contribute up to
30 % of the functional hyperaemia response in the hindlimb of anaesthetized cats.
The vasodilator properties of adenosine have been
recognized since 1929 (Drury & Szent-Gyorgyi, 1929). It
was not until 1964 that adenosine was considered an
important factor in the regulation of blood flow to skeletal
muscle (Imai, Riley & Berne, 1964). Since then, the release
of adenosine into the venous effluent of contracting canine
skeletal muscle has been demonstrated when perfused at
low blood flow (Belloni, Phair & Sparks, 1979), at resting
flow (Bockman, Berne & Rubio, 1975), at 150% of resting
blood flow (Ballard, Cotterrell & Karim, 1987a), and
during free perfusion (Bockman, Steffen, McKenzie,
Yachnis & Haddy, 1982; Karim, Ballard & Cotterrell,
1988). Goonewardene & Karim (1991) demonstrated that
removal of adenosine produced during muscle contraction
using adenosine deaminase reduced the freeflow vascular
resistance response by 11 %. However, this may be an
underestimation of the overall contribution of adenosine,
since the administered adenosine deaminase is restricted to
activity in the vascular compartment. Therefore adenosine
deaminase will not remove adenosine produced by, and
released from, skeletal muscle cells and acting at
abluminally located adenosine receptors. In addition, the
product of adenosine deaminase, inosine, has vasodilator
properties and can also potentiate the vascular actions of
adenosine (Collis, Palmer & Baxter, 1986; Ngai, Ibayashi,
Meno & Winn, 1987). Ballard, Cotterell & Karim (1987b)
estimated that adenosine actually contributed up to 40 % of
the vasodilatation response in dogs. This degree of
contribution of adenosine was also found using the P,
purinergic antagonist, 8-phenyltheophylline (8-PT) in cat
gracilis muscle (Poucher, Nowell & Collis, 1990).
Adenosine can reduce vascular tone by inhibiting
sympathetic tone to resistance vessels through inhibition of
noradrenaline release from nerve terminals (Hedqvist &
Fredholm, 1976; Verhaeghe, Vanhoutte & Shepherd, 1977;
Cotterrell & Karim, 1982) mediated by the Al adenosine
receptor subtype, or alternatively, by action directly at A2
adenosine receptor subtypes located on the blood vessels
(Hargreaves, Stoggall & Collis, 1991; Ueeda, Thompson,
Arroyo & Olsson, 1991; Nally, Keddie, Shaw & Collis,
1991; Martin, 1992b). The A2 receptor has been further
subdivided into two classes (Daly, 1985) based upon the
observation that some A2 receptors have EC50 values for
J Physiol. 492.2
S. M. Poucher
496
adenosine in the high nanomolar range (A2A receptor
subtype) rather than in the micromolar range (A2B receptor
subtype) (Premont et al. 1979; Londos, Wolff & Cooper,
1982). Both A2 adenosine receptor subtypes have been
demonstrated on vascular preparations in functional
studies (Ueeda et al. 1991; Martin, 1992b).
The aim of the current study was to determine the
contribution of the adenosine A2A receptor subtype to the
functional hyperaemia response of the cat hindlimb.
METHODS
General preparation
Cats (2-1-3-6 kg, male, n = 35) were anaesthetized with sodium
pentobarbitone (Sagatal, Rh6ne Merieux, Harlow, UK;
30 mg kg-' i.p.). The animals were secured in a supine position and
following a tracheotomy the animals breathed spontaneously.
Anaesthesia was maintained throughout the whole of the
experiment by the continuous infusion of sodium pentobarbitone
(0 13 + 0 008 mg kg-' min-' i.v.). Arterial blood samples were
withdrawn periodically from the brachial artery throughout each
experiment and the pH, Po2 and Pco2 measured (Corning 280 blood
gas analyser, Medfield, MA, USA). Metabolic acidosis was
corrected by intravenous injection of 1'0 M NaHCO3. Rectal
temperature was recorded and maintained at 3758 + 0 1 ¡ãC by
means of a thermostatically controlled heating blanket (Harvard
Instruments, Edenbridge, Kent, UK).
Systemic arterial blood pressure was recorded through a catheter
in the right common carotid artery and hindlimb perfusion
pressure was recorded at the bifurcation of the abdominal aorta
through a catheter inserted in the left femoral artery. All pressures
were measured using strain gauge manometers (PDCR 75, Druck
Ltd, Barendeecht, Netherlands) attached to DC bridge amplifiers
(MT8P, Lectromed, St Peter, Jersey). Strain gauge manometers
were calibrated with a column of mercury at the start of each
experiment. Pulse rate was derived electronically from the
systemic arterial blood pressure.
The aortic bifurcation was exposed through a mid-line abdominal
incision. Hindlimb blood flow was measured using a wrap-around
electromagnetic flow probe (Carolina Medical Instruments, King,
NC, USA; 5 mm circumference) placed on the left external iliac
artery. Zero blood flow was determined by mechanical occlusion of
a snare placed on the artery distal to the flow probe. The flow
probe was calibrated in situ at the end of the experiment with the
animal's own blood before the animal was killed with an overdose
of anaesthetic.
The left sciatic and femoral nerves were exposed, crushed and
stimulating electrodes placed around them distal to the site of
crush. Contraction of the left hindlimb was mediated by
supramaximal stimulation of the sciatic and femoral nerves at 3 Hz
(10 V, 0-1 ms pulse duration) for 20 min. Combined tension
produced by the tibialis anterior (TA) and extensor digitorum
longus (EDL) muscles was measured using an isometric strain
gauge (Grass FTC 10, Quincy, USA). Blood pressure, pulse rate,
blood flow and muscle tension were recorded on an 8-channel pen
recorder (Graftech Linearcorder Mk 8 WR3500, Nantwich, UK).
Antagonism of adenosine receptors was assessed as the inhibition
of the decrease in diastolic blood pressure following the
administration of the adenosine receptor agonist 2-chloroadenosine
(2-CADO) over the range 0 3-300 jug kg-' iv., in the absence and
presence of adenosine receptor antagonist. All cats were treated
with drug vehicle before the first contraction period. Upon
completion, the cats were given one of the following: vehicle (50%
polyethylene glycol 400 (PEG 400), 50% 0-1 M NaOH, group I);
the non-selective adenosine receptor antagonists theophylline
(10 mg kg-' i.v., in physiological saline solution, group II) and
8-PT (5 mg kg-' iv., in 50% PEG 400, 50% 0-1 M NaOH, group
III); and the A2A selective adenosine receptor antagonist
ZM 214385 (0 3 and 1.0 mg kg- i.v., in 50% PEG 400, 50% 01 M
NaOH, groups IV and V, respectively).
Protocol
Following stabilization of the preparation and, if required,
correction of the animal's acid-base balance, a dose-response
curve to 2-CADO was determined. When systemic haemodynamics had recovered, the sciatic and femoral nerves were
stimulated for 20 min and the functional hyperaemia response
measured. Blood flow was also measured for 20 min during
recovery following cessation of stimulation. The hyperaemia
responses during muscle contraction and recovery were measured
as vascular conductance (calculated as the mean hindlimb blood
flow divided by the mean hindlimb perfusion pressure). The area
under the dose-response curve for conductance responses was
calculated for the contraction period. The compound to be studied
(or vehicle) was then given and the functional hyperaemia
response repeated. To assess the efficacy of the adenosine receptor
antagonists, 2-CADO was administered after recovery from the
final contraction period of the hindlimb, as used previously
(Poucher et al. 1990). The decrease in diastolic blood pressure is
mediated by vascular adenosine receptors inducing vasodilatation;
since in this preparation there are no reductions in heart rate at
the doses of 2-CADO used, reductions in cardiac output are
unlikely to be involved. The sensitivity of the diastolic blood
pressure response to intravenous 2-CADO is greater than that of
hindlimb blood flow. Therefore lower circulating concentrations
of the agonist are required, which should reduce the risk of
adenosine receptor downregulation. Efficacy of the different
adenosine receptor antagonists was determined from the dose of
2-CADO required to produce a 30 mmHg reduction (ED30) in
systemic diastolic blood pressure before and after antagonist.
Drugs
2-CADO, theophylline and 8-phenyltheophylline (8-PT) were
supplied by Sigma; ZM 241385 (4-(2-[7-amino-2-(2-furyl)
[1,2,4]triazolo [2,3-a] [1,3,5]triazin-5-yl amino] ethyl) phenol) was
synthesized by Geraint Jones and Peter Caulkett at Zeneca
Pharmaceuticals.
All results are expressed as the means + S.E.M. Statistical analysis
was undertaken using Student's t test for paired and unpaired
data.
RESULTS
Arterial pH and blood gases
The mean values of arterial pH, PCO2 and Po2 were
7.39 + 0 01, 36-9 + 05 mmHg and 888 + 1P3 mmHg,
respectively.
Functional hyperaemia response
In group I, before administration of drug vehicle mean
systemic blood pressure was 122 + 8 mmHg and did not
J Physiol. 492.2
Adenosine receptors and functional hyperaemia
497
Table 1. Effects of theophylline, 8-PT and ZM 241385 on baseline haemodynamics and potency
against adenosine receptor-mediated responses
MABP
(mmHg)
Group I (Drug vehicle, n = 9)
Pre
Post
Group II (10 mg kg-' theophylline, n = 5)
Pre
Post
Group III (5 mg kg-1 8-PT, n = 4)
Pre
Post
Group IV (0 3 mg kg' ZM 241385, n= 5)
Pre
Post
Group V (PG0 mg kg-' ZM 241385, n= 5)
Pre
Post
Pulse rate
(beats min-1)
Conductance Dose shift
Blood flow
(ml min-' kg-') (ml mmHg-' kg-')
120 + 9
116+9
224 + 11
216+12
8-9 + 1P6
9 0 + 1*4
0'07 + 0-01
0 07 + 0-01
123 + 15
119 + 9
195 + 18
212 + 15*
7-1 + 1-4
7-9 + 1P7
0-06 + 0-001
0 07 + 0-01
17-2
118+11
117 + 12
224+18
212 + 19
5-9 + 241
5-11 + 1.1
0-05 + 0-01
0-04+0-02
56-0
118+11
121 + 9
219+14
212 + 14
10-7 + 2-0
9-7 + 1P2
0 09 + 0.01
0-08 + 0-01
8-5
111 + 9
116 + 8
194 + 11
182 + 10
10-4 + 1P5
8-9 + 0-4
010 + 002
0-08 + 0'01
17-2
1P02 (n = 4)
Drug vehicle used was 50% PEG 400, 50% 041 M NaOH. MABP, mean arterial blood pressure.
Haemodynamic values were obtained pre- and 10 min post-administration of either vehicle or compound.
The efficacy of each compound, determined pre- and 50 min post-vehicle or compound (i.e. after the final
contraction period) is given in the column labelled dose shift. Each value is the mean + S.E.M. * P< 0 05,
Student's t test for paired data.
vary during the period of muscle contraction. Resting
tension in the EDL and TA muscles was set at
1P3 + 0-12 kg (100 g)-f, which increased to a peak value of
11'1 + 0-83 kg (100 g muscle mass)-' following 1 min of
hindlimb muscle isometric contraction. From this point,
the tension produced by the muscle began to decline over
the next 5 min of contraction, but remained steady for the
remainder of the contraction period. Hindlimb blood flow
at rest before contraction was 8 6 + 1 7 ml min-' (kg body
mass)-1. This increased during the contraction and reached
a peak level at 5 min (33 0 + 4-4 ml min-' kg-') and
remained 3-4-fold higher than resting values at 20 min
contraction (29'2 + 3*7 ml min-' kg-'). On cessation of
contraction the hindlimb blood flow declined over the
recovery period to a level not significantly different from
that measured before contraction (8 9 + 1P6 ml min-' kg-').
Administration of drug vehicle produced a transient
depressor effect on mean arterial blood pressure, pulse
rate and hindlimb conductance. However, by the time the
second period of contraction was commenced these had
recovered and there was no difference in any of the
haemodynamic parameters measured (Table 1). The peak
isometric tension produced by the EDL and TA muscles
was 8-3 + 0-68 kg (100 g)-1, which was lower than that
produced during the first contraction period (P < 0-01).
However, from 5 to 20 min onwards there was no difference
in the active tension produced by the muscle (Table 2).
No difference was observed in the functional hyperaemia
response during both contraction periods (Table 2). This
was also demonstrated by the area under the hindlimb
conductance dose-response curves, which was 3 458 + 0 425
and 3-332 + 0'485 ml mmHg-' kg-', respectively, for the
first and second contraction periods (5 5 + 3 7 % decrease,
not significant).
Effect of compounds on baseline parameters
Administration of drug vehicle (PEG 400, 041 M NaOH), or
the adenosine receptor antagonists 8-PT and ZM 241385
had no long-term effects upon baseline haemodynamic
parameters (Table 1). In contrast, theophylline
administration produced a transient reduction in diastolic
blood pressure (from 110 + 14 to 74 + 10 mmHg,
P < 0X01) and pulse rate (from 195 + 18 to 224 + 17 beats
min-', P < 0X001). The effect upon pulse rate persisted such
that it remained elevated during the second contraction
period (222 + 10 beats min-', P < 0 05; Table 2).
Potency of adenosine receptor antagonists
Potency of each of the adenosine receptor antagonists was
assessed by comparing their efficacy at blocking the effects
of 2-CADO on diastolic blood pressure. Administration of
theophylline, 8-PT and ZM 241385 resulted in a rightward
shift of the 2-CADO dose-response curve (Fig. 1 and
Table 1). The compound producing the greatest shift in the
response was 8-PT (5 mg kg-') which resulted in a 56-fold
J Physiol.492.2
S. M. Poucher
498
Table 2. Effects of theophylline, 8-PT and ZM 241385 on the hindlimb response to isometric contraction
MABP
(mmHg)
Tension
Blood flow
Conductance
(kg (100 g)') (ml min-' kg-') (ml mmHg-' kg-')
Pulse rate
(beats min-')
Peak tension
(kg (100 g)-')
237 + 9
226 + 11*
11.1 + 0'8
8-3 + 07**
66+07
6-3 + 0-6
29 2 + 3 7
27-5 + 3-0
0-22 + 0 03
0-23 + 0-03
211 + 19
222 + 10
12'7 + 1P0
10-4 + 0.9*
8 6 + 1P1
8-3 + 1 0
26 6 + 3 8
224 + 2'6
0 23 + 0 03
0-18 + 0.02*
231 + 14
223+16
14 1 + 0 9
106+1.5**
8.8 + 1.0
8-1+1 0
26 6 + 3 8
18.8+2.5*
0 23 + 0'02
0.16+0.01*
231+14
227 + 11
112+112
8-4 + 0.8*
7-2+10
7-1 + 0-9
386+3-4
324 + 2.2*
0-31+0-02
025 + 0.02*
210 + 10
201 + 9*
11 6 + 1 1
8-7 + 08**
74+08
7-0 + 0-7
45 3 + 3 5
32-5 + 2.2*
0 40 + 0 05
0-28 + 0.04*
Group I (Drug vehicle, n = 9)
Pre
124 + 8
Post
118 + 8
=
Group II (10 mg kg-' theophylline, n 5)
Pre
125 + 16
Post
125 + 7
Group III (5 mg kg-' 8-PT, n = 4)
Pre
116 + 11
Post
119+8
Group IV (0 3 mg kg'- ZM 241385, n = 5)
Pre
123+9
Post
128 + 7
Group V (1 0 mg kg-' ZM 241385, n = 5)
Pre
118 + 9
Post
122 + 8
Drug vehicle used was 50 % PEG 400, 50 % 0-1 M NaOH. MABP, mean arterial blood pressure. With the
exception of peak isometric tension, values were taken at 20 min isometric muscle contraction pre- and
post-vehicle or compound. Each value is the mean + S.E.M. * P < 0 05; ** P < 0.01, Student's t test for
paired data.
shift. The smallest shift (8-5-fold)
ZM 241385 (0 3 mg kg-').
was
observed following
Effect of adenosine antagonists on functional
hyperaemia responses
The effects of the non-selective adenosine receptor
antagonists theophylline and 8-PT and the A2A selective
adenosine receptor antagonist ZM 241385 on functional
hyperaemia are summarized in Figs 2 and 3 and Table 2.
Effect of theophylline and 8-PT. In group II, as for
group I, the peak but not the steady-state muscle isometric
tension was reduced during the second contraction period
following theophylline (Table 2). In contrast, the functional
hyperaemia response, was reduced following theophylline
(Table 2 and Fig. 2). Assessment of the total conductance
response to the hindlimb during contraction showed the
area under the curve to be reduced by 27-0 + 6O0%, from
3-68 + 0 45 to 2-62 + 0-29 ml min-' (kg)-' mmHg-' preand post-theophylline, respectively (P < 0.05, n = 5).
In group III (8-PT), similar effects of adenosine receptor
antagonism were found. The peak muscle tension was
reduced during the second contraction period (Table 2). The
70
cm
I
E
E
60
Figure 1. Efficacy of adenosine receptor antagonists in the
anaesthetized cat
a)
(a)
50
U)
a)
C0
0
0
The hypotensive effects of increasing doses of the adenosine receptor
agonist 2-CADO (iv.) were investigated 10 min before (pre) and
50 min after (post) administration of either vehicle or adenosine
receptor antagonist. El, control first dose-response curve, n = 15-23;
*, control second dose-response curve, n = 4; O>, post 0 3 mg kg-'
ZM 241385, n = 5;*, post 0 mg kg-' ZM 241385, n = 5; post
10 mg kg-' theophylline, n = 5; A, post 5 mg kg-' 8-PT, n = 4.
40
.0
co
30
coU)
~0
a)
20
A,
CU
a)
0
a)
a)
10
0
0
0-1
1
10
100 1000 10000
2-CADO (ug kg-1)
J
499
Adenosine receptors and functional hyperaemia
Phy8iol. 492.2
reduction in the functional hyperaemia response, as
observed with theophylline, also occurred following 8-PT
(Table 2 and Fig. 2). Assessment of the total conductance
response to the hindlimb showed the area under the curve
to be reduced by 30 0 + 2-0%, from 3-82 + 031 to
2-67 + 0-16 ml min-' (kg)-' mmHg-' pre- and post-8-PT,
respectively (P < 0-01, n = 4).
Therefore with the non-selective adenosine receptor
antagonists theophylline (17 2-fold shift of 2-CADO
dose-response curve) and 8-PT (56*0-fold shift), the same
degree of reduction in the functional hyperaemia response
of the cat hindlimb was observed.
Effect of ZM 241385. In the low-dose group (group IV),
the EDL and TA muscle isometric tension profile was the
same as for groups I-III (Tables 1 and 2). Hindlimb blood
flow at rest was 10-2 + 2-1 ml min-' kg-'. This increased
during muscle contraction to reach a steady state within
5 min, and was 38-6 + 3-4 ml min-' kg-' at the end of
contraction (a 3-8-fold increase over blood flow at rest). On
cessation of hindlimb contraction the blood flow declined to
a level not significantly different from that measured before
contraction (10f7 + 2f0 ml min-' kg-'). ZM 241385 (0 3 mg
kg-') had no effect on baseline parameters (Table 2).
Following compound administration, peak isometric muscle
tension was reduced (Table 2) and the time to peak tension
was increased from 0 5 to 1i0 min. However, from 3 min
onwards there was no difference in the active tension
produced following vehicle or compound. This difference in
peak response between the first and second hindlimb
contraction periods was the same as observed in the vehicletreated group. Therefore the reduction in peak tension was
unlikely to be due to an effect of the compound. In contrast
to the control group, however, the hyperaemia response
was significantly attenuated following the low dose of
ZM 241385 (Fig. 3). During the contraction period the
hindlimb blood flow response was reduced following
compound at each of the timepoints measured from 1 to
20 min contraction (P < 0 05). Similarly, hindlimb vascular
conductance was reduced, compared with vehicle, from
1 min (0X263 + 0X015 vs. 0-210 + 0-012 ml min-' kg-'
mmHg-1, P < 0 005) measured through to 20 min
(0-314 + 0-018 vs. 0-254 + 0-015 ml min-' kg-' mmHg-',
P < 0 005). Assessment of the area under the hindlimb
vascular conductance dose-response curve during
contraction showed that the total vascular responses were
4-63 + 0 57 and 3-67 + 0 43 ml kg-' mmHg-' before and
after 0 3 mg kg- ZM 241385, respectively (P < 0-01). This
represents a reduction of 20 1 + 1-5% following adenosine
receptor antagonism (dose ratio, 8 5).
ZM 241385 at 1 0 mg kg-' (group V) resulted in effects
similar to those of the lower dose of antagonist (Fig. 3,
Tables 1 and 2) upon EDL and TA muscle tension, hindlimb
blood flow and vascular conductance at rest and during
contraction. Area under the hindlimb conductance
dose-response curves during contraction was 5f87 + 0 75
and 4f27 + 0f64 ml kg- mmHg- pre- and post-compound,
respectively (P < 0 02). This was a 27-1 + 4-8% reduction
of the vascular response following adenosine receptor
antagonism (dose ratio, 17 2).
B
A
0-5 -
0-4
8
-
c
C
0-5
-
0-4
-
0-3
-
0-2
-
_
0
OE
C ,
: _:
oO E
0-3 -
0
E
',-
, 0Ea
-M_-
Cu'_
"
E(0
0-2
-
0-1
-
._U
,C
I
2-'
c
0-1 -
0
0
-5
5
15
25
Time (min)
35
45
-5
5
15
25
35
45
Time (min)
Figure 2. Effects of theophylline and 8-phenyltheophylline on hindlimb vascular conductance
A, effect of theophylline (10 mg kg-', n = 5) on hindlimb vascular conductance during isometric
contraction at 3 Hz for 20 min. o, drug vehicle; *, theophylline. B, effect of 8-PT (5 mg kg', n = 4) on
hindlimb vascular conductance during isometric contraction at 3 Hz for 20 min. o, drug vehicle; *, 8-PT.
Each point is the mean + S.E.M.
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