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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