Angiotensin II receptors negatively coupled to adenylate cyclase in rat aorta

BIOCHEMICAL

Vol. 117, No. 2, 1983 December

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 420-428

16, 1983

ANGIOTENSIN II RECEPTORS NEGATIVELY COUPLED TO ADENYLATE CYCLASE IN RAT AORTA' Madhu B. Anand-Srivastava2

Clinical Research Institute 110 Pine Ave West, Montreal, Received

October

27,

of Montreal, Quebec, Canada

1983

SUMMARY: Angiotensin II (AII) inhibited adenylate cyclase from rat aorta in a concentration dependent manner. The maximal inhibition ( %20%) was observed at IO ,.,M. The inhibitory effect of angiotensin II was dependent on monovalent cations such as Nat or Lit and was blocked by saralasin, an antagonist of angiotensin. Guanine nucleotides such as GTP or GMP-P(NH)P were also required to elicit the inhibition by angiotensin II. In addition, angiotensin II also inhibited the stimulation exerted by catecholamines. These data suggest that angiotensin receptors are present in aorta which are negatively coupled to adenylate cyclase.

Angiotensin of vascular

tropic that

a vasoactive

and nonvascular

effects the

II,

of angiotensin

with the receptor

sites

tissues

II has been recently

Very recently,

of angiotensin

active

drugs

through

the

II on carbohydrate

produce changes

could demonstrate

(4-9).

(IO).

tissues

their

effects

in CAMP (11) that

the

metabolism

effects

there

The existence

smooth

used:

GMP-P(NH)P,

0006-291X/83 $1.50 Copyright 0 I983 by Academic Press, Inc. All rights of reproduction in any form reserved.

420

that the effect

evidence that

in aorta

or heart

Foundation

guanyl-5’-yl(&y-iminoldiphosphate;

sin II.

and liver

angiotensin

in these tissues

This work was supported by grants from the Quebec Heart Fonds de la Recherche en Sante du Quebec. 2 Canadian Heart Foundation Scholar. Abbreviations

cyclase by

cortex,

through

receptors

II vaso-

muscle and heart contraction

was performed

1

of angiotensin

wnich demonstrates

is considerable

of angiotensin

by its interaction

adrenal

was mediated

and chrono-

have demonstrated

of adenylate

in kidney,

on vascular no study

inotropic

is mediated

An inhibition

demonstrated

Although

positive

Various studies

an evidence was presented

in the liver

receptors

II on target

vasoconstriction

shown to produce

muscle and exhibit

on the membranes (3).

has been shown in several

(7-9).

smooth

has been

on mammalian heart muscle (1,2).

effect

angiotensin

peptide

which

are mediated

and the

AII,

angioten-

Vol. 117, No. 2, 1983

through to

adenylate

investigate

receptor

cyclase/cAMP if

binding

MATERIALS

BIOCHEMICAL

angiotensin studies

system. II

are

AND BIOPHYSICAL

The present

receptors

coupled

RESEARCH COMMUNICATIONS

studies

demonstrated

to adenylate

were

in aorta

cyclase

therefore

undertaken

(3,12,13)

by

system.

AND METHODS

Preparation of particulate fraction Aorta were dissected out and quickly frozen in liquid nitrogen. The frozen aorta were pulverized to a fine powder using a percussion mortar cooled in liquid nitrogen. The powdered aorta were stored at -70' Aorta were homogenized using motor-driven teflon-glass homogenizer in until assayed. a buffer containing 10 mM Tris-HCl, 1 mM EDTA and 1 mM DTT, pH 7.5. The homogenate was centrifuged at 16,000 x g for 10 min. The supernatant was discarded and the 1 mM EDTA and 1 mM DTT, pH 7.5 and pellet was finally suspended in 10 mM Tris-HCl, used for the determination of adenylate cyclase activity. Adenylate cyclase activity determination. Aden late cyclase activity was determid by measuring LJL 1 AMP formation from [,-3 3 PI ATP as described previously (14). ?Fpical assay mediumPcoktained 50 mM glycylglycine, pH 7.5 0.5 mM Mg ATP [,32Pi ATP (l-l.5 x 106 CPM), 5 mM MgC12 (in excess of the ATP ¢ration), !OO mM NaCl, 0.5 mM CAMP, 5 U adenosine deaminase per ml, 1 mM DTT, 10 ,.,M GTP, and ATP regenerating system consisting of 2 mM creatine phosphate, 0.1 mg creatine kinase per ml, and Incubations were initiated by 0.1 mg myokinase per ml in a final volume of 200 ,,l. the addition of the particulate fraction (50-100 pg) to the reaction mixture which had been thermally equilibrated for 2 min at 37°C. Reactions were conducted in Reactions were terminated by the addition of 0.6 ml triplicate for 10 min at 37°C. of 120 mM zinc acetate. CAMP was purified by co-precipitation of other nucleotides with ZnC03 by the addition of 0.5 ml of 144 mM Na2C03 and subsequent chromatography by the double column system as described by Salomon et al (15). Under the assay conditions used adenylate cyclase activity was linear with respect to protein concentration and time of incubation. talline

Protein was determined essentially as described bovine serum albumin as standard.

by Lowry

et al

(16),

with

crys-

Materials. Adenosine deaminase (EC 3.5.4.5), GTP, GMP-P(NH)P (guanyl-5'-yl-B-y-imino Creadiphosphate), ATP, cyclic AMP were purchased from Sigma, St.Louis, Missouri. tine kinase (EC 2.7.3.2) and myokinase (EC 2.7.4.3) were purchased from Boehringer Mannheim, Canada. [,32PlATP was purchased from Amersham and angiotensin II and saralasin were from Peninsula Laboratories, Inc., California. RESULTS Effect

of Angiotensin

angiotensin

II

receptors

various

concentrations

resuits

are

shown

on adenylate

coupled

cyclase.

to adenylate

of angiotensin in Fig.

1.

In order

cyclase

II on adenylate

Angiotensin

II

inhibited

to determine

in rat

aorta,

cyclase

the

the

presence

effect

of

was studied

adenylate

cyclase

of

and the activity

in

a concentration dependent manner, the maximal inhibition (2. 20%) was obtained at -5 IO M. The inhibitory effect of AI1 on adenylate cyclase was reversed by about 90% -5 by saralasin (10 M), an antagonist of angiotensin, as shown in Table I. These data suggest pled

the

presence

to adenylate

of angiotensin

receptors

cyclase. 421

in rat

aorta

which

are

negatively

cou-

Vol. 117. No. 2, 1983

BIOCHEMICAL

AND BIOPHYSICAL

I

I

10-g

0

I

I

I

10-e

10-7

10-6

ANGlOTENSlNll Figure

1

Effect

of

activity

I

I

(;;-5

various concentrations of angiotensin II on adenylate cyclase rat aorta. Adenylate cyclase activity wasdetermined in the of 100 mM NaCl and 10 PM GTP as under "Methods". Inset showsthe inhibition of adenylate cyclase activity by various concentrations

in

presence percent of angiotensin II. Values are the means + tions from one of three experiments.

Effect

RESEARCH COMMUNICATIONS

of sodium chloride on angiotensin II

SEMof triplicate

induced inhibition

determina-

of adenylate cyclase.

The requirement of sodium has been demonstrated for the inhibitory

response of adeny

late cyclase to a-adrenergic

(7),and adenosine

agents in renal cortex

in myocardial sarcolemma (18).

It was of interest

adenylate cyclase by angiotensin

Table

I

Effect

of

(17) and liver

to determine if the inhibition

II was also dependent on the presence of sodium.

saralasin

[Sar

18 Ala I-angiotensin II

on

angiotensin induced inhibition of adenylate cyclase in rat aorta Adenylate cyclase activity pmol CAMP(mgprotein. 10 min)-1

Additions None

117

Angiotensin II (AII)1(10-5M) AI1

96 +2

+ saralasin (IO M)

Adenylate NaCl and the mean ments.

cyclase activity

f 1

114

?2

wasdetermined in the presence of 100 mM

10 PM GTP as given under "Methods". The values f SEM of triplicate determinations from one of

422

represent two experi-

of

BIOCHEMICAL

Vol. 117. No. 2, 1983

0

AND BIOPHYSICAL

50

100

150 NoCl

Figure

2

2 shows the

vity

in the

effect

presence

as is

was also

observed

(data

not

tiuanine

shown

100 mM NaCl and then

effect

adenylate

cyclase

in the

in the

inset.

of LiCl,

activity. in the

manner

for

binding

effect

however

hormone

protein.

inhibition

However

it

was less

acti-

cyclase the percentage

at higher

of AII

of adenylate

concentrations A small

absence

on adenylate potent

inhibition

of guanine

receptors A small

are inhibition

These coupled

cyclase

of GTP or GMP-P(NH)P

concencyclase

than

NaCl

by AII.

Fig.

data

Maximum suggest

of adenylate

inhibition

cyclase

by AI1

in a concentra-

(%20%) was observed

angiotensin

cyclase

3

responsive

cyclase

was increased

that

to adenylate

423

on AI1

(% 5%) of adenylate nucleotides

by GTP or GMP-P(NH)P.

10 IJM of GTP or GMP-P(NH)P. other

The inhibitory

presence

of various

observed

dependent

like

declined.

was maximum at 100 mM NaCl and decreased

requirement

shows the

at

up to

cyclase

shown).

nucleotide

(10T5M)

250

concentrations of NaCl on adenylate -5 of 10 M angiotensin. Basal adenylate

and absence

by angiotensin

trations

tion

of various

was increased

inhibition

200 (mM)

Effect of various concentrations of NaCl on adenylate cyclase activity in rat aorta. Adenylate cyclase activity was determined in the absence (o-m) or presence (A-A) of 10 PM angiotensin II as under "Methods". IO PM GTP was present in the assay mixture. Inset shows the dependence on NaCl of inhibition of adenylate cyclase by angiotensin II. Values are the mean SEM of triplicate determinations from one of two experiments.

Fig.

activity

RESEARCH COMMUNICATIONS

II

by guanine

by angiotensin

receptors nucleotide in the

Vol. 117, No. 2, 1983

BIOCHEMICAL

AND BIOPHYSICAL

0

4

6

GUANINE

Figure

absence

3

of washed

Effect

of

In

nucleotides

some to

agonists

might

on the

angiotensin

receptors

in

proterenol,

epinephrine

shown

20 @MJ

be

due

to

the

presence

of

endogenous

GTP

in

adenylate

II.

rat

of

aorta,

on

Interaction

of

Adenylate pm01 CAMP

other of

adenylate were

angiotensin

cyclase (mg protein.

interaction

hormone

effect

experiments

their

and

some

the

dopamine

These

II

cyclase

presence

and

Table

Table

in addition

receptors

various

catecholamines

cyclase

was

carried

II

with angiotensin

(AII)

out

with

activity 10 min)-1

in

some

such

studied the

and presence

agonists

Percentage inhibition by angiotensin II

Additions

None AI1

in

particles.

determine

are

order

16

Dependence on guanine nucleotides of inhibition of adenylate cyclase by angiotensin II in rat aorta. Adenylate cyclase activity was determined in the presence of 10 PM angiotensin II, 100 mM NaCl and increasing concentration of GTP (A-A) or GMP-P(NH)P (o-o). Values are the means of triplicate determinations from one of three experiments.

guanine

aorta

12

NUCLEOTIDES

RESEARCH COMMUNICATIONS

(10

PM)

Isoproterenol

(50

Isoproterenol

t AI1

Epinephrine

(50

Epinephrine

t AI1

Dopamine

(100

Dopamine

+ AI1

NM) (10

PM)

LIM) (IO

PM)

PM) (10

uM)

Adenylate cyclase activity IO PM GTP as given under triplicate determinations

96

f 7.1

79

f 3.2

229

* 9.1

206

* 5.5

207

kl1.6

187

klO.3

152

klO.5

134

k14.6

was determined in the presence "Methods". The values represent from one of two experiments. 424

18

to.1

11.2

11.9

of 100 mM NaCl and the mean f. SEM of

the of

II

to as

iso-

results 100

mM

Vol. 117, No. 2, 1983

Table

III

BIOCHEMICAL

Effect

of

some

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

antagonists on adenylate cyclase activity

by epinephrine,

norepinephrine

and

Adenylate cyclase CAMP (mg protein.

pmol

rat

aorta

activity 10 min)-1 Dopam i ne 100 IIM

Additions

Basal

Epinephrine 50 PM

None

7622

263+1B

224*9

126+1

76+1

93% 5

8624

62k4

75+3

2122 8

179t5

87k4

65r2

186+ 9

159k2

7024

Propranolol

(10

Phentolamine

NaCl.

(10

aM) uM)

Norepinephrine 50 PM

cyclase

activity was determined as given under "Methods". NaCl was assay mixture. The values represent the mean + SEM of triplideterminations from one of two experiments.

Adenylate omitted

cate

PM)

(10

Flupentixol

ted

in

dopamine

stimulated

from

the

Isopreterenol

adenylate

(50 PM), epinephrine

cyclase activity

(50 PM) and dopamine

by about

(100

PM) all

115 and 40% respectively.

140,

stimula-

This stimula-

-5 tion (1)

in the presence

was decreased the

presence

angiotensin vities

of adrenergic

and dopaminergic

was also able to inhibit

in vascular

smooth

M angiotensin

of IO

muscle

II.

These

suggest

in rat aorta

receptors

the hormone-stimulated

and (2)

that

cyclase acti-

adenylate

in other

as has been reported

data

tissues

nonvascular

(7-9). To investigate some antagonists and the results

the nature of catecholamine on catecholamine-stimulated

are

shown

in Table

Propranolol

epinephrine-stimulated

enzyme activities

(~20-25%)

by phenotolamine.

nylate tion

was observed

cyclase activity by epinephrine

40% in Table II) which increases

was inhibited

adenylate

III.

in aorta,

receptors

cyclase activity inhibited

effect

of

was studied

epinephrine

by about 90-95% whereas a small

and norinhibition

On the other hand dopamine-stimulated by all

three antagonists.

and dopamine (%250% and 66% respectively was due to the fact that

basal activity

the

NaCl was omitted

and thereby

The higher

adestimula-

as compared to 150% and from the assay mixture

decreases the percent

stimulation.

DISCUSSION The data

adenylate

presented

cyclase

demonstrates

in rat aorta.

the

presence

The inhibition 425

of angiotensin

of adenylate

receptors

coupled to

cyclase by angioten-

Vol. 117, No. 2, 1983 sin

II

BIOCHEMICAL

AND BIOPHYSICAL receptors

suggests

that

the

angiotensin

nylate

cyclase

in

rat

aorta.

reports

obtained

with

where

angiotensin

tion

also

by angiotensin

a well

known

angiotensin Lit,

liver

II

Nat was more potent

it

presence Lit

of monovalent

cortex

(8)

in other

of adenylate

adenylate inhibition

systems

(7-9).

However

angiotensin-induced Similar the

presence

stable

GTP analog,

by angiotensin presence

the

rent

than

cyclase

cyclase mechanism that

cyclase

by angiotensin.

by which

reports

in other

were

also

able

Although

of other to the

been demonstrated, hormones.

studies

in the

previously

(20). which

cyclase

of adenylate

in other

to have

we have demonstrated

the

presence

we further

show that

the

muscle

little

enzyme prepared 426

the

data

the

the

inhibicyclase

inhibition

suggests

that

in

may be diffe-

by the

increased

are

in agreement

stable

GTP analogs

(21,22). of smooth

muscles

or no sensitivity

of adenosine-sensitive from

a

be due to the

cyclase

systems

cells

on GTP, but

might

our

in a variety

on the

of adenylate

be mediated

of

angiotensin-

The observed

demonstrate

cyclase

smooth

inhibition

However

found

vascular

cyclase

inhibition

GTP in exhibiting

adenylate

enzyme has been

in cultured

acti-

that

of GMP-P(NH)P

and may not

investigators

the

dependent

particles.

inhibits

is

(7).

nucleotides

presence II

systems

adenylate

presence the

Recently

cyclase

inhibit

washed

angiotensin

of GTP as suggested

the

of guanine

GTP in aorta

cyclase

by hormones,

of

The small

absence

which

by t such as Na or

we have demonstrated

as that

inhibi-

been reported t t than Na or K in

was not only

was as potent

by angiotensin

observed

with

present

adenylate

in the

the

has been shown to be dependent

studies,

(8),

have

cyclase

cyclase

present

GMP-P(NH)P

of some endogenous

hydrolysis

late

In the

observed

of adenylate aorta

of adenylate

of aorta

of adenylate

adenylate

and neurotransmitters

of GTP (19). inhibition

tion

stimulation

by hormones

induced

of liver

cortex

of adenylate

was shown to be more potent

inhibition to the

cyclase

Li

previous

cyclase

cations

t previously

the

by saralasin,

of monovalent

in the

with

Furthermore

was antagonized

cations

to ade-

and adrenal

activity.

in inhibiting

agonists

coupled

in agreement

The inhibition

than

and a-adrenergic

are

cyclase

since

on the

negatively

kidney

adenylate

of angiotensin.

II was dependent

by angiotensin

results (7),

was specific,

The requirement

vity.

membranes

inhibited

antagonist

however

These

are

RESEARCH COMMUNICATIONS

rat by this

aorta

(23)

method

has to adeny-

and in the is greatly

BIOCHEMICAL

Vol. 117, No. 2, 1983

responsive the

to epinephrine,

presence

norepinephrine,

of adrenergic

The inhibition

tive

also,

adenylate

and dopamine

and glucagon-stimulated

that

by several

angiotensin

II

CAMP levels investigators

inhibited

The physiological

cyclase activities.

and suggests

in aorta.

receptors

II has been reported

we demonstrate

RESEARCH COMMUNICATIONS

isoproterenol

and dopaminergic

of epinephrine

cyclase by angiotensin our studies

AND BIOPHYSICAL

and adenylate (8,24,25).

In

catecholamine-sensi-

significance

of this

effect

remains to be established. It is concluded aorta

that

from these studies

which are negatively vasoconstriction

cyclase/cAMP

that angiotensin

coupled to adenylate

elicited

by angiotensin

receptors

are present

in rat

cyclase and it can be postulated may be mediated

through

adenylate

system.

ACKNOWLEDGEMENTS I wish to thank Dr. Marc Cantin for constant encouragement and Marie-France Nolin, Georges Lepage and Pierre Dupuis for technical assistance. The secretarial help of Vivianne Jodoin is greatly appreciated. REFERENCES 1. ::

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