BIOCHEMICAL

Vol. 179, No. 1, 1991 August

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 85-89

30, 1991

Inhibition

of Bovine

Brain

Phosphodiesterase Angiotensin R. K. Sharma, Department

of Medical

July

Ligands and G. J. Moore

Biochemistry, Alberta,

cGMP

and Non-Peptide

Receptor .I. R. Smith

Calgary, Received

Calmodulin-Dependent

By Peptide

University Canada

of Calgary,

T2N 4Nl

9, 1991

Inhibition of a purified 60 KDa bovine brain calmodulin-dependent cGMP phosphodiesterase (PDE) was investigated for' a number of peptides and nonThe peptide peptides which are known to bind to angiotensin (ANG) receptors. antagonists sarilesin and sarmesin had K, = 120 and > 200 PM respectively, and the peptide agonists ANG II and ANG III had K, - > 200 and 45 PM respectively. Non-peptide ANG receptor antagonists related to DuP 753 exhibited K, values in For both peptide and non-peptide antagonists, inhibitory the same range. activities in the PDE assay reflected the order of antagonist potencies at ANG receptors in the rat isolated uterus assay and binding affinities at ANG receptors in rat uterine membranes, suggesting that molecular recognition factors are similar for both ANG receptors and cGMP PDE. The vasodilatory and blood pressure lowering effects of compounds related to DuP 753 may be due in part to inhibition of cGMP PDE. The differential effects of ANG II and ANG III at target tissues may relate in part to the marked differences in cGMP PDE inhibition associated with these two peptides hormones. 0 1991 AcademicPress, Inc.

Sarilesin analogues tissues

([SariIle']ANG

of ANG II (1).

Recently

imidazoles

(Fig.

to have blood class

have

We thought peptide effects intracellular

Materials

l),

of

of non-peptidic

shown

levels

found

to act

activity to

ligands these

in that

ligands

(2);

certain

from

in

are

to

and of this

artery

peptide

assay,

of

substituted

renal

of both

part

are

compounds

bovine

and blood

be due

II) a variety

at ANG receptors

cGMP PDE inhibition vasodilatory

in

which

as antagonists

the activities

might

of cGMP resulting

compounds,

cGMP PDE from

our the

at ANG receptors

in rats

inhibit

to investigate

possibility

some of

been

([Sar1Tyr(Me)4]ANG

Sarmesin

a group

of interest the

and

as antagonists

lowering

been

ANG receptor

II) act

have

pressure also

it

investigate

which

in

(3).

and nonorder

to

pressure

lowering

an increase

in

the

PDE inhibition.

and Methods

Peptides use in this study were either purchased from Peninsula or were synthesized in this laboratory by methods described previously (4). Non-peptide ligands were synthesized essentially according to published procedures (2) and

85

0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, 1~. All rights of reproduction in arty form reserved.

Vol.

179, No. 1, 1991

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIGURE

1.

integrity and purity were established by NMR spectroscopy and HPLC (4). Rat isolated uterus bioassays were conducted as described previously (4). Displacement of lz51-ANG II from rat uterine membranes was carried out according to described methods (5). Bovine brain calmodulin-dependent 60 KDa cyclic nucleotide phosphodiesterase isozyme was prepared as described by Sharma et al. this enzyme has lo-fold higher affinity for cGMP than for CAMP. Assay of (6); phosphodiesterase activity was measured as previously described (7).

Results Figure for

2 shows the concentration

a number

Figure rat

of

2 are

isolated the

uterus

assays

membrane.

compartments, that

receptors inhibition. showed

in

may only uterus

Similarly, the

represent

same order

have

on the

binding

in all

hand

86

assays.

require

of

molecular diffuse

intracellular

The data

in Table

potency

at

as is

observed

The agonist

the

a receptor-

to

investigated

in

In Table

their can

from

ligands

assays. on

access

assays

antagonists three

K, derived

which

gain

same order

two peptide

of potency

binding

other

to

of PDE activity for

of these

dependent

to agonists.

the and

activities

a class

order

be applicable

bioassays the

in

the

groups

ligands

inhibition Values

membrane

ligands

antagonists rat

uterine into

mechanism

which

non-peptide

with

divided

Peptide

internalization

for

ligands.

1, together and in rat

been

Non-peptide

cytoplasmic based

in Table

have

dependence

and non-peptide

shown

ligands

1, properties.

peptide

1 show

angiotensin for

in this peptides

PDE study ANG

Vol.

179,

No.

1, 1991

BIOCHEMICAL

I

AND

BIOPHYSICAL

I

I

I

I

I

,

I

I

I

I

40

60

60

100

120

140

180

160

200

$g/ml)

Inhibition of phosphodiestease activity by peptide and non-peptide ligands. 0 Compound A, OCompound B, OCompound C, ASarilesin, o Sarmesin, A ANG II, HANG III

FIGURE 2.

and ANG III,

to the

COMMUNICATIONS

20

INHIBITOR

II

RESEARCH

on the other

two uterus

hand,

had opposite

effects

in the PDE assay

compared

assays.

Discussion The present

findings

effects

vitro

their inhibition it

be noted

receptors

than

are

by the cell if

presumably influence

This

Thus

may present

an artificially

be

thereafter

able

to

high

directly events

taken

if

without

complex

membrane

the

affinity

need

for

for

of several

ANG

orders

particularly which

concentration

cell

However,

receptor-transduction

account,

to PDE.

cGMP PDE

receptors.

in affinity into

coupled the

its

may elicit

involves

much higher

a ligand-receptor

traverse

intracellular

have

significant are

antagonists which

from

difference

less

internalized.

ANG receptor a mechanism

of ANG II

considerations

is

by

ligands

1).

become

may

the complex

that

vivo

ANG receptor

PDE (Table

compartmentalization ligands

in

to displacement that

for

magnitude

illustrate and

in addition

should

of

&

is

if

internalized

of sequestered Non-peptide by

ligand

ligands

passive

the

will

diffusion

a receptor

and

internalization

mechanism. The order

of potency

for

PDE and ANG receptor

assays.

assay

emphasizes

importance

(Fig.

1) on the

peptide

antagonists

the

imidazole

ring

sarilesin

the non-peptide The absence

antagonists

of activity

of the hydroxymethyl for and

PDE inhibition. sarmesin 87

is

compound

and/or

chloro

The order also

the

for

suggests

same in substituents

of potency common

cGMP

C in the PDE of the

recognition

Vol.

179, No. 1, 1991

Table

1.

BIOCHEMICAL

Activities uterine

rat

Ligandr

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

of peptide andnon-peptide ligands in membrane and brain cGMP phosphodiesterase

Bioassay'

Binding

Assay3

rat

isolateduterus, assays

cGMP phosphodiesterase4

(ID,,,N

(PA,)

WI >PM)

A

7.9

20

100

B

6.5

100

150

C

5.6

>200

Sarilesin

8.5

1

120

Sarmesin

7.5

30

>200

(100%)

1

>200

(10%)

6

ANG II

Agonist

ANG III 1

Agonist

See

2

Fig.

1 and

text

for

structural

inactive

45

details

Rat isolated uterus contraction assay; the pA, is the negative logarithm of the dose of antagonist required to reduce the response to an ED50 dose of ANG II to that of an ED50/2 dose of ANG II; in the absence of factors relating to uptake and metabolism, pA, - -log K,.

3

Displacement of concentration =

specifically of ligand

ID 50 4

See

Materials

features

for

peptide

and

Methods

each

group

both

ANG and PDE.

between

suggests

that This

sites

evolutionary

divergence. was

inhibitor

binding

removed

Another

by causing

muscle

why

ANG

III

but

has

only

muscle

PDE after has 10%

17%

of

of

the

ANG III

relaxation binding

potency

on the other affinity

of

ANG

of II

88

in

the

ANG rat

of

imidazole when

that

activator

and

study

was

actions

at ANG receptors, of cellular at uterus

This rat

the

the agonist

contradictory

hand. II

result

affected

present

due to elevation

internalization, the

the

action

the

of one another.

couldhave

due to its

Possibly

not

of PDE observedhereinwas

that

contraction

from

the

included

was

illustrating

at

equate

as the

studies

independently

deriving

may

perhaps

inhibition

buffer

of within

a relationship

which

unrecognized.

related,

of

affinities similarly

to establish

used in these

inhibitor

the

operate

element

PDE operate

finding

that

and PDE correlate

previously

assay

seem, therefore,

and smooth

by inhibiting

the

the most potent

would

appears

level

membranes;

radioligand.

properties

and PDE are

of brain

interesting

smooth

one hand,

a level

The

from

sites

that It

to

(7).

finding

transduction

The PDE assay

activator

imidazole

observation

finding

a signal

uterine of

details.

ANG receptors

recognition

of the ANG receptor

as an enzyme

2 for

The

for

unexpected process

Fig.

binding.

molecular

and

receptor-transduction

ANG III.

and

antagonists

a receptor

binding

section

PDE and ANG receptor

and non-peptide

rat

bound rZ51-ANG II from causing 50% displacement

uterine bioassay

on the

cGMP levels might

explain receptors

(Table

1).

Vol.

179,

This

No.

discrepancy

which

BIOCHEMICAL

between

affinity

that

ANG III

has suggested

smooth more

muscle potent

PDE (data actions Arg

1, 1991

tissues inhibitor

not

shown).

residue

action.

Finally,

may be relevant readily

it that

by ANG III

than

suggested

of ANG effects the brain

than

is

ANG II

effects

(9), [Sar'

that

cGMP PDE used

it

(10,

in these

III,

not also

element

ANG III,

in brain

in certain

Ile']ANG

an important

to previous

Ile']ANG

does

as on PDE];

COMMUNICATIONS

relate

[Sar'

ANG II with

RESEARCH

may also

analogue

[as well

peptides

has been

mediator

inhibit

is consistent

at receptors

of angiotensin

primary

could

The ANG III

This

BIOPHYSICAL

and potency

of ANG III

to ANG III

be the

(8).

AND

inhibit III

vascular which

is

our

brain

having

a

opposing

suggests

that

the

of PDE inhibitory

rather

than

11).

In this

studies

work

is

ANG II, regard,

inhibited

might it more

ANG II.

Acknowledgments Canada,

This work was supported by grants from the Medical Research the Alberta Heart Foundation and Bristol-Myers Squibb.

Council

of

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

11.

Matsoukas, J.M., Goghari, M.H., Scanlon, M.N., Franklin, K.J. and Moore, G.J. (1985) J. Med. Chem. 28, 780-783. Duncia, J.V., Chiu, A.T., Carini, D.J., Gregory, G.B., Johnson, A.L., Price, G.J., Wong, P.C., Calabrese, J.C. and Timmermans, P.B. (1990) W.A., Wells, J. Med. Chem. 33, 1312-1329. Booth, R.F., Lunt, D.O., Lad, N., Buckham, S.P., Oswold, S., Clough, D.P., Floyd, C.D. and Dickens, J. (1990) Biochem. Pharmacol. 40, 2315-2321. Matsoukas, J.M., Cordopatis, P., Belte, U., Goghari, M.H., Ganter, R.C., Franklin, K.J. and Moore, G.J. (1988) J. Med. Chem. 31, 1418-1421. Scanlon, M.N., Koziarz, P. and Moore, G.J. (1990) Gen. Pharmacol. 21, 5965. Sharma, R.K., Adachi, A.-M., Adachi, K. and Wang, J.H. (1984) J. Biol. Chem. 259, 9248-9254. Sharma, R.K. and Wang, J.H. (1979) Adv. Cyclic Nucleotide Res. 10, 187-198. Kwok, Y.C. and Moore, G.J. (1984) J. Pharmacol. Exp. Ther. 231, 137-141. Tobrizchi, R., Moore, G.J. and Pang, C.C.Y. (1988) Life Sciences 43, 537563. Wright, J.M. Mizutani, S., Murray, C.E. Amir, H.Z. and Harding, J.W. (1990) J. Hypertens. 8, 969-974. Hanley, M.R. Cheung, W.T., Hawkins, P., Poyner, D., Benton, H.P., Blair, T.R. and Goedert, M. (1990) Ciba Found. Symp. 150, 23-38. L ., Jackson,

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