Inhibition of the partially purified canine lung angiotensin I converting enzyme by opioid peptides

Biochemical Pharmacology. Vol. 29. pp. 3115-3118. @Pecgamon Press Ltd.1980. Printed in&cat Britain.

INHIBITION

OF THE PARTIALLY

PURIFIED CANINE LLmG ANGIOTENSIN BY OPIOID PEPTIDES

Gary E. Sander*,

Walter

Patrick

E. Lorenz,

The angiotensin

I converting

angiotensin

and inactivates

bradykinin,

umbilical

cell cultures

(met-enk)

or leucine

reported

Cheung

(leu-enk),

the opioid peptides

of met-enk

was competitively reported

rat brain; both cleave C-terminal

chloride

contains

enkephalinase Arregui

activity

calculated

from methionine

and kinins.

SQ20881, although

thus

Benuck

and Marks

and leu-enk;

SQ142.25 (3).

metallopeptidases angiotensin

on the basis of differential

by the peptide

these

from both met-enk

leu-enk,

Erdos

enkephalin

as ACE inhibitors,

of two distinct

from met-enk,

(1).

(ACE) from human

by the ACE inhibitor

ACE and a distinct,

and Iversen have demonstrated

(S-LPH: 61-65),

enzyme

phe-arg

II,

from

I, and the

activation

these authors

by

theorize

that

very similar,

(4).

a 91 amino acid pituitary met-enk

removed dipeptides

However,

inhibition

both the classical

the angiotensins

II,

to form angiotensin

as an enkephalinase;

may function

the isolation

to as kininase

dipeptide,

dipeptide

inhibited

dipeptides

hip-his-leu.

ions and differential

the brain

with

his-leu

I converting

thus functioning

from rabbit brain

et al. have recently

ACE substrate

of a C-terminal

that the angiotensin

1980)

also referred

dipeptide

that the opioid peptides

that ACE isolated

synthetic

the C-terminal

again by removal

enkephalin

linking

the hydrolysis

S. Verima

15 September

(EC 3.4.15.1),

can also remove a C-terminal

have hypothesized

metabolically

enzyme

I by removing

et al. (2) have demonstrated --

authors

ENZYME

Department of Clinical Physiology, Division of Medicine, Reed Army Institute of Research, Washington, DC 20012, U.S.A. (Received 27 May 1980; accepted

activates

and Pritam

I CONVERTING

peptide

containing

can competitively

Ki of 0.78 PM.

of this enzyme preparation

that the opioid precursor the sequences

inhibit

by S-endorphin,

of B-endorphin

an ACE purified

These authors were unable met-enk,

B-lipotropin

(S-LPH),

(B-LPH: 61-91) and

from human brain, with a

to demonstrate

significant

or D-ala'-met-enk

inhibition

at concentrations

up

to 40 pM (5). We report that B-endorphin,

met-enk.

and leu-enk

competitively

inhibit

the ACE from

canine lung. METHODS

AND MATERIALS

Enzyme preparation. general methodology

A particulate

ACE fraction was prepared

of Sander and Huggins

*Present Address: Department of Medicine, Avenue, New Orleans, LA 70118, U.S.A.

(6).

from canine lungs using

Fresh lungs were perfused

Tulane University 3115

the

free of blood

School of Medicine,

1430 Tulane

3116

with iced saline, dissected free of hilar structures, homogenized in 50 mM Tris-HCl buffer (pH 7,4), and centrifuged at 2300 g for 30 min.

The resulting supernatant fraction was

centrifuged at 50,000 g for 30 min, and the pellet washed twice with the Tris buffer. The final pellet was resuspended in the Tris-HC1 buffer and used as the source of ACE.

Substrate

hydrolysis by this fraction was chloride ion dependent and completely inhibited by both bradykinin (33.3 uM) and EDTA (16.7 nM), indicating that the hip-his-leu hydrolysis was due solely to ACE activity. ACE assay.

(14C)Hip-his-leu(sp. act. 2-4 mCi/mmole), obtained from the New England

Nuclear Corp. (Boston, MA), was used as ACE substrate in all experiments; no unlabeled substrate was added. All reagents were made up in the assay buffer, which was 50 mM Tris-HCS (pH 7.5, 0.3 M NaCl). Each reaction mixture consisted of 10 ~1 of enzyme preparation (containing 48 ug protein), appropriate substrate and opioid concentrations,and buffer to a final volume of 300 i_il.All components of the reaction mixture were preincubated at 37O for 30 min, and the reactions initiated by addition of substrate to the enzyme-inhibitormixture. Reactions were run for 30 min at 37', and then stopped by addition of 1.0 ml of 0.1 N HCL. One ml of ethyl acetate was added to each tube, and the tube was shaken on a vortex mixer for 5 set and then centrifuged at 1000 pi for 10 min.

Five hundred ~1 of the ethyl acetate (upper)

layer, containing the (14C)hippuric acid, was then added directly to a vial containing 10 ml of scintillation fluid. Activity was expressed as nmoles (14C)hip releasedamin-l*(mgprctein)-1. @-Endorphin,met-e& , and leu-enk were purchased from Sigma Chemical Co. (St. Louis,

MO).

RESULTS AND DISCUSSION The ACE from canine lung is inhibited by @-endorphin,met-enk, and leu-enk, as indicated in Table 1.

B-Endorphin-inducedinhibition is first evident at concentrationsof 5 PM, and

Table 1.

Inhibition of angiotensin I converting enzyme by opioid peptidesn Concn

Met-enk

33.3 160 333

0.36 1.7 3.6

12 20 30

Leu-enk

33.3 160 333

0.36 1.7 3.6

11 23 38

5.0 9.7 24 48

0.05 0.10 0.26 0.52

12 28 38 57

B-Endorphin

IiS Ratio

% Inhibition

Inhibitor

*All reaction mixtures contained-)hip-his-leu in a final concentration of 94 PM. The X/S ratio represents the molar ratio of inhibitor to substrate in that particular reaction. The % inhibition is expressed relative to control reactions containing no inhibitor. Each data point represents the mean of two experiments, with each experiment performed in duplicate.

.

prelurunarycommmications

inhibition

by met-enk

tration was 94 uM.

and leu-enk

The kinetics

in Fig. la; these experiments from 5.8 to 29.2 nmoles weaver-Burk

at 33.3 uM.

In these experiments

of ACE inhibition

were performed

per reaction

plot of ACE inhibition

3117

by 8-endorphin

the substrate

and met-enk

concen-

are illustrated

with final hip-his-leu

concentrations

(19.3 PM to 97.3 uM).

Fig. lb depicts

mixture

by two concentrations

ranging a Line-

(33.3 uM and 66.7 uM) of leu-enk.

08

40

0.6

3.0 _1 v

v 2c

I .c

0

IO

20

30

0

20

40

S (a)

Fi-5.

1.

pattern

of 146 PM.

experiment

(b)

calculated

is indicative

of competitive

The data shown represent

was performed

and S-endorphin

a minimum

Ki of 118 UM for met-enk of Arregui

and Iversen

inhibition

(7), as contrasted

is correspondingly

used was isolated from the canine Thus,

the results

with a calculated

from a typical

(Lineweaver-Burk

Ki for

experiment.

identical

results.

Each Met-enk

plots not shown), with a

and of 32 uM for 8-endorphin. to demonstrate

the use of a much higher hip-his-leu

they utilized

inhibition,

of two times, with virtually

also inhibit ACE competitively

The failure reflect

6C

(a) Rate of release of (14C)hi-,puric acid by canine lung ACE as a function of the initial substrate (hip-his-leu) concentration, in the absence of inhibitor ( l ), with 66.7 uM met-enk ( o ), and with 24 uM 8-endorphin ( A ). S is expressfd as total nrnzies of substrate present per reaction mixture and v as nmoles*min*(mg protein) . (b) Lineweaver-Burk plot of the hydrolysis of hip-his-leu by ACE in the absence ( a ) of leu-en!:, and with 37.3 uM ( A ) and 66.7 pM ( o ) leu-enk.

The inhibition leu-enk

60

1 *103 s

inhibition

concentration

from human brain and may differ

may

(1 r&l) in the assay system which

with the 94 UM concentration

more likely to be observed.

with these peptides

in our system.

Furthermore,

in activity

Thus, the

the ACE which

and substrate

they

specificity

lung ACE.

S-endorphin,

met-enk,

and leu-enk

competitively

inhibit

the ACE from canine lung;

FwhiIlalycommunications

3118

this pattern

is consistent

thus competing

for the active site on the enzyme.

for ACE, followed

by leu-enk

based on the results than B-endorphin. may reflect

structural

concentration

of leu-enk

normally

displays

better

inhibit

angiotensin

present

in human plasma.

to the natural

with ACTH following

after adrenalectomy

(13).

substrate

used to inhibit (9,lO).

However,

of ACE activity.

has been demonstrated

The intravenous

of S-endorphin

administration

and heart rate

by high endogenous

levels,

by interacting

Ki of 0.6 nM (8).

concentrations

of the

2.02 to 4.19 nM (12),

from the pituitary

of S-endorphin

that, at least in certain physiological

and bradykinin

less

in

in the 2.5 nM range would

bility must be considered

8-endorphin

I,

levels rising as high as 2.5 nM in rats

to alter both blood pressure

opioid

angiotensin

the hip-his-leu

I/S ratios in excess of the 0.52 I/S ratio which we have demonstrated

inhibition

this

the ACE are in excess of the

has been shown to be released

Thus, concentrations

affinity

considerably

SQ20881, which has a reported

stress, with plasma

however,

than met-enk;

the ACE, they display

present -in vivo

affinity

position.

I, 8.48 to 67.8 pM (ll), and bradykinin,

8-Endorphin

a higher

substrate

(94 pM) was also far in excess of the physiological

substrates

as ACE substrates,

the greatest

would display

at the C-terminal

of these opioids

of these peptides

natural

provide

similarity

do competitively

the concentrations

concentrations

parallel

to be a slightly

for the ACE than does the nonapeptide

Furthermore,

B-Endorphin

it presumably

have the leucine residue

these opioids

can function

We did not use S-LPH in our experiments;

and Iversen,

Leu-enk would appear

since both peptides

affinity

and met-enk.

of Arregui

a greater

Although

with the fact that the enkephalins

may influence

Hence the possi-

situations

with the lung ACE, and in this way participate

57%

and the enkephalins

(14,15).

the metabolism

to produce

characterized

of angiotensin

I

in cardiovascular

control.

ACKNOWLEDGEMENTS:

We wish to thank Carolyn

Umstott

for her excellent

technical

assistance.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

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