Evidence for a similar receptor site for binding of [3H]leukotriene E4 and [3H]leukotriene D4 to the guinea-pig crude lung membrane

Vol.

122,

August

No.

16,

BIOCHEMICAL

3, 1984

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

1984

949-954

EVIDENCE FOR A SIMILAR RECEPTOR SITE FOR BINDING OF ]3H]LEUKOTRIENE [3H]LEUKOTRIENE D4 TO THE GUINEA-PIG CRUDE LUNG MEMBPANE John Allergic

ieceived

Disease

June

B. Cheng and Robert

Center Creighton

E4 AND

G. Townlq

and the Departments of Medicine and Pharmacology, University, Omaha, Nebraska 68178

8, 1984

To determine whether the action of leukotriene E4 is mediated by its cross reaction with leukotriene D4 receptor sites, we compared [3H]leukotriene E4 and [3H]leukotriene D4 binding activities in selected tissues as well as their competition results in the guinea-pig crude lung membrane. We demonstrated good correlation of [3H]leukotriene E4 and [3H]leukotriene D4 binding activities among the tissues studied. A significant correlation was FPL-55712 and demonstrated between the ability of leukotriene C4, D4 and E arachidonic acid to inhibit lung 13Hl leukotriene E4 and 14' Hlleukotriene D4 binding. These correlations suggest that leukotriene E4 binds to a site which is similar to or close to the leukotrine D4 receptor. Leukotriene peripheral smooth

(LT)

airways

airways

is

unclear,

binding

unlikely

since

LTC4

receptor

cell

line

its

cross

assess

to

sites

(9).

to

be the

result

reaction

inhibit

with

the

binding

lung

in of

both

to induce

uterine

and vascular

effect

1)

its

(6-8),

and

The first

brain

tissues

and

radioligands

in

on the at lung

/or

3)

possibility with

are

its seems

[3H]LTC4

(8) and a smooth

features we used

LTE4

interaction

in competing

receptor,

of

LTD4 receptors,

LTE4 binding

selected

guinea-pig

site.

of lung

LTD4

isolated

of:

ineffective

whether

activity

with

of

the potent

LTE4 receptor

in homogenates

To determine

inability for

reaction

a distinct

contraction

The reason

could

cross

its

LTE4 is relatively

the binding

agents

potent

despite

(4-5). but

2) its

LTC4 receptors: direct

(l-3)

response

muscle

E4 produces

for muscle

compatible

with

L3H1LTE4 and

i3H]LTD4

the

of

ability

the guinea-pig

to

several

crude

lung

membrane. METHODS Sources and Preparations of Chemicals: Synthetic LTB4, were gifts from Dr. J Rokach (Merck Frosst Canada, Inc.). Dr. R.C. Murphy (Univ. of Colorado at Denver). Arachidonic Abbreviations:

LT,

Leukotriene;

AA, arachidonic

acid;

LTC4, LTD4 and LTE4 FPL-55712 was from acid and L-serine

GP, Guinea

Pig.

Vol.

122,

No.

3, 1984

BIOCHEMICAL

were purchased from Sigma Co. Scientific Co. (Fair Lawn, NJ). Ci/mmol) and (14,15[3H]LTD4) Nuclear Co. (Boston, MA).

AND

BIOPHYSICAL

RESEARCH

(St Louis, MO). Sodium borate (14, 15-L3H]LTE4) (specific (35.9-40.3 Ci/mmol) were gifts

COMMUNICATIONS

was from Fischer activity: 35.7 from New England

LT solutions were prepared and stored according to the procedures provided by Merck Frosst Canada, Inc. We determined the actual concentrations of the LT stock solutions by UV spectrophotometry at 280 nm for LTC4, LTD4 and LTE4 and 270 nm for LTB4, and corrected accordingly. FPL-55712 was freshly prepared in distilled, double deionized water to a stock solution of 10m2 M. Serial dilutions were made with the deionized water. The arachidonic acid solution was made daily and diluted in absolute ethanol. The ethanol used had no effect on lung [3H]LTE4 and [3H]LTD4 binding. To prevent the conversion of LTC4 and LTE4 to LTD4 and LTF4, respectively, by the membranebound enzyme, y-glutamyl transpeptidase (lo), we included the serine-borate complex in every assay tube. The final concentration of the serine-borate complex consisted of 5 mM L-serine and 10 mM sodium borate according to previous reports (8, 11). Under the assay conditions there is minimal degration of LTD4 in the lung membrane preparation (5,12). Tissue Crude Membrane Preparations: Hartlq guinea pigs (200-350 g) were stunned by a blow on the back of the neck , exsanguinated and lung, heart, small intestine, stomach, spleen, brain and uterus were removed. Lungs from Sprague-Dawley rats (100-300 g) were similarly removed and bovine lungs were prepared according to the previous method (13). The crude membrane preparation of each tissue was isolated by Polytron homogenization and differential The protein concentration was determined by the Lowry's centrifugation (13). method with bovine serum albumin as the standard. L3H1LTE4 and L3H]LTDp Binding Assays: The r3H]LTE4 binding assay was performed in a 300 ul volume containing the membrane preparation (1 mg/ml), 50 mM Tris-HCl buffer (pH=7.0 at 25OC), the serine-borate complex, and [3H]LTE4 (3-6 nM) in the presence and absence of 4.8 uM LTE4. The mixture was incubated at 25OC for 30 min. For the L3HlLTD4 binding assa the conditions were identical to those described above except that l-2 nM [3' HlLTD4 and 1.0-3.6 uM LTD4 were Free and bound radioligand was separated by the rapid used. Each assay was carried out in duplicate. filtration method (13). Specific [3H]LTE4 binding, defined as binding in the absence of LTE4 minus binding in the presence of 4.8 uM LTE4, was about 60% of lung total r3H]LTE4 binding. We used 1.0-3.6 uM LTD4 to define non-specific [3H]LTD4 binding. Specific [3H]LTD4 binding was about 80% of its total binding in the lung homogenate. The IC50 value is the concentration of an agent that reduces either radioligand binding by 50%. Statistics: To analyze the data statistically, we perform&1 single linear In Fig. regression to test significance of a regression (Fig. 1 & 2). 2, we also test whether the slope of the regression line is similar to 1.0 by the Theil test for the slope coefficient (14), and to evaluate whether the demonstrated IC50 value (Yi) determined from from the L3H1LTE4 binding study differs significantly from a predicted value (j;) expected at the given IC50 value (Xi) from the [3H]LTD4 binding study. The difference is considered to be significant if p < 0.05.

RESULTS AND DISCUSSION We

measured

homogenates

and

l-2 found

nM [3a LTD4 only

the

binding following 950

capacity 7 tissues

in

22 different exhibited

tissue demonstrable

Vol.

122,

No.

specific were

r3H)LTD4

contained heart,

lung,

which

More

significantly,

these

tissue

binding

sites

small

intestine.

for

a further

1) .

Under

contained

is similar

not

find

a

This

the

had little

the

distribution

that

well

that

1

z

\ 1

Fig.

1.

having

--

and (8),

sites.

13H1LTD4 binding binding

[~HILTD~ guinea-pig

intermediate

distributed

binding

in both

BINDING

the

stomach

binding

r3H1LTE4

of

brain

values uterine whereas

to (Fig.

or

r3H!LTE4 lung

data (see

and

points Fig.

homogenates [3HlLTE4

homogenates.

o GPLUNG . GP SMALL INTESTINE A BOVINE LUNG A GP HEART x RAT LUNG 0 GP STOMACH D GP SPLEEN

t3H)LT0,

of

values

evenly

13HlLTC4

activity

160

in

of

lung

by guinea-pig

[3HlLTD4

contained

guinea-pig

specific binding

the

us from

of fit

that

3-6

and guinea-pig

activity

that

was between

conditions,

of

with

homogenate

lung

and bovine

Guinea-pig

followed

rat

tissues

concentration:

homogenates.

binding

the relative

precluded

exclusively

i3HlLTD4

intestine,

tissue

of goodness

assay

small

correlated

in an amount

test

13H1LTE4

These

and spleen,

(L3H1LTE4

membrane

COMMUNICATIONS

data).

stomach

activity

of

to

we found

We could

1).

guinea-pig

RESEARCH

unpublished heart,

tissue

activity

homogenates

also

binding

in these

highest

bovine

and spleen,

L3HlLTE4

determined

the

(15,

BIOPHYSICAL

intestine,

small

Specific

then

AND

activity

lung,

lung.

was

BIOCHEMICAL

binding

guinea-pig

and rat nM)

3, 1984

(fmol/mg

I /

.--

PROT 1

Relationship between specific 13Hl LTD4 and L3H1LTE4 binding activities in the crude membrane preparation of indicated tissues. Each tissue preparation (1.0 mg/ml) was incubated with 3.0-6.0 nm [3H1Ll'E4 (or 1.0-2.0 nM (3H1LTD4), 10 mM MgC12 and the serine-borate complex in the presence and absence of 4.8 uM LTE4 (or l-O-3.6 UM LTD4 at 25OC for 30 min. GP: Guinea Pig. Mean + SE of the number of experiments indicated. 951

and

Vol.

122,

No.

The binding

3, 1984

fact

BIOCHEMICAL

that

activitiy

indicates

the for

that

We therefore

lung

chose

competition

previous

arachidonic if

we analyzed inhibit

are

the

LTE4

binds

and

for

at

COMMUNICATIONS

least

higher

3-fold

any other

membrane

the action

preparation

of

of LTD~

for

(3.6-20~10-~

order

10V5 M had no effect

the

of potency

the

lung

M)

tested and

LTE~.

subsequent

M) ) arachidonic

on both

arachidonic

_)

r3HlLTE4

potency

acid

to Based

[3H]LTE4

binding M) :, LTC4

acid

?? LTC4

[3H]LTD4

and

agents

preparation. of

To

binding,

these

(2.5-72.0~10-~

the

and

8).

[3H]LTD4

of

membrane

M)

(3,

for

inhibition

study,

FPL-55712

binding

site

_) LTD4

(1.3-4.7~10-~ M)

LTE4,

abilities

for

(2.8-4.4~10-~

LTE4

(4.8-6.6~10-~

to the

in the

competition

2.

LTD4,

[3H]LTD4

similar

1.4-7.0~10-~

[3H]LTD4 M)

- FPL-55712

inhibiting

binding

M) _) FPL-55712

FOK the

LTC4,

between

[3H]LTE4

range:

that

to a site

the rank

(IC50

(9.6-1OOx1O-8

shown

interrelationship

on the IC50 value,

(1.8-3.9~10~~

order

was

LTD4

(2.7-7.0~10-~

(4.3-13.5~10~~ binding

in

M)

M). the

LTB4 at

guinea-pig

membrane.

As

shown

inhibition

in

Fig.

of lung

2, we compared

[3H]LTD4

correlation.

Further

line

significantly

to be not

to reduce the

site

lung

have

capable

[3H]LTE4

[3H]LTD4

lung

has

13H]LTE4 than

target

guinea-pig

RESEARCH

membrane

and

is a major

studies

acid

determine

M) -

lung

[3H]LTD4

the

BIOPHYSICAL

experiments.

Our

was

guinea-pig

both

its

AND

lung

airway

binding.

Similarly,

FPL-55712

binding.

The potency

binding DeBaven

sites when

consistencies

binding,

in this they

LTC4,

The

ICSO value

study used

strongly

of the is

revealed

that

Consistent

1.0.

indicate

LTD4

with

in

equal

952

guinea-pig

of the regression their

than and

1.0 uM. L3W LTE4

[3HjLTE4

result

of

These lung

of

inhibited

less

with

(16).

ability

in the presence

[3H]LTD4

recent

for

a significant

to or

competing the

agents

effectively

both

radioligand the

these

with

that

LTE4

OK

was

LT agonists

as the

of

the slope

decreasing

compatible

L3H]LTD4 suggest

in

values

and demonstrated

results

either

was effective order

from our

IC50

binding

of the data different

complex

[3H] LTE4

[3H]LTE4

analysis

[3H]LTD4

serine-borate

and

the

for Pong

findings

membrane

lung and and LTE4

Vol.

122,

No.

BIOCHEMICAL

3, 1984

LTD, A LTE, A FPL I AA

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

l

8 $9’

/ 9

8 -LOG

Fig.

2.

with

LTD4 receptor

tissue the

a site

comparison

competition

which

of

study

site

evidence

for

FPL-55712

effectively

contraction

which LTE4

(17) demonstrated

inability

consistent

K,(M)

is

-iNHIBITION OF(‘H)LTCI,

BINDING

pharmacologically

[3H]LTD4

provide

indistinguishable

[3H]LTE4

is

similar

binding

and

its

the first to

or

time

close

to

to the LTD4 receptor

inhibits

both

binding

of both

in the brain

inability

binding

of the potency

for

of LTE4 to directly with

and

and our comparison

receptor

the

COMPETITOR’S

, 4

5

from

the

site.

homogenates

[3H]LTE4

, 6

7

Relationship between the IC50 values of LTC4, LTD4, LTE4, FPL-55712 and arachidonic acid (AA) for inhibition of 13Hl LTD4 and 13H1LTE4 binding in the guinea-pig lung preparation. The lung membrane (1.0 (or 1-2 nM c3H]LTD4), 10 mg/ml) was incubated with 3-6 nM 13H)LTE4 the serine-borate complex, and indicated agent in the mM wlc12, presence and absence of 4.8 uM LTR4 (or 1.0-3.6 UM LTD4) at 25OC for 30 min. The concentration range for the LT agonists was 1.0-4.8~10-~~ to 1.0-4.8~10-~ M, 10s7 to 10e3 M for FPL-55712, and acid. The dotted line indicates a line 10-6 to 10m3 M for arachidonic where the slope is 1.0. Statistical analysis demonstrated the significance of a regression (p <0.03), and that the slope of the regression line did not differ significantly from 1.0 (p > 0.4, Theil test for the slope coefficient). The IC50 value of LTE4 and LTC4 from the [3H]LTE4 binding study was within the 95% confidence interval (p >O.OS). Mean + SE of the number of experiments indicated.

interacts

Our

55712

and

LTD4-

orders

of

evidence

that

the

uterine with

guinea-pig 953

LTD4

is provided

Little homogenate the

in selected the

LTE4 binds

by the

binding is

finding

to a

that lung

activity

consistent

contraction

for

Further

peripheral

LTC4 receptor

uterine

agents

receptor.

LTE4-induced

radioligands.

interact

to induce

and

activities

of with

(6-g),

and is (4,

also

Vol.

122,

unpublished airways

No.

3, 1984

data). by LTE4 is

BIOCHEMICAL

Conversely, likely

mediated

AND

BIOPHYSICAL

the potent

contraction

by its

cross

reaction

RESEARCH

COMMUNICATIONS

of guinea-pig with

peripheral

the LTD4 receptor

sites.

ACKNOWLEDGEMENTS We thank Mr. M. Ewer and Dr. K.J. [3H] LTD4 , Dr. J. Rokach for contributing FPL-55712.

O'Brien for LTs and Dr.

supplying R.C. Murphy

[3H]LTE4 and for providing

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 ‘. 11. 12. 13. 14. 15. 16. 17.

Lewis, R.A., and Austen, K.F. (1981) Nature 193, 103-108. Dahlen, S.E. (1983) Acta Physiol. Stand. Suppl. 512, 1-51. Cheng, J.B., and Townley, R.G. (1984) Biochen. Biophys. Res. Commun. 118, 20-26. Weichman, B.M., and Tucker, S.S. (1982) Prostaglandins 24, 245-259. Krilis, S., Lewis, R.A., Corey, E.J., and Austen, K.F. (1983) Proc. XI Intl. Congress of Allergology and Clin. Immunol. pp.3-9, MacMillan Press Ltd., London. Pong, S-S., DeHaven, R.N., Kuehl, F.A., Jr., and Egan, R. (1983) J. Biol. Chem. 258, 9616-9619. Hogaboom, G.K., Mong, S., Wu, H-L., and Crooke, S.T. (1983) Biochem. Biophys. Res. Commun. 116, 1136-1143. Cheng, J.B., and Townley, R.G. (1984) Biochem. Biophys. Res. Commun. 119, 612-617. Krilis, S., Lewis, R.A., Corey, E.J., and Austen, K.F. (1983) .J. Clin. Invest. 72, 612-617. Samuelsson, A. (1983) Proc. XI Intl. Congress of Allergology and Clin. Immumol. pp. 24-28, MacMillan Press Ltd., London. Proc. Natl. Acad. Sci. (U.S.A.) 75, Tate, S.S., and Meister, A. (1978) 4806-4809. Bruns, R.F., Thomsen, W.J., and Pugsley, T.A. (1983) Life Sci. 33, 645-653. Cheng, J.B., and Townley, R.G. (1982) Life Sci. 30, 2079-2086. Wolfe, D.A., and Hollande, R.M. (1973) Nonparametric Statistical Methods. pp. 200-205, John Wiley & Sons, Inc., New York. Townley, R.G., Cheng, J.B., Cheng, E., Krokos, K., and Jedruska-Witt, S. (1984) Am. Rev. Resp. Dis. 129, A2 (Abstract). Acad. Sci. (U.S.A.) 80, pang, s-s., and DeHaven, R.N. (1983) Proc. Natl. 7415-7419. Krell, R.D., Tsai, B.S., Berdaulay, A., Barone, M., and Giles, R.E. (1983) Prostaglandins 25, 171-177.

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