Characterization of specific binding sites for PAF in the iris and ciliary body of rabbit

Vol.

160,

No.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BIOCHEMICAL

1, 1969

Pages

April 14, 1969

CHARACTERIZATION IN THE M.T.

Domingo,

OF SPECIFIC IRIS

AND

P.E.

Chabrier,

N . J - Van

BINDING

CILIARY

BODY

J.L.

Haeringem

lnstitut

Henri

PAF

Delft,

N-L.

Verbeij,

P. Braquet*

Beaufour

1, avenue

des Tropiques

91952

ULIS,

LES

FOR

OF RABBIT

Van

and

SITES

250-256

France

Received January 13, 1989 The protective effect exerted by BN 52021 a specific PAP-receptor antagonist in experimentally induced ocular inflammatory disorders led us to investigate the possible presence of specific receptors for PAP in rabbit iris and ciliary body. Two classes of P?IF binding sites were found in isolated iris and ciliary process of pigmented rabbit eyes : a high affinity site Kdl = 4.9 + 0.47 nM, Bmax, = 3.17 + 0.50 pmoles/mg protein, a low affinity sites K& = 11.6 + 0.33 nM, Bmax, = 12.46 + 2.3 pmolesjmg protein for iris. The specific binding was not affected by lyso-PAP the biologically inactive precursor and metabolite of PAF, up to 10-6M . inhibition by unlabelled PAP demonstrated a biphasic curve partially a&agonized by BN 52021. The present results demonstrate the presence of specific binding sites for PAP in rabbit eyes which could mediate the action of this mediator in eye inflammatory processes and explain the protective effect observed with BN 52021.

Q 1989

Academic

Platelet rylcholine)

factor

(PAF,

is an ether-linked

released

and from

endothelials

platelets

cells

exocytosis

muscle

in and

Il.

neutrophils

2, (5).

appropriately and

PAF

also

induces

in various 3).

PAF

(6).

It induces

neutrophils,

increases

inflammatory,

is synthetized

macrophages

stimulated.

platelets

In vivo

contraction

failure

when

involved

disorders

(41,

(8)

1 -O-alkyl-2-acetyl-sn-glycerol-phospho-

phospholipid

respiratory

thrombocytopenia.

(7)

and

aggregation and

leukopenia

vascular

permeability,

bronchoconstriction

hypotension,

and

smooth and

renal

(9,101. The

of its

availability

proposed One

potent

and

aggregation * To

Inc.

activating

cardiovascular

and

Press,

whom

0006-291X/89

of specific

role

such (11)

antagonists

in pathophysiological

compound,

selective

PAF

the

PAF and

correspondence

has

models

ginkgolide

antagonist been should

for

be addressed.

250

the

investigation

of inflammation.

BN 52021, PAF

demonstrated

$1.50

Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

has allowed

induced to exert

has been platelet

described and

a protective

as a

neutrophil effect

in

BIOCHEMICAL

Vol. 160, No. 1, 1989

various

experimental

cardiac

models

anaphylaxis Recently

infiltration (18).

and and

breakdown

rabbit

the

blood

Furthermore, deleterious

vivo

electroretinogram These PAF binding

sites

in eye

led

sites

inflammation,

in

induced

amplitude

eye.

‘H-PAF

and

BN 52021

the

and

ciliary

pulmonary

and

irradiation rats the of

the

the

in two

leukocyte

immune

keratitis (IOP)

and

(19). dramatic

beta-wave

and of

the

(20).

possible

present

block

pressure

by BN 52021 The

to by

intraocular

us to examine

MATERIALS

shown

after laser diabetic

inhibited

rabbit iris

been

of

the

(12.13)

(16,171.

cornea,

rise

in

is also

observations using

acute

shock

has

in the

decrease

(ERG)

recognition

52021

aqueous barrier alloxan-induced

in

ex

endotoxin ischemia

BN

formation

prevent

of the

brain

eye,

edema

to

including

(14,15),

in

AND BIOPHYSICAL RESEARCH COMMUNlCATtONS

study of the

presence

of

characterized main

areas

specific PAF

implicated

body. AND

METHODS

Materials

Synthetic tritiated PAF (3H-PAF) with a specific activity of 59.5 Ci per mmole was purchased from New England Nuclear (DuPont de Nemours, France). Unlabelled PAF and lyso-PAF were purchased from Calbiochem (France), in solubilized in ethanol and stored at - 20° C. BN 52021 was solubilized heparin (Roche) were purchased from Coger DMSO. Pentobarbital (Clin-Midy), Industries (France). Tissue

preparation

Pigmented rabbits (either sex) were injected with heparin (1 000 UI per rabbit) 5 minutes prior to sacrifice by an overdose of pentobarbital. Eyes were enucleated and placed into ice cold 50 mM tris-HCl buffer pH 1.4, containing 10 mMMgCl,, 2 mNEDTA, 50 UI per ml trasylol and lo-'M PMSF. The perfused prior to dissection in order to remove the eyes were arterially blood of anterior segment. The iris and ciliary body were carefully dissected out and homogenized using a dounce homogenizer. The homogenates were filtered through 2 layers of gauze and centrifugated twice at 40 000 g for 20 minutes. The pellets were resuspended in the homogenization buffer and assayed for protein content using BIORAD protein assay reagent and bovine serum albumin as the standard. The

binding

assay

40 to 70 ug of protein was added to a final volume of 1 ml of 10 mMtris HCl buffer, containing MgCl, 5 mM, BSA 0.025 %, trasylol 50 UI per ml, PMSF 10-4M, pH 7 in plastic tubes containing 3H-PAF (lo-'1) with or without 10-6M unlabelled PAF (for nonspecific binding), lyso-PAF or the PAF-receptor antagonist BN 52021. The incubation was carried out for 40 minutes at 25O C. The unbound ligand was separated from the bound by immediate filtration through Whatman GF/B fiber-Filters presoaked 24 hrs in the binding buffer and using the BRANDEL vacuum system (Brandel, Biomedical Research and Development Laboratories Gathersburg MD/USA). The assay tubes and filters were washed 3 times with 3 ml of precooled binding buffer. The filters were placed into polyethylene vials containing 10 ml of liquid scintillation fluid (instagel, Packard). The radioactivity was measured using a 45 % efficiency LEB counter. The specific binding was calculated as previously described (21). Binding data were analyzed by Scatchard method using the computerized programm of VINDIMIAN et al. (22). 251

Vol. 160, No. 1, 1989

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

In some experiments the stability of the tritiated ligand was analysed after 40 minutes incubation and extraction from the reaction mixture using the solvent system, chloroform : methanol, 2 : 1 and separation on T.L.C. silica plates (Prolabo SIL Gz5 UV 250) using a chloroform : methanol : ammonia, 70 : 35 : 7. system. In our binding conditions intact 3H-PAF represented 93 % of the total radioactivity recovered from the plates. RESULTS Binding

of 3H-PAF

- Saturation ciliary

study

body

and

a function protein

both

in

concentration

was

linear

body,

and

and

ciliary

of

the

specific

biphasic

of

‘H-PAF

saturation nM

- Competition was

assessed

PAF

receptor

a

(Figure

second

study

: The

step the

BN

52021,

in

to

of the

about

first binding

compete

to iris PAF, with

% of of

as

the

total

increasing

demonstrated

reached

observed

of unlabelled to

20

10D8M)

step

and

binding

of 10 to 90 ug of

function

x

iris

specific

range

a

1.4

in cornea,

The

the

was

was

ability

found

two.

as

1) . The

specificity

by determining antagonist

8 10”’

was

latter

binding

(from

isotherm

and

binding

in the

iris

Study

concentrations

specific

characterized

of protein

binding.

4-5

: ‘H-PAF was

a maximum

at

10.8

and

ciliary

lyso-PAF

‘H-PAF

a at nM. body

and

binding.

A ll10 9. z_ i-f E

B7.

0 II 2.7 3.9 45 5.4 6.3 7.2 9.1 9 89 10.9 11.712.9 “H-PAF (nM ) Figure 1 The binding 13.5 nM. performed BSA 0.025

in iris at 25OC. - Typical saturation isotherm for 'H-PAP binding was performed over radioligand concentration range of 1 nM to Each point is the average of triplicate and n = 4. The assay was 50 UI per ml and in tris 10 mM, MgC12 5 mM, PMSF 10-4M, trasylol % pH 7 buffer. 252

the As

BIOCHEMICAL

Vol. 160, No. 1, 1989

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

100

[ANTAGONIST 1 Figure unlabelled average

shown

2

-

Inhibition

PAF (O-O), triplicate

of

in figure

A biphasic binding

2,

partially

Scatchard competition

plot studies

(1-3

nM) binding and lyso-PAF ; n = 4.

no displacement

displacement was

of 'H-PAF BN 52021 M) determinations

curve displaced analysis demonstrated

of

was observed

MgCl,

at

with

was observed

with

(60 %I by

BN 52021.

the

binding

data

a curvilinear

3H-PAF Figure3 10 mM, buffer, n = 4.

to (w).

BOUND

lyso-PAF

unlabelled from

PAF both

distribution

by the

up to

106M.

and

‘H-PAF

saturation (Figure

and 3).

(nM )

Scatchard analysis of 'H-PAF binding to iris 5 mM, PMSF 10-‘M, trasylol 50 UI per ml, 25V. Each point is the average of triplicate

253

iris homogenate Each point is

homogenate in tris BSA 0.025 % pH 7 determinations ;

A

Vol. 160, No. 1, 1969

BIOCHEMICAL

a

10

I5

20

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

25

30

35

40

45

56

50

60

(Min)

TIME

Figure 4 - Typical association and dissociation kinetic curve of 'H-PAF binding in iris or ciliary body. The experiment was performed at 25°C in tris 10 mM, MgCl? 5 mM, PMSF 10-4M, trasylol 50 UI per ml and bovine serum albumin 0.025 % pH I buffer.

high

affinity

and for

site

a low iris

affinity

- Kinetic

site

study

(Kd2

: As

of ‘H-PAF

within

the C.

mixture

+ 0.47

nM Bmax,

= 11.6

shown

(l-3

first

by

nM)

25 minutes

An

excess

at

30

reversibility was

= 4.9

+ 0.33

nM,

= 3.17

2 0.5

pmole/mg

Bmax2

= 12.46

+ 2.3

protein pmole/mg

; n=5).

binding 25O

(Kd,

of

min

and

45

binding

after

45 min.

kinetic and

of reaction

unlabelled

of the

observed

the

to iris

and

PAF

(1

of

the

min (about

curve

ciliary

(Figure

body

remained 000

fold)

time

41,

reached

specific state

stable

up

to 60 min

added

to

the

course

50 %) at 30 min.

the

a steady

showed

whereas

reaction a

no

at

partial

reversibility

DISCUSSION The here

3H-PAF

binding

demonstrate The

70-80

the

percent but

for

lung

human

brain

tissue

Specific PAF,

over

experiments, binding

in the

of PAF in

is comparable tissue

or

the

crude

binding the

range a

two

characteristics.

rabbit

iris

tissues

platelet low

to the

20-25

preparation

is low

membrane

specific

that

ciliary

body

reported

sites.

in these

rabbit

possible

and

recognition

to the

(24)

nonspecific

on

binding

binding

I21 1. It is also

concentrations increased

presence

of specific

% of specific

conditions,

studies

binding

used

to the buffer

binding

which

for

compared the

same

(30-40

% specific melanin,

in

these

8)

described

described

is present tissues,

for

in high

might

have

binding. as a function of step

8.10-l’

of increasing to

14.10-‘M

saturation

Results

have

isotherm,

of saturation 254

concentrations shown

from

suggesting

experiments

of

were

labelled

replicated

heteregenous confirmed

by

BIOCHEMICAL

Vol. 160, No. 1, 1989 displacement

studies

Displacement

by

where

unlabelled

scatchard

analysis

revealed

described

by a two

component

In

the

Kd, = 4.9 component protein.

iris,

the

the

two

PAF

presented

binding

a

curvilinear

steps

easier

to

inhibition

distribution,

computerized

binding

were

a biphasic

describe.

curve.

which

The

was

better

analysis.

characteristics

of

the

first

component

were

= 3.17 pmol/mg protein and for the second -+ 0.47 nM and Bmax, Kd2 = 11.6 + 0.33 nM associated with Bmax2 = 12.46 pmol/mg

Similar

nM Bmax

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

results

= 3.41

1

were

obtained

2 1 pmol/mg

and

for

Kd2

ciliary

= 24.4

body

with

+ 0.91

Kd,

= 5.7 + 0.09

nM Bmax2

= 16.6

+ 0.51

pmol I mg . In our

study,

experiments 52021,

using up

differed

from

that

did

maximum only

PAF

partial

the

course

45

This

dissociation. site

associated

medium

was

The several

not

significantly

presence

inflammatory conjunctiva

Given pressure

and

the

due

to the

(19).

PAF

is

also

suggest

study

rabbit

iris

inflammation.

and

ciliary

How

these

inflammation distinct

same receptor

types site

by its

serum chick

PAF

however

binding

two

these

different

to be clarified. 2.55

many in

rat

by to

of

into

upon

a

inhibit

in

intraocular the

laser

aqueous irradiation

stimulation eye

with

function

and

binding

site

mediated.

main

of

rise by

retina

of

whether

in

(18).

barrier

body,

classes

the

since

antagonized

proteins

aqueous

the

to

in

PAF

reported

the

may be receptor

two

time

binding

PAF of

keratitis

implicate

effect

or

a

expected

of

also

inhibits

of

blood

results

and

associated

of PAF 3 H-PAF

was

response was

subclasses

two

be

injections

of two

receptor

remains

dose

coexistence

processes, of

low

also

embryo

These the

participation

in immune

leakage of the

that

shows

the

52021

drug

subsequent

(25).

and

Our

edema

the

noticeably

experiments,

since

in eyes

inflammatory

cornea1

BN

assay.

sites

Local

BN

produced

ligand

in our

(23).

disruption

neurotransmitters pathology

and

to

In kinetic

to disapearence bound

binding

marked

preventively,

humor

two

a

infiltration

the

eye.

and

‘H-PAF

BN 52021

appeared

curve.

demonstrated the

antagonist

leukocyte

during

in

elicitated

PAF-receptor

in

have

processes

of

specific

PAF.

to inhibit

with

60 % and

degradated

of PAF

studies

obtained

may be attributed

a captation

of

failed

%) was observed at 35 min of the 3 the H-PAF binding was resistant

incubation

property

analog

by competition

monophasic and only partial. In fact, 3 H-PAF binding in these tissues. The

about I50

assessed

inactive

competition

binding

min

with

recent

was

of the

of the

after

:

it was

was

Lyso-PAF

curve

completely

step

dissociation

biologically

since

obtained

first

binding

antagonist.

inhibition

inhibit

inhibition

with

The

of PAF

not

of the

the

receptor

to 10m6M.

52021

specificity

lyso-PAF,

a specific

binding BN

the

areas

of PAF involved

in

sites

interact

with

two

subclasses

conformational

ocular PAF

represent states

of the

Vol. 160, No. 1, 1969

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ACKNOWLEDGMENT The

authors

wish

to thank

Mr

Moumen

Mohamed

for

technical

help.

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