3H-mepyramine binding to histamine H1-receptors in guinea pig lung: Characteristics and regulation by ions

Life Sciences, Vol. 28, pp. Printed in the U.S.A.

3

H-MEPYRAMINE

705-713

BINDING

Pergamon

TO HISTAMINE

H -RECEPTORS IN GUINEA 1 AND REGULATION BY IONS

CHARACTERISTICS

M. Lombroso, Institute

of P h a r m a c o l o g y

and

Sarto (Received

PIG LUNG:

S. Nicosia

Pharmacognosy,

University

21, 20129 Milan,

in final

Press

of Milan,

Via

A.

Del

Italy.

form December

i, 1980)

Summary The antagonist tors of

in

binding

(Na + >

[ 3 H I -Mepyramine

guinea

sites.

Li + >

exhibit

a

lung.

biphasic

decreasing

and

number

reveals

decrease

steady-state

cations

increasing

higher

two

binding

(Mg ++ , Ca ++ , Mn ++ , Ba ++ )

binding at low + Na decreases

levels.

H -recepi classes

analysis

concentrations both

affinity

of binding sites. Dissociation curve shows two + Na accelerates the rate of dissociation of the

and

component.

at

cations

divalent

curve,

it

is used to label histamine

Scatchard

Monovalent

K + ), while

and

nents,

pig

GTP

does

not

affect

the

binding

of

the

composlower 3 H-

antagonist

Mepyramine.

lungs

The presence and airway

H receptors 2 receptors in a

debated

the

role

of histamine smooth muscle

are

involved

various

question. of

[ Hh-mepyramine to

phenomena Direct

histamine

f3~

in

H 1 and H 0 receptors has been demonstrated in (~i). H ~receptors mediate contraction, while i relaxation (2,3); however, the role of these such

binding

receptors (3H-Mep),

in an

as

the

studies

anaphylactic might

different antagonist

prove

response

useful

is

still

in u n r a v e l i n g

physiopathological

conditions.

of

been

histamine,

has

shown

label

(5-9),

H -receptors specifically in intestinal smooth muscle (4), brain i and a number of peripheral tissues (i0). In all these instances, how-

ever, nothing is mentioned about the modulation of the r e c e p t o r - l i g a n d interaction by those agents known to modify the binding to other receptors, such i as guanyl nucleotides and ions . Here beled with

we describe the characteristics and modulation 3 H-Mep in guinea pig lung parenchima. Materials

i_Pyridinyl-51

While this work ions on histamine

3HI-Mep

(27

of

H -receptors i

la-

and Methods

Ci/mmole,

was in progress, Chang binding in brain ~.

New E n g l a n d

& Snyder

(19)

Nuclear)

reported

0024-3205/81/060705-09502.00/0 Copyright (c) 1981 Pergamon Press Ltd.

migrated

as

an effect

a

of

706

3H-Mepyramine Binding in Guinea Pig Lung

single

band

-water

in thin layer c h r o m a t o g r a p h y

(15:9:6,

The salts

v:v:v).

and EDTA were reagent

Preparation

of membrane

with

on

in

a blow ice-cold

through lung trate

the

and

the

pulmonary

grade

buffer, with

mM

the

same

in

glass-teflon

homogenizer

and

at

15,000

gel

the

EDTA

g

M

Milan,

(500-700

gr)

immediately

buffer.

The

external

15

7.4,

(1:30, nylon

min

the

and

London.

were

stunned

and in

screen.

pellet

with Tris-maleate in 1/8 of the initial volume (S-a mg prot/ml). preparation was made freshly every time, since binding does

placed

perfused

lobes

w:v)

through

acid-

B,

removed,

pH

buffer

&

Italy.

mM,

same

for

from

2

filtered

x

Erba,

were

+

in ethanol-acetic

was

pigs

lungs

50

homogenized

centrifuged

guinea

The

excised,

was

from Carlo

Male

bled.

artery

on silica

dihydrochloride

fraction.

head

Tris-maleate

were

driven

Mepyramine

Vol. 28, No. 6, 1981

of the a

motor

The

fil-

resuspended The membrane not survive

freezing. Bindin$

assay.

Assays

membrane

preparation

various

concentrations

O°C

for

kinetic

mepyramine

of

was

fiber

of ice-cold

was

by

a at

volume

30°C

or

250 ~i.

The

buffer

with

for different times at -5 M unlabeled

of 2 x i0

under

followed

vacuum

by rapid

of

from

filters

a 200 H I

washing

5 to i0 ml did not affect

and counted

of

Tris-maleate

or presence

filtration

with

total in

binding.

CF/C)

extracted

(0.6 ml)

5 min

absence

(Whatman

Washing

in

incubated

for

the

terminated

buffer.

Radioactivity

in

out

was

non specific

filters

with Lumasolve

carried H-Mep

studies,

to calculate

Incubation on glass

were

(50 3~i)

by incubation

aliquot

with 2 x 4 ml

specific

binding.

for 40 min at 40°C

in Lipoluma. Results

Specific

binding

up to 0.8 mg/ml

binding

3

H-Mep

was

linear

with protein

concentration

at least

(data not shown).

Steady-state fic

of

binding

increased

as

a

over

function a

wide

3

of

range

H-M~p

of

is shown

H-Mep

in Fig.

IA. Speci-

concentrations;

similar

results were obtained w h e ~ S n o n sp2~ific~ binding was measured with unlabeled m e p y r a m i n e varying from 10 to i0 M. Analysis of the same data by Scatchard plot

(Fig.

IB)

reveals

In replicate pmol/mg protein, low affinity

two classes

of binding

sites.

experiments K and maximum binding (n,) were respectively,D1for the high affinity class;

binding

sites

were

K

D2

=200-aO0

nM and n :12-90 2

The above mentioned parameters have been obtained r e g r e s s i o n and have a correlation coefficient > 0.85. GTP

5 x i0

The Hill (Fig. 2) gave A number

-5

M did not affect

plot of the data a slope of 0.999. of cations

modified

steady-state relative

to

the amount

binding the

of

3

high

H-Mep

by

5-10 nM and 1-5 values for the

pmol/mg

protein.

unweighted

linear

(data not shown). affinity

bound

binding

sites

at equilibrium

Vol. 28, No. 6, 1981

3H-Mepyramine Binding in Guinea Pig Lung

B

A

6

707

016 I

OJ2 - ~ e

"6 E 4 ¢L v 008

nO

oo ~ .

o

/ 0

10-o

10 -°

10-'

2 4 6 3H-Mep bound (pmole / mg prot)

0

['H'Mep], M

3H-Mepbinding

Specific concentration.

to B: Scatchard

8

FIG. 1 guinea pig lung membranes. A: function of 3H-Mep plot; 0--0: no additions. K and K =8.28 and 180 DI

D2

nM, n l and n 2 = 1.38 and 14.9 pmol/mg prot. @--@: +NaCI 50 mM. KD1and KD2=13.3 and 265 nM, n and n = 0.69 and 8.15 pmol/mg prot. The experiment shown is 1 2 representative of 5 similar ones.

0

.=

.

°1_o4 0 _1

-0.:

o12

'

o.'6

'

;

Log [3H-mepyromine] free, nM

FIG. 2 Hill plot. The line was drawn with least square method (correlation coeff. 0.998). K calculated as antilog of the abscissa value where ordinate value D~ = 0 gives 9.17 riM. Data from the same experiment of Fig. i.

708

3H-Mepyramine

Binding

in Guinea Pig Lung

Vol.

28, No.

6,

1981

180

160 ¸

140 o

L20 =

,oo,

80.

60 ¸

40.

//

7

........

,'0

lion],

FIG.

........

I%0

mM

3

E f f e c t ofd i f f e r e n t 3 c o n c-9 e n t r a t i o n s of NaCI, CaCI2 and MgCI 2 on specific binding. H-Mep was 5xlO M. Values are means of 3 d e t e r m i n a t i o n s + S.E.

TABLE Effect

of Cations

on

3

I H-Mepyramine

Binding

Bound Additions

mM

p m o l / m g prot.

Variations over control

2.32

+ 0.06

1.00

20

0.97

+ 0.07

0.42

20

1.86 + 0.04

0.80

5

8.71 + 0.01

1.60

i00

3.02 + 0.02

1.30

5

3.32 + 0.01

1.43

i00

0.70 + 0.02

0.30

+

Li +

K ++

Ba ++

Ba ++

Mn ++

Mn 3

H-Mep was

5xlO

-9

M. Values

All ions were added

are the mean of 3 d e t e r m i n a t i o n s

as chloride.

3

+ S.E.

H-Nep

Vol. 28, No. 6, 1981

(Table with

I

and

the

II,

order

with

an

indicated

that due

presence

of high

binding

to

in Guinea

Pig Lung

709

3). Monovalent cations reduced steady-state binding, + + + potency Na > Li > K , while divalent ions exhibited a ++ ++ Mg and Ca maximally increased binding at 7 and 1 mM, + maximum inhibition attainable with Na was approximately

apparent K = 13 mM + I Na decreased both

however,

Binding

Fig.

of

biphasic pattern. respectively. The 70%,

3H-Mepyramine

the

low

amount

of

concentrations

(Fig.

3).

Scatchard

affinity

and

analysis

number

of

(Fig.

binding

IB)

sites;

bound radioactivity, data obtained in the + did not permit accurate calculation of

of Na

parameters.

+ The effect of simultaneous addition of Na and a stimulatory concentration ++ of Mg is shown in Table II: the binding parameters of the high affinity site were

intermediate The

as

between

actions

exemplified

of these by the

the same potencies The

in

less

were performed Fig.

4

salts

finding

were that

in the presence

independent MgCI A and

were seen also for NaC~,

association

occurred

those obtained

of

than

3

H-Mep

1 min

(IO-8M)

was

from

nature the

ion alone. of the anion,

same

potency;

and

Na SO (data not shown). 2 4

extremely

(data not shown).

the

MgSO 4 had

NaF,

of either

rapid

at

30°C:

For this reason,

saturation

kinetic

studies

at O°C.

shows

the

association

time-course

3

of

H-Map

in

the

absence

and

presence of 20 mM NaCI. Maximum binding was attained in a p p r o x i m a t e l y 3 and 2 + min, respectively, and Na did not markedly affect the initial velocity of the process. 3 of bound H-Map shows a biphasic nature (Fig. 5); the from the intercept on the ordinate axis) dissociated with

The dissociation major amount (~ 90%, a t

i/2

anger

dly

of a p p r o x i m a t e l y

half-life

NaCI 20 mM dissociating

accelerated

the

(tl/2 did

9.5

not affect component or

dissociation

Interactive

sac,

a small

percentage

Effects

significantly either its percentage. On

of

of

the

slower

( ~ 10%) displayed

K

None + Na i0 mM ++ Mg 1 mM + ++ Na i0 mM + Mg 1 mM is representative

DI

the half-life the contrary,

component

TABLE II 3 Cations on H-Mep

Additions

The experiment chloride.

while

~ i min).

(nM)

(tl/2 ~21

High

n

I

Affinity

(pmol/mg

6.09

2.14

i0.01

1.82

4.23

2.40

8.19

1.92

of

two

similar

ones.

All

of the rapi+ Na markedly

sec).

Binding

prot)

ions

were added as

a

710

3H-Mepyramine Binding in Guinea Pig Lung

Vol. 28, No. 6, 1981

25" /

20' c

°-15-

+

+ 20

mM

i c

I0.

0 m

05-/

0

0 T i m e (mm)

FIG. 4 3 Association time-course of H-Mep (Io-SM) in the absence (0-0) and presence (0-0) of NaC1 20 mM. The experiment shown is representative of 4 similar ones.

I

C8

,--& i °~

-2

_J

0

'

0"30

Ii00

'

I:30 '

'

2'00

Time (min)

FIG. 5 3 D i s s o c i a t i o n time-course of H-Mep (IO-8M) in the absence (0-0) and presence (O-e) of NaCI 20 mM. The experiment shown is representative of 4 similar ones.

Vol. 28, No. 6, 1981

3H-Mepyramine Binding in Guinea Pig Lung

711

Discussion Guinea

pig

lung

has

been

widely

at the level of the respiratory tors

has

been

inferred

used

system.

as

a model

to study histamine

The existence

of both H

and H

role

recep-

I 2 pharmacological studies, using selective agonists in guinea pig lung have been Recently, H I receptors 3

from

and antagonists (2,3). revealed also by direct

binding

studies

with

H-Mep

(i0).

The

validity

of

~2

the

antagonist

mainly

by

The

H-Mep

inhibition

results

classes

as a selective of

of

of blnding

its

this

label

binding

by

investigation

sites

in guinea

for H

receptors

i number

a

show

of 3

that

has been assessed

specific

H-Mep

pig lung parenchima.

drugs

labels

two

(4,10). different

The high and low affi-

nity classes displayed K s in the range of 5-10 nM, and 200-400 nM, respectiD vely. While distinct classes of binding sites had been demonstrated already by

Chang

ferent

et

al.

(i0),

the characteristics

from

the

data

we

present

sites

with

workers, no

a class

sites

binding;

used

they

maleate)

triprolidine

of

the

definitively

mentioned

different

instead

incubation

affected

discrepancies.

As

fact,

seem to be quite

according

to

= 0.9 nM exists in guinea DI -7 of I0 M were revealed. The

K in the order D might reside in the

composition

of such sites In

K

discrepancies rent

with

of

here.

of

buffer

the

way

unlabeled but

below,

Na

pig

lung,

co-

while

for

non

mepyramine.

dif-

and

reason

evaluating

(Na+-K + phosphate

results,

discussed

of

Chang

such

specific

The

instead

diffe-

of Tris-

cannot account for the above + and to a much lower extent

+

K , decreased

or

A

curved

from

the

the steady-state Scatchard presence

interpretation being

almost The

is

existence nature

ted

rapidly,

very

the

identical

biphasic

demonstrated

plot

of

arise

either

classes

one

is

of

demonstrated

different

binding

dissociation

while

sites

curves;

i0% dissociated

more

tempting

to speculate

component

corresponded

to

the

affinity

the

affinity

We

sites

according

modulated

by

steady-state increase the slower

in

high

represented

to the data

demonstrate

by

here

that

the

cooperativity

That Hill

further

that

class

of

approximately

from Scatchard the

is

slowly.

is

of sites,

negative sites.

the

latter

coefficient

binding

supported

90% of the total

it

high

from

binding

with unity.

the

here,

might

multiple

correct

of

of

binding.

of

Even the

amount

the

if it has not been slowly

binding

5-i0%

by

dissocia-

dissociating

sites.

of the

In fact,

total

number

plots. 3

H-Mep

to H

receptors

can

be

monovalent

and divalent cations, but not by STP. i Na + inhibited 3 binding of H-Mep; the mechanism seems to be through a dramatic 3 the rate of dissociation of H-Mep from the component showing

dissociation

rate.

3 On the other hand, divalent cations have a more complex effect on H-Mep binding, being stimulatory at low levels and inhibitory at higher levels. ++

Mg by Na

~pposes

the

decrease

in

affinity

and

number

of

binding

sites

elicited

.

An effect of cations on binding observed: in the case of.~ -adrenergic

to various receptors had been already (11,12) and~3-adrenergic (13,14) recep-

712

3H-Mepyramine

tots, both

only

agonist

agonist

and

Binding

but not

antagonist

antagonist

ceptors

was modified

whether

antagonist

in Guinea Pig Lung

binding

by monovalent

binding

binding

was

to opiate and

affected.

(15)

divalent

to cholinergic

Vol.

and

On

the contrary,

dopaminergic

cations;

receptors

28, No. 6, 1981

(16) re-

it is still

is decreased

debated + (17,

by Na

18). In this tors

in

guinea

however, not

at

onl~

pig

least

by

case of

ions

the

interaction

lung resembles

with

opiate

but

also

of the antagonist

binding

receptors

by

GTP,

to opiate

ion dependence

of

H

receptors,

1 brain receptors

while

this

H-Mep with H

and

dopaminergic

(15), antagonist

of antagonist since

labeled with

3

this

binding

recep1

sites;

binding was affected

has not

been

observed

is not an intrinsic

phenomenon

H-Mep

does

in the

is

of

some

interest

that

tions very close to those present of binding

phenomenon. affected

by

However,

in different

ions it

occur

characteri-

in

guinea

pig

+

Mg

and

Na

were

in extraoellular

in vitro appears

not

(19). ++

It lation

3

H-Mep binding.

The stic

respect,

may

that

reflect the

effective

fluids;

at concentra-

therefore

a physiologically

various

classes

of

the modu-

significant

receptors

are

ways by the same ions. Acknowledsements

The

skillful

technical

assistance

of Mr.

Ermes Tonoli

is gratefully

ack-

nowledged. References i. G.V.

ROSSI,

Histamine

& Scientific

Books,

Receptors,

2. N. CHAND and L. DeROTH, 3. S.S.

(T.O.

New York and London Pharmacology

YEN and W. KREUTNER,

Life Sci.

Yellin

ed.),

pp.

I,

SP

Medical

(1979).

19 185-190 25 507-514

(1979). (1979).

4. S.J. HILL, J.M. YOUNG and D.H. MARRIAN, Nature 270 361-363 (1977). 5. R.S.L. CHANG, V.T. TRAN and S.H. SNYDER, Eur. J. Pharmacol. 48 463-464

(1978). 6. V.T.

TRAN,

6290-6294

R.S.L.

CHANG

and

TRAN

and

7. R.S.L. CHANG, (1979).

V.T.

8. T.T. QUACH, A.M. DUCHEMIN, 60 3 9 1 - 3 9 2 ( 1 9 7 9 ) . 9.

S.J.

S.H.

SNYDER,

Proc.

Natl.

Acad.

Sci.

USA,

75

(1978).

H I L L and J . M .

10.

CHANG, V . T . 437-442 (1979).

11.

H. GLOSSMANN and 67-73 (1979).

S.H. C.

YOUNG, B r . TRAN and

R.S.L.

P.

PRESEK,

ROSE

J.

SNYDER,

Neurochem.

and J.C. SCHWARTZ,

Pharmac.

S.H.

J.

68 6 8 7 - 6 9 6

SNYDER,

J.

1653-1663

Eur. J. Pharmacol.

(1980).

Pharmacol.

Naunyn-Schmiedeberg's

32

Arch.

Exp.

Ther.

209

Pharmaeol.

306

1 2 . H. GLOSSMANN and R. HORNUNG, g u r . J. P h a r m a c o l . 61 4 0 7 - 4 0 8 ( 1 9 8 0 ) . 13. L.T. WILLIAMS, D. MULLIKIN and R.J. LEFKOWITZ, J. Biol. Chem. 253 2989 ( 1 9 7 8 ) . 1 4 . S . J . BIRD and M.E. MAGUIRE, J . B i o l . Chem. 253 8 8 2 6 - 8 8 3 4 ( 1 9 7 8 ) .

2984-

Vol. 28, No. 6, 1981

15. 16. 17. 18.

3H-Mepyramine Binding in Guinea Pig Lung

713

A.J. BLUME, Proc. Natl. Acad. Sci. USA, 75 1713-1717 (1978). T.B. USDIN, I. CREESE and S.H. SNYDER, J. Neurochem. 34 669-676 (1980). J-W. WEI and P.V. SULAKHE, Eur. J. Pharmacol. 62 345-347 (1980). L.B. ROSENBERGER, H.I. YAMAMURA and W.R. ROESKE, J. Biol. Chem. 255 820-

823 (1980). 19. R.S.L. CHANG and S.H. SNYDER, J. Neurochem. 34 916-922 (1980).