Vol. 81, No. 3, 1978 April 14,1978

BIOCHEMICAL

AND BlOPHYSlCAl

RESEARCH COMMUNICATIONS

Pages 710-717

MUSCARINICBINDINGTD MOUSEBRAINRECEPTOR SITES Yoel Kloog and Mordechai Sokolovsky The George S. Wise Center for Life Sciences Department of Biochemistry, Tel-Aviv University Tel-Aviv,

Received

February

10,

Israel

1978

In mousebrain the binding of C3H]-Atropine to the muscarinic receptor seemsto be a simple mass-action determined process as gauged both by approach to equilibrium kinetics and binding at equilibrium. In contrast, using isotopic dilution technique,dissociation measurementsindicate the existence of two receptor-ligand complexes. It would appear that association and dissociation rates of binding of the muscarinic antagonists atropine, scopolamine, N-methyl-4-piperidyl benzilate (4NMPB)and 3-quinuclidinyl benzilate (QNB) decrease with increasing affinity based on comparisons of kinetic binding data. The differences between the association rate constants are small whereas those between the dissociation rate constants differ markedly. This kinetic behavior is similar to the well-known time profile of antimuscarinic activity in isolated tissues. These phenomenaare discussed in terms of possible isomerization of the receptor-ligand complex, as has been proposed recently for [3H]-scopolamine and c3H]-4NMPBbinding. Atropine generally behaves as a competitive at muscarinic receptors;

its onset and offset

antagonist of acetylcholine

of antagonism are slow. It has

been suggested that the rates of onset and offset of atropine blockade are determined by the rates of binding to and dissociation from the muscarinic receptor

(l-3). This communication describes studies on the binding properties The main of the muscarinic receptors of mousebrain using C3H]-atropine. features of the kinetic behavior of the system are reported here and compared to those of its direct analogue scopolamine and the highly potent antagonist N-methyl-4-piperidyl benzilate (4NMPB)(4-6). EXPERIMENTAL C3H]-atropine 2.6 Ci/mmole, [3H]-scopolamine 1.7 Ci/mmole and C3H]-N-methylpiperidyl benzilate (4NMPB)6 Ci/mmole were prepared by a catalytic tritium exchange as described elsewhere (7). The chemical and radiochemical purity was determined by analytical thin-layer chromatography (Merck Silica 60 plates, 0.25 mmthickness) in two solvent systems: n-butanol, acetic acid, water (4:1:1), and chloroform, acetone, diethylamine (5:4:1). c3~] drugs moved as a single peak, identical to the authentic, unlabeled compoundin these two systems. The purity was C3H]-atropine and [3H]-scopolamine > 99%, and c3H]-4NMPB> 97%. Male ICR mice (20-25 gr) were decapitated, brains were removed (within 2 minutes) and homogenized in ice cold 0.32M sucrose using a motor-driven 0006-291X/78/0813-0710$01,00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

710

Vol. 87, No. 3, 1978

BIOCHEMKAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

teflon pestle (950 rpm) in a glass homogenizer to yield a 10%homogenate (w/v). The entire homogenatewas centrifuged for 10 min. at 1OOOxg(Sorvall RCZ-B), the pellet discarded and the resultant supernatant fluid (Sl) was used for assays of binding as described elsewhere (4, 5). 50 ~1 of S1 fraction were incubated at 25' in 2 ml modified Krebs Henselite solution (25 mMTris HCl instead of bicarbonate), pH 7.4, containing the labeled ligand. After various times of incubation ice cold Krebs solution (3 ml) was added and the contents were passed rapidly through a glass filter (GF/C, Whatman25 mmdiameter) by suction. The filters were washed 3 times (3 ml of ice cold Krebs solution). All procedures were completed within less than 10 sec. and all binding experiments were performed in triplicate together with triplicate samples containing 5x10m5Mof unlabeled ligand. The filters were placed in vials containing 5 ml of scintillation liquid (33 ml Triton X-100, 66 ml toluene, 5.5 gr PPO (Packard) and 0.1 gr dimethyl-POPOP (Merck), maintained at 25O for 30 min. and the radioactivity then assayed by liquid scintillation spectrometry (Packard Tricarb model 2002, 31%efficiency). Specific binding is defined as the total minus the non-specific binding i.e., binding in the presence of 5~10-~M of unlabeled ligand. Approach to equilibrium kinetics were analyzed by assuming a simple bimolecular reaction. A best fit calculation method was employed and curvefitting was carried out with a CDC6400 computer using program D506 (MINUIT) from the CERNcomputer 6000 series, program library I. The observed time course of dissociation was fitted to the sumof two first order exponentials, pertaining to fast (k-2) and slow (kel) steps using a best fit calculation method (5). RESULTS The binding of C3H]-atropine at 25' was studied as described recently for t3H]-4NMPB and [3H]-scopolamine (5) using S1 fraction of mousebrain homogenate. The binding capacity of the preparation for these drugs is very similar: 1.55, 1.68 and 1.62 pmole/mg protein for C3H]-atropine, r3H]-4NMPB and [3H]scopolamine respectively when calculated at the present conditions.* Equilibrium

binding studies were performed at a range of concentration

from 0.25 to 20 nM, shown in Fig. 1 for specific and non-specific binding of C3H]-atropine. Specific ligand binding (Fig. 1, upper) exhibits the hyperbolic shape typical for saturation phenomena(half saturation at 1.3 nM ligand). Non-specific binding is much lower and varies linearly with ligand concentration (Fig. 1, lower). The binding data when replotted in a double reciprocal form gave a straight line (Fig. 1, insert), therefore [3H]-atropine binding reflects a single population of binding sites at the concentration range investigated. Binding was investigated further the time course of association of r3H]-atropine tions, corrected for non-specific binding. The fitting, assuming a simple bimolecular reaction

kinetically. Fig. 2 shows at three ligand concentrasolid lines represent curve scheme. The samekinetic

parameters k,=2.9x108M-' min-1 and kWl=0.45 min-' accomodate the association data at all C3H]-atropine concentrations investigated. The dissociation * In our previous work we determined the binding capacity to be 0.62 and 0.68 pmole/mg protein for c3H]-4NMPBand [3H]-scopolamine respectively for those experiments;

711

Vol. 81, No. 3, 1978

Fig. 1.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Binding of C3H]-atropine at 25“ as a function of concentration. 0.05 ml S1 samples were incubated with varying concentrations of [3H]-atropine for 10 min. at 25' in 2 ml Krebs solution (pH 7.4). Lower curve: nonspecific binding (in the presence of SX~O-~M atropine). Upper curve: specific binding. Each point is the mean of triplicate samples whose standard error was less than 5%. Insert: Double reciprocal plot.

1

1

I

I

I

I

I

2

4

6

8

IO

12

Time

Fig. 2.

,

u

tmin)

Time course of C3H]-atropine binding at 25'. S1 samles (0.05 ml) were incubated in 2 ml modified Krebs solution with P3H]-atropine for various periods of time at 25'. Specific binding was determined as described in "Method9'. Each experimental point is the mean of triplicate sampleswhose standard error was less than 5%. The kinetics were measured with C3H]-atropine at the following concentrations: (o-•)0.75 nM, (o-0)1.0 nM, (A-A)l.5 nM.

712

Vol. 81, No. 3, 1978

8lOCHEMlCAL

AND 8lOPHYSlCAL

I

I

I

I

I

2

4

6

0

10

Time

Fig.

3.

constant

tmin)

deduced

from the ratio

at equilibrium

(1.5

k-,/k,

is

very

the dissociation

of [3H]-scopolamine

is

of C3H]-atropine also

[3H]-scopolamine

and C3H]-4NMPB

from

The data

linearity.

can,

similar

to the

Kd value

as

nM and 1.3 nM respectively).

An isotopic dilution technique is suitable dissociation of ligand-receptor complex. Fig. for

I

'12

Dissociation of C3H]-atropine and [3H]-scopolamine at 25’. S1 sam les (0.05 ml) were incubated with 6.0 nM(o-p), 0.6 nM(A-A) for 10 min. and 3.7 nM [3H]-scopolamine c3H!i - atropine (o-o) for 30 min. in 2 ml modified Krebs solution. 5x10m5M unlabeled ligand was then added and the samples were filtered immediately (zero times) , and at the times indicated. Specific C3H]-drug binding was determined as described in text. Each experimental point is the mean of triplicate samples whose standard error was less than 5%.

determined

plot

RESEARCH COMMUNICATIONS

given

and the for

at 25“ however,

713

to follow

same plot

comparison.

(5) the be fitted

the

course

3 demonstrates

first

for plot

of

first

order

dissociation

As in the order

with

the

case of

deviates

the sum of two first

Vol. 81, No. $1978

order

exponential

order

rate

terms,

constants

corresponding

with

viz:

for

double

were

thus

to trapped,

al=0.35

than

uptake complex

it

with

the

structures”

reaction this

(8)).

the

first

min-’

of “closed

was terminated

results

in osmotic

In these

was similar

seems that

labeled

the

and a2=0.65.

buffer; system

where

k-,=0.13

The dissociation

rather

[SH]-CABA

experiments;

attributed

sites

min-l,

due to the presence

of ligand-receptor

original

exp(-k-2t),

are k-,=2.87

performed.

water

by the

dissociation

exp(-k-lt)+a2

artifacts

distilled

(as confirmed

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of binding

possible

experiments

al

atropin

fractions

To avoid control

8lOCHEMlCAt

to that

biphasic

shock

experiments measured

the in the

dissociation

cannot

be

ligand. DISCUSSION

The binding

of C3H]-atropine

equilibrium

can be described these

first

sight

ments

showing

(Fig.

3).

dict

results

the

are not

the possibility

receptor

should

Fig.

complex with

ligand

complex

observed

the possibility

conditions

used here

solely

its

Here receptor step

sequence

(R) involves would

the

sites

rates

and R*L forms

the

the

(where

only

kl=2.9x10*M-1

had been reached, It

worth

adding

association

and dissociation entrophy

in macromolecules.

binding

of c3H]-4NMPB

is

Compared

after

the

estimation

frequently

and [3H]-scopolamine

714

min-l).

proposed

is of

On the other the RL

reactions

of the

associated

this

between

backward

to

the associa-

formation

entrophy

4NMPB yielded

to the model

combined (L)

and that Thus,

equilibrium

of e.g.

the

to the

and kml=0.45

hence,

be monophasic. for

(RcR*L)

leading

reflect that

reactions value

changes

would

the

of [3H]-atropine

reactions.

measured here

should

step

min -’

of dissociation,

Under

by ligand;

can account

on the reactions

seems to

se.

occupied

binding

an isomerization

RL and R*L. Such a positive

R+L&m*L that

eliminating

of receptor-

per

dissociation

is

after

thus

technique

fully

there

As shown in

dissociation

association-dissociation

depend

is

are

if

complex

dilution

to contraof ligand-

of occupancy.

cooperativity

cooperativity,

reaction

than

the RL complex hand,

receptor

it is assumed tacitly

much slower tion

an isotopic

sites

Moreover,

of dissociation

The biphasic

At

measure-

appear

or 6.0 nM are similar,

of negative

on negative

The minimal data.

using

the

sites.

the degree

process.

of binding

kinetics

the rate

and at

dissociation

of [3H]-atropine-receptor

of heterogeneiety.

contradict based

with

rate

the

binding

then

0.6 nM C3H]-atropine

the possibility

with

determined

populations

of association

sites,

change

3, the dissociation

incubation

in keeping

of two preexisting

of binding

to equilibrium

mass-action

of two different

the measurements

a heterogeneiety

by approach

by a simple,

existence

Indeed,

measured

of both from

the

AS= +40 eu.

with

conformational

recently

(4, 5) the present

for

one is

the

8lOCHEMlCAL

Vol. 8 1, No. 3, 1978

simplified.

In the

former

type

differences

between

suggests

itself

account

for

their

of a hydrogen It

transformation

binding

structural

different

binding

be noticed

that

consistent

with

a combination Moran that

the

quite

that

with

the

rate

of the bind

labeled

of loss

that

muscarinic

antagonists

tions

isomerization

enables

tion

from

This

obviates

isolated

the direct

whereas

lead

conclusion

various

study sites

studies

of labeled

antagonists

antagonist

is

applied

with

out.

and

correlate

They suggest receptor

here

is

proper

in excellent

prveiously

(10-16).

in subcellular

bind

seem to support

population

Most

preparations

most of the agonists

experiments

the

of binding

ligand-receptor

receptor-ligand

muscarinic

to conclu-

sites.

interactions

The

rate

minimal,

that

if

any,

bath,

dissociation equilibrium

than

to and dissocia-

limitations

of access. with

(9 and references measured

those

therein).

and atropine

(Table

by direct

of antagonism,

binding where

the

(2, 6).

2. Increasing affinity and association rates (Table 1) e.g. at 0.6nM

715

prepara-

when working

constants

are much greater

to reach

of association

of antagonism

1. kinetic

may thus

to subcellular

of QNB, 4NMPB, scopolamine

to an organ

decrasing

complex

encountered

rates

(17-20).

interactions.

antagonists

of their with

and measuring

required

reported

studied

many of the difficulties binding

time

observed

of the muscarinic-receptor-ligand

The kinetic

correlates

for

of membrane

the receptor

to the

be ruled

by BHM is reversible

reported

to a homogeneous

of labeled

tissues

cannot

of atropine, scopolamine and 4NMPB on the other hand by a simple mass-action process.. Biphasic dissociation

be a common feature The binding

behavior

site.

atropine

Our kinetic

have been described

The slow

bimo-

at the moment

the acetylcholine

studies

site

bind

biphasic dissociation cannot be explained rates

for

binding

antagonists

of these

This

that

from the tissue.

one being

value

(15,16).

biphasic.

of the response

or regulatory

high-affinity

sites

are

be emphasized

of ligand

of action,

equilibrium

to a single

multiple

formation

seems to be a simple

of response

of recovery

constant

with

the

on BHM (PhzCHO CHzCHzNMe CH2CH2Cl) QP rat

an allosteric

The binding agreement

might

of all

and isomerization

inactivation

rates

BHM has two sites

and the other

the

the

two distinct

well

that

(9) working

observed

possibility

including pattern

reactions should

interactions

structural

change.

binding

association

It

models.

of allosteric

and Triggle

jejunum

sion

either

slight

epoxy group)

perhaps

kinetic

drugs is similar, i.e. whereas dissociation reaction,

lecular

(the

conformational

the

of R*L in a non-

and scopolamine,the

features

antimuscarinic is

are only

difference

bond and concomitant

should

there

of atropine

this

dissociation

of R* to R, to result

Since

curve.

the molecules

that

RESEARCH COMMUNICATIONS

scheme we had to assume rapid

to R* and L and irreversible Michaelis-Menten

AWD BlOPHYSlCAL

ligand

is

40 min.

for

QNB

1)

Vol. 81, No. 3, 1978

TABLE I.

Btoctwc~~

KINETIC PARAMETERSFOR BINDING OF MIJSCARINIC LIGANDS

association k,(M-'

ligand

AND BIOP~~YSICAL RESEARCH ~~MMUNKATION~

min-')

dissociation experiments

experiments k-l(min-l)

k-I/kl(nM)

t,I

@inI

QNB a

2.0x108

0.012

0.06

57

4NMPB

2.3x108

0.08

0.34

12

Scopolamine

2.2x10*

0.15

0.68

10

Atropine

2.9x10*

0.45

1.55

2 b

a

Data taken

from Ref.

14 measured at 3S0 in rat brain

preparation

(at 35'), 23 min. for scopolamine (25') and about 9 min. for atropine (25'). Such correlation was also established for muscarinic antagonism (1, 6, 21). These data suggest

that

although

there

are factors

other

than association

and dissociation from the receptors which determine the rates of onset and offset of antagonism, the events at the receptor sites are dominant. Similar conclusions

have been reached recently

by Bolton

(22) by a different

route

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

Paton, W.D.M. (1961) Proc. Roy. Sot. Lond. B 154, 21-69. Paton, W.D.M. and Rang, H.P. (1965) Proc. Roy. Sot. Lond. B 163, l-44. Rang, H.P. (1966) Proc. Roy. Sot. Lond. B 164, 488-510. Kloog, Y. and Sokolovsky, M. (1977) Brain Res. 134, 167-172. Kloog, Y. and Sokolovsky, M. (1978) Brain Res. in press. Rehavi, M., Yaavetz, B., Kloog, Y., Maayani, S. and Sokolovsky, M. (1978) Biochem. Pharmacol. in press. Kalir, A., Maayani, S., Rehavi, M., Elkavetz, R., Pri-Bar, I., Buchman, 0. and Sokolovsky, M. (1978) Eur. J. Med. Chem. in press. Redburn, D.A. and Cotman, C.W. (1974) Brain Res. 73, 550-557. Triggle,D.J. (1976) In: Chemical Pharmacology of the synapse. (Triggle,D.J. and Triggle,C.R. Eds) pp. 233-416, Academic Press, NY. Alberts, P. and Bartfi, T. (1976) J. Biol. Chem. 251, 1543-1547. Beld, A.J. and Ariens, A.J. (1974) Eur. J. Pharmacol. 25, 203-209. Farrow, J.T. and O'Brien, R.D. (1973) Mol. Pharmacol. 9, 33-40. Schleifer, L.S. and Eldefrawi, M.E. (1974) Neuropharmacol. 13, 33-63. Yamamura, H.I. and Snyder, S.H. (1974) Proc. Nat. Acad. Sci. 71, 1725-1729. Birdsall, N.J.M. and Hulme, E.C. (1976) J. Neurochem. 27, 7-16. Massoulie', J., Godwin, S., Carson, S. and Kato, G. (1977) Biomedicine 27, 250-259.

716

to

Vol. 81, No. 3, 1978

17. 18. 19. 20. 21. 22.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Maelicke, A. and Reich, E. (1976) Cold Spring Harbor Symposia on Quantitative Biology Vol. XL, 231-235. Weber, M. and Changeux, J.P. (1974) Mol. Pharmacol. 10, 15-34. Raftery, M.A.J., Schmidt, J., Clark, D.G. and Wolcott, R.G. (1971) Biochem. Biophys. Res. Comm.45, 16-22. Cuatrecasas, P.and Hollenberg, M.D. (1975) Biochem. Biophys. Res. Corn. 62, 31-41. Inch. T.D., Green, D.M. and Thompson, P.B.J. (1973) J. Pharm. Pharmac. 25, 359-370. Bolton, T.B. (1977) Nature, 270, 354-356.

717