Antimuscarinic effects of chloroquine in rat pancreatic acini

Vol. 137, No. 2, 1988

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

June 13, 1986

Pages

664-669

ANTIMUSCARINICEFFECTSOF CHLOROQIJINE IN RAT PANCREATICACINI Yoshiaki Habara, John A. Williams,

and Seth R. Hootman

Departments of Physiology and Medicine, University of California San Francisco, CA 94143 Received April

4, 1986

Chloroquine inhibited carbachol-induced amylase release in a dosedependent fashion in rat pancreatic acini; cholecystokininand bombesininduced secretory responses were almost unchanged by the antimalarial drug. The inhibition of carbachol-induced amylase release by chloroquine was competitive in nature with a Ki of 11.7 uM. Chloroquine also inhibited L3~]~methylscopolamine binding acinar muscarinic receptors. The IC5o for chloroquine inhibition of ts [ H]N-methylscopolamine binding was lower than that for carbachol or the other antimalarial drugs, quinine and quinidine. These results demonstrate that chloroquine is a muscarinic receptor antagonist in the exocrine pancreas. 0 1986 Academic Press, Inc. Rat pancreatic

acini release digestive

enzymes in response to various

Amongthese are acetylcholine

secretagogues. which initiate

their

biological

, cholecystokinin,

responses by binding to specific

the acinar cell plasma membrane. Although it is generally secretagogues interact stimulus-secretion

with distinct

coupling are

polyphosphatidylinositides,

The antimalarial

generally

(8).

(7).

distribution

In evaluating

noted a selective

and include the breakdown of

eimilar

to effect

used for its

the turnover

and action of insulin

effect

ability

of the antimalarial

the binding,

0006-291X/86St.50 Copyrighf 0 I986 by Academic Press, Inc. All rights oj reproduction in any form reserved.

664

acini

secretion,

we

drug on carbachol-

Evidence is here presented that

muscarinic antagonist and that this effect

considered in studies using this agent.

to

of receptors and

in mousepancreatic

of chloroquine on pancreatic

induced secretion in rat pancreatic acini. chloroquine is a specific

calcium and changes

For example, chloroquine affects

the effects inhibitory

accepted that these

the subsequent steps in

drug chloroquine is frequently

other membraneproteins

receptors in

(1-6).

increase lysosomal pH and therefore

intracellular

receptors,

an increase in intracellular

in protein phosphorylation

and bombesin

must be

BIOCHEMKAL

Vol. 137, No. 2, 1986

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

MATERIALS-ANDMETHODS The following acre purchased: HEPES(N-2-hydroxyethylpiperaxine-N'-2ethansulfonic acid), carbachol, chloroquine (7-chloro+[+diethylamino-lmethyl-butylamino]-quinoline) diphosphate salt, atropine sulfate, quinidine HCl monohydrate and soybean trypsin inhibitor type I-S (Sigma); bovine serum albumin fraction V (Miles Laboratory); minimal Eagles amino acids (GIBCO); collagenase (Cooper Biomedical); bombesin (Bachem). quinine sulfate dihydrate (Aldrich Chemical); and [3H]N-methy18copolamine ([3H]NMS), Protosol and Carboxyl-terminal octapeptide of CCK (CCKS) Omnifluor (New England Nuclear). w8s a gift from Squibb. HEPESIbuffered Ringer solution (HR) containing 0.1 mg/ml of soybean trypsin inhibitor and 1.0 mg/ml of bovine serum albumin enriched with minim81 Eagles amino acids and 2 mMglut-amine was used throughout the experiment (9,101. The mediumwas oxygenated with 100% O2 and pH was adjusted to 7.4 with NaOH. Pancreatic acini were prepared as described previously (9). Briefly, a fasted male Sprague-Dawley rat wes exsanguinated after cervical dislocation and the whole pancreas was removed, trimmed of excess fat and lymph nodes and injected with 5 ml of purified collagenase solution (75 U/ml HR). Injected glands were incubated for two 30 min periods with 5 ml of the collagenase solution for a total of 60 min at 37'C with sheking. The tissue was then dissociated mechanically by pipeting through a siliconized pipet. Isolated acini were collected after filtrating through a 100 Urnnylon meshand were rinsed twice with HR. Acini were preincubated for 30 min at 37'C prior to measurementof amylase release. Acini were stimulated with carbachol, CCKSor bombesin in the presence or absence of chloroquine for 30 min at 37'C. Amylase released into the incubation mediumwas analyzed by the method of Jung (11). The release over 30 min W8Sexpressed as a percentage of the initial acinar amylase content. The ability of chloroquine to interact with pancreatic acinar musarinic receptors ~8s characterized using the specific muscarinic antagonist [ 3H]NMS (85 Ci/mmol) 86 described in a previous report (IO). Acini were incubated with 100 PM [3~1~~~ in a total volume of 5 ml of HR in the presence of varying concentrstions of several substances including atropine, chloroquine, Carb8Cho1, quinine and quinidine for 120 min at 37OC. The binding reaction was then stopped by pouring the mediumonto WhatmanGF/A glass fiber filters in 8 v8cuunl filtering manifold and rinsing three times with 5 ml of chilled 0.9% NaCl. Filter8 were placed in scintillation vials, extracted for several hours in a cocktail consisting of 10% Protosol in Omnifluor, and counted for radiO8Ctivity in 8 Packard Tri-Carb liquid scintillation spectrometer at efficiencies of 45-50s. RESULTS --Effect

of choroquine --on the secretory At high concentrations,

release but this effect The most noticeable inhibition

chloroquine slightly

was not statistically

effect

induced amylase release was first

significant

amylase release. noted at

8

In contrast

response was not significantly

1).

and dose-dependent

Inhibition

of carbacholof

30

being almost completely

to carbechol,

affected 665

(P > 0.05) (Fig.

chloroquine concentration

the response decreased sharply,

abolished at 1 mMchloroquine. secretory

decreased basal amylase

of chloroquine was a selective

of carbachol-stimulated

uM and thereafter

response --in acini

the CCK8-induced

by chloroquine except for a

Vol. 137, No. 2, 1986

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

24-

20-

8

16-

Jp i?g 8

12z

Q E

a

4-

02

o-

0

0

10.5

3x10.5

1

10.4

Chloroquine

Fig.

Effect

1.

3x10-4

of chloroquine

pancreatic eclni. (A)

or

10-3

(M)

0

lo-7

10.6

on maximally

(m)

lo-4

10.3

Chloroquine

(IA)

(M)

amylaserelease in ret uMcarbechol (a), 300 pM CCKs

stimulated

Acini were stimulated by

3 nM bombesin

10-5

Carbachol

10

for 30 mfn at 3'7'C in the absence or presence

of

varying concentrations of chloroquine. Opencircles indicate base1release in the absence

of secretagogue.

the Initial

acinar amylaaecontent.

Amylaee release

in uhich

duplicate

measurementa

Fig.

A) Effect

of chloroquine

2.

was expressed

a8 a percentage

of

Values are the mean+ SE of 3 experiments

were performed. carbachol-induced

on

amylase release.

Acini

were stimulated with varying concentrations of carbachol in the presence of chloroquine (0, 30, 100, 300 uM). Values which duplicate measurements were carried

are the mean of 3 experiments in B) Schild plot of data for out. ratio (DR) taken of amylaeerelease. Values for dose

chloroquine inhibition from (A).

small decrease at millimolar bombesin, by contrast, concentrations concentration

was inhibited

slightly

and this small inhibition range of chloroquine tested.

of chloroquine concentration To further

characterize

to the right

chloroquine concentration

Thus neither similarly

maximal

of chloroquine,

release,

a kinetic

The carbachol dose-response for amylase

in a concentration

dependent manner when

was increased from zero to 300 uM. The Schild plot

This data indicates a competitive

amylase

dose-response

action of chloroquine,

obtained from these results showed a linear relation (Fig. 2B).

CCKS-nor bombesin-

to carbachol.

carbachol'induced

the inhibitory

experiment was performed (Fig. 2). release was shifted

2

at low chloroquine

persisted unchanged throughout the

stimulated amylase release was inhibited

Effect --

The response to

chloroquine concentration.

yielding

a Ki of

modeof inhibition.

which was obtained at 3

uM

11.7

vM

However,

carbachol in the absence

decreased at the highest chloroquine concentration

tested.

BIOCHEMICAL

Vol. 137, No. 2. 1986

oJ , , 10-10 10-0

AND BIOPHYSICAL

,

1 lo-’

10-a

hug

RESEARCH COMMUNICATIONS

,

,

t

,

I

10-a

lo.5

10-d

10-s

10-Z

concentration

(M)

substancesof [~H]NMS binding to aclni.

Inhibition by various were incubated in HR containing

Fig. 3.

100

pM [3H]NMS for

120

min

at

37’C

Acini the

41

presence of varying concentrations of atropine (a), chloroquine (m), carbachol (A), quinine (0) and quinidine (a) and plotted as a percentages of IlWlXiLlKXU. Values are the mean of 3 experiments in which duplicate measurements were carried

out.

This non-competitive

inhibition

may be related

to the small inhibition

other secretagogues observed at the highest concentrations

--Effect

of chloroquine.

of chloroquine z L3H]NMSbinding Since a selective

by

of

inhibition

of the carbachol-induced secretory

chloroquine was found, a direct

antimalarial

study of the inhibitory

effect

drug on muscarinic antagonist binding was carried

in Fig. 3, chloroquine competitively

inhibited

[3~]~~~ binding.

of the

out.

As shown

The IC50 for

chloroquine inhibition

(10

than that for atropine

(1.5 nM), though it was lower than that for carbachol

(50 vM).

Specific

In addition,

and structurally

these cinchona alkaloids but with inhibitory

seven thousand times higher

[%]NMS binding to isolated acini was completely inhibited

by 1 mMchloroquine. antimalarial

IJM)

was approximately

response

higher IC50’s

we tested the effects

of the other

related drugs, quinine and quinidine.

showeda similar than chloroquine.

inhibitory

effect

Both exhibited

Both of

on [~H]NMS binding

almost identical

effects. DISCUSSION

This study demonstrates that chloroquine is a muscarinic antagonist the exocrine pancreas.

Carbachol-stimulated 667

amylese secretion

in

is selectively

Vol. 137, No. 2. 1986

inhibited

BIOCHEMICAL

RESEARCH COMMUNICATIONS

by chloroquine over the range of IO PM to 1 mM.

curve for carbachol stimulation

a straight

line.

to the right

by the antimalarial

a specific

muscarinic antagonist,

drug over the range of

1

uM to

has been stated that the presence of at lea& one positively

nitrogen

ion in cholinergic

muscarinic receptor

(12).

charged

agonists is essential for binding to the However, when considering the structures

possession of one or more benzene(s). scopolamine, QNB(Fquinuclidinyl

of a

biological quinoline

change after

response. in its

benzilate)

ligand-receptor

and propylbenzilylcholine

binding required to ellcite

Thus, it is not surprising

drugs, quinine and quinidine,

binding in pancreatic

acini,

from a structural

which is also used to treat

also inhlbited

but with reduced potency. cardiac arrhythmia,

a

nitrogen and a

point of view that chloroquine could act as a muscarinic antagonist. other antimalarial

mustard

(13) which may prevent the

Chloroquine also possesses an ionizable

structure.

the

In may cases drugs such as atropine,

have one or more benzene rings and/or quinoline conformational

mM.

1

of muscarinic antagonists , they reveal a second commonfeature,

series

by

and a Schild plot of this data generated

The binding of [%I]NMS,

was also inhibited

The dose-response

of amylase release was shifted

increasing chloroquine concentration

It

AND BIOPHYSICAL

The

[3H]NMS

Recently quinidine,

has been shown to block

r3H]QNB or [3H]4-NMPB binding to muscarinic receptors of cardiac muscle and serveral brain regions (14-17).

Both of the cinchona alkaloids,

is a stereoisomer of the other, possess a very similar chloroquine. inhibit

Therefore,

pancreatic

both alkaloids

exocrine secretion

induced by cholinergic

drugs.

However,

except at very high doses and

but also CCKS-and bomeain-induced

amylase release (data not shown). Thus among the drugs tested, chloroquine revealed a selective

to

might also have been expected to

quinine and quinidine were not inhibitory decreased not only carbachol-induced

structure

one of which

inhibitory

effect

only

on muscarinic receptor-

mediated amylase release at lower concentrations. Since chloroquine is known to alter used for investigating

regulatory

lysosomal functions,

mechanismsof cellular 668

It is now widely

receptors.

For

Vol.

137,

example, complexes

chloroquine

inhibits

since However,

alkaloids. antimuscarinic

studies

the process

As an antimalarial

(8,18).

therapeutically

receptor

BlOCHEM~CALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS

No. 2. 1986

effect

it

causes

drug,

fewer

as demonstrated of this

of degradation chloroquine

side effects

of insulin-receptor is often

than other

in the present

drug must be taken into

studies,

used

cinchona the direct

consideration

both in

and therapeutics. ACKNOWLEDGEMENT

This work was supported by NIH grant AM32994 and an NIH International We also thank Margaret Brown and Research Fellowship (No. G50111 to Y.H.). Phyllis Munowitz for their technical assistance. REFERENCES 1. 2.

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