Cholinergic effects of histamine-H2 receptor antagonists partly through inhibition of acetylcholinesterase

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  • GastroenterologiaJaponica Vol. 21, No. 3 Copyright 9 1986 by The Japanese Society of Gastroenterology Printed in Japan

    ~Or ig ina l Ar t i c lew



    Mitsuru AON0, M.D.*, Motoyuki MORIGA, M.D.**, Kazuhiko MIZUTA, M.D.** and

    Hiroaki NARUSAWA, M.D. * *

    9 Department of Clinical Nutrition and **First Department of Internal Medicine, Kyoto University School of Medicine, Kyoto 606,Japan


    The effects of histamine Hz-receptor antagonists on acetylcholinesterase and pseudocholinesterase ac- tivity were studied. All H tested inhibited both enzyme activities dose-dependently. The potency of inhibitory activity of H~-antagonists on acetylcholinesterase estimated from median inhibi- tory dose were in the following order of decreasing activity: ranitidine > TZU-0460 > cimetidine > YM- 11170, whereas that on pseudocholinesterase were TZU-0460 > ranitidine > cimetidine > YM-11170.

    As the effects derived from the inhibition of acetylcholinesterase by H2-antagonists may affect intesti- nal motility, we studied ileal muscle contractions. Ranit idine had the most potent stimulating effect on contraction, the pattern of which was similar to physostigmine and was blocked by atropine and mor- phine. YM-11170 had a weak action on muscle contraction and cholinesterase activities.

    Key Words: A cetylcholine, Acetylcholinesterase, H2-antagonist, Guinea-pig ileum.

    I n t roduct ion

    Several histamine H2-receptor antagonists

    have been developed and used clinically for the treatment of peptic ulcers. They exert powerful

    inhibitory effects on gastric acid secretion in

    animals and humans. They usually have no

    effects on gastrointestinal motil ity, however, it

    is possible that some of them may affect the

    gastric emptying and the lower esophageal

    sphincter pressurel-S). Bertaccini et al.1) have

    reported that ranit idine exerts cholinergic-l ike

    Received October 11, 1985. Accepted January 13, 1986. Address requests for reprints to: Mitsuru Aono, M.D.,

    Department of Clinical Nutrition, Kyoto University School of Medicine,'Sakyo-ku, Kyoto 606, Japan.

    This study was supported in part by grant from the Japa- nese Ministry of Education, Science and Culture (No. 59570300).

    effects. Ranit idine provokes atropine-sensitive

    contraction of smooth muscle preparat ions of

    several different animal species. There are

    some reports4-O that cimetidine and ranit idine

    inhibit human erythrocytes and gastric acetyl-

    cholinesterase (ACHE) as well as human serum pseudocholinesterase (PChE).

    In the present study, the effects of Hg_-

    antagonists on AChE and PChE were ex-

    amined. We compared the effects of new H2- antagonists, TZU-04607) and YM-11170s,*),

    with those of cimetidine and ranit idine. From

    the result of ACHE, the effects of H2-antago-

    nists on contraction of the ileal muscle prepara-

    tion were investigated.

    Mater ia ls and Methods

    Cholinesterase assay

  • 214 M..40NO ET AL. Vol. 21, No. 3

    Human erythrocyte AChE was obtained from heparinized blood. The erythrocytes were washed 5 times with saline and finally resus- pended in distilled water for hemolysis. Assays were performed after dilution in 0.1 M phos- phate buffer, pH 8.0. Human serum was used for PChE assay.

    Cholinesterase activities were measured at 25~ by a spectrophotometric method using acetylthiocholine as a substratel~ In the stan- dard procedure, to 2 ml of enzyme solution in 0.1 M phosphate buffer, pH 8.0, 0.8 ml ali- quots of 0.33 mM 5,5'-dithiobis-(2-nitro- benzoic acid) (DTNB) in 0.1 M phosphate buffer, pH 7.0 with or without 0.013 mM pro- fenamine (Yoshitomi Pharmaceutical, Osaka) as a PChE inhibitor were added. After a 15- rain preincubation at 25~ the reaction was started by the addition of 0.2 ml of 1 mM acetylthiocholine. The enzymatic reaction was determined by the increase of absorbance at wave length 405 nm in a photometer equipped with a chart recorder.

    Experiments with histamine-Hz-antagonists and other drugs were carried out by adding 0.5 ml of drug solution that had been diluted with 0.1 M phosphate buffer, pH 8.0, to 1.5 ml of enzyme solution. Ileal muscle preparation

    Male guinea pigs, weighing 300 to 400 g, were killed by a blow on the head, and the ileum was isolated and placed in Tyrode solu- tion. Tissue specimens about 3 cm in length were suspended in a 10-ml organ bath with Tyrode solution kept at 32~ and gassed with 95% 02 and 5% CO2. The Tyrode solution had the following composition (per liter): NaC1, 8.0 g; KCI, 0.2 g, CaC12, 0.2 g; MgCI2, 0.1 g, NaHzPO4, 0.05 g; NaHCO3, 1.0 g and glucose, 1.0 g. Response to drugs were re- corded isotonically under a tension of 1.0 g.

    In some experiments ileal longitudinal muscle was gently separated from the under-

    lying circular muscle. The dissected muscle strip was stimulated electrically by the method of Patonal). The electrodes were made of plati- num and an intraluminal electrode was used as the anode. Rectangular pulses of 0.1 msec duration were used at a frequency of 0.1 Hz and at a voltage of 5 V. Agents used

    Acetylcholine chloride (Daiichi, Tokyo), morphine hydrochloride (Dainippon, Osaka), physostigmine sulfate (Wako, Osaka), hexa- methonium chloride (Nakarai, Kyoto), beta- zole hydrochloride and chlorphenylamine malate (Shionogi, Osaka), diphenhydramine hydrochloride (Tanabe, Osaka), atropine sul- fate and pyrilamine malate (Sigma, St. Louis, MO) were used. Cimetidine was a gift from SKF Fujisawa, Tokyo. Ranitidine was a gift from Glaxo Japan, Tokyo. YM-11170 was a gift from Yamanouchi, Tokyo. TZU-0460 was a gift from Teikokuzoki, Tokyo. The chemical structure of Hz-blockers used are shown in Fig. 1.



    I H~N N--CmN

    Ranit, idine ,-CH=SCH=CH=NHCNHCH= CHNO=

    ~NSO=NH= /==~CH=SCH=CH=C,~.NH

    YM-11170 ~ 'N ' (Famot.idine)

    N=N~ ~N H=N/C

    Fig. 1. The chemical structure of the histamine Hz- receptor antagonists used.

  • June 1986 Cholinergic Effects of H2-blockers 215


    Cholinesterase assay Cimetidine inhibited both AChE and PChE

    activities in a dose-dependent manner. The

    median inhibitory dose (ID-50) was 6.3 x 10-4 M for erythrocyte AChE and 2.6 x 10-3 M for serum PChE shown in Fig. 4. In order to deter- mine the type of cholinesterase inhibition by cimetidine and inhibition constant, saturation experiments with increasing substrate concen-

    Fig. 2.

    trations were carried out. The double recipro- cal plots obtained from the experimental data were linear in all cases examined, as shown in Fig. 2. Both Km and Vmax were affected by the agent. These results are consistent with an inhibition of the mixed type, which presents characteristics common to both competitive and non-competitive inhibition. The inhibitory constants (Kt) were 1.5 x 10-4 M for AChE and 1.9 x 10 -4 M for PChE, as calculated by Dixon-plots. Fig. $ shows that histamine-


    Z/v 6.6 lO'4N CIMETIDoIflE DOSE 1/v 6.6 10"4M ~, IO-W~ 1 xlO'r 3. xlO'4N 1'6 10- M

    IDIHE( ) 6 10"5M

    10" 100

    INE( ) 5 j

    / - / ~ / Ii " i ! I 1/10-3 1/10-~ 1/2.5x10-5 1/10-] 1/10-4 1/2.5x10-$ Iv[ "1

    (l/S) (l/S)

    Inhibition of erythrocyte AChE (left) and serum PChE (right) by cimetidine: double reciprocal plots. Acetylthiocholine in a range of concentrations (S): 0.025-1 mM; was used. V=A absorb- ance/min. The concentration of cimetidine are given at the range from 6.6 x 10- ] to 6.6 x 10- 2 mM. The points on the graph represent the mean values of 3 separate experiments performed in duplicate.

    A I-r

    50 -

    Fig. 8.

    loo - o - -~ t r ~ ~ - ; ~ ~ - - ~ - - +

    ~ " . " " ,~ . . Hlstid,n. (0) 9 ". ~ Hlstltrnine (&)

    9 " . ".~\ Eetezole (~) \~ ~\ Oiphenhydrarnine

    ~ ~, on AChE (0) ~ (~ PChE (0)

    ', ~ Chlorpheflylarnine on AChE (FI)

    o., , I{ . . . . . . . 0 i0 -8 i0 -7 i0 -6 10 .5 10 -4 i0 -3 i0 -2

    DOSE (MOL)

    Inhibition of erythrocyte AChE and serum PChE by histamine-related compounds.

  • 216 M. AONO ET /IL. Vol. 21, No. 3




    n e

    o 9 P~OOAOL~

    I() -7 10-* 10 -= 10 -4 10-* 10 -= 10 -~ r oo~ (MOL)

    TZU-0460 ETC't'M( m'rmTY (~)

    100 ~--I I "~~m'rv

    50 L ~ SE

    0 6 I~_. 10-" 16-" 10-" 1~-' 10-" 1~-" Tzu-o~o oo~ (MOL)

    Fig. 4.

    % Ranitidine


    6 I I 16-, lg '-o lo-, 16-" lo-' lo-,

    YM-11170 100 o If

    0 ~ rt 10~? 10~,


    F~tXX~0t . I~S~

    I to'-" 1~" to" 10 ~"

    W4-1W/0 ~ OWL)

    Inhibitin of erythrocyte AChE and serum PChE by several Hz antagonists. ID-50 of agents on AChE is shown by the dotted line.

    related compounds such as histidine, histamine and betazole hydrochloride, which have an imidazole ring, had no influence on either cho- linesterase. Histamine Hi-blocking agents, such as chlorphenyramine and diphenhydra- mine, inhibited this activity, especially that of PChE.

    Other Hz antagonists such as ranitidine, TZU-0460 and YM-11170, also inhibited ac- tivities of both cholinesterases (Fig. 4). Raniti- dine was the most potent inhibitor of AChE among them. Its ID-50 was 1.3 x 10-6 M. YM- 11170 was a weak inhibitor of both AChE and PChE. Ileal muscle preparation

    Acetylcholine (0.05 aM) elicited an immedi- ate phasic contractile response followed by tonic contraction, whereas 29 aM of ranitidine increased contractile activity gradually, and enhanced acetylcholine-induced contractions as shown Fig. 5. Cimetidine, TZU-0460 and YM-11170 at a concentration of 26 and 30/zM

    respectively had no effect on acetylcholine- induced contraction. However, at a concentra- tion of 300 pM they slightly stimulated the con- traction; the effect was identical to that seen with 2.9 aM of ranitidine. Ranitidine and physostigmine showed similar patterns of con- traction.

    Fig. 6 shows the effect of Hi-antagonists and atropine sulfate on ranitidine-induced contrac- tion. Diphenhydramine inhibited the contrac- tion induced by ranitidine. Another HI- blocker, pyrilamine, had no effect. The gan- glionic blocker, hexamethonium, had no effect on ranitidine-induced contractile activity. Prior treatment with morphine abolished the ranitidine-induced activity. Exogenous acetyl- choline-induced contractile activity was com- pletely maintained despite the presence of mor- phine. Fig. 7 shows the effects of H2-antago- nists on the contraction of the longitudinal muscle preparation of isolated guinea-pig ileum using electric field stimulation. Hv

  • June 1986 Cholinergic Effects of H=-blockers 217

    Ach Ach Ach Ach

    Ranitidine( 29 )

    Ach Ach ACh Ach Ach

    Cimetidine TZU-OZ,60 ( 40 ) ( 26 )


    Ranitidine (29)

    Fig. 5.

    5 rain.


    t Ranitidine( 29 ) Physostigmine( 0.1z )

    Ach Ach Ach Ach Ach


    YI,6-11170 (3o) (29)


    J ~anitidine C~metid~r~

    ( z.9 ) (4o0) (26o)

    ~ 0mm I~an~idine ~M -11170

    ( z9) (3oo)

    Contraction of isolated guinea-pig ileal preparation by H2 antagonists was measured isotonically. ACh: Acetylcholine 0.05 ltM, parentheses indicated the concentration of agents (pM). Asterisks indicate the washing out of incubated solution in the preparation.

    .5 rain . .~

    Diphenhydramine( 039 ) DPH( 039 ) Pyritamine Pyrilamine [~( o'~)PH~ ) I ~ ( " Pyrilam~ ) Atropine sulf. (DPH> . 1 * I ( 0.1S ) I (0~) [ ~ * I "'1 ..a~t 0.88 ,

    I ]lOmm

    I I I | I | ~anitidine Ranitidine Ranitidine Ranitidine Ranitidine Ach Ach ( ~Z7 ) ( a27 )

    Hexarr~thoniumic6( 37 ) , (C6:37 )1

    t I Ranitidine Ranitidine

    i , , l] I

    Ach( 0.05 ) Ranitidine Ranitidine Ach( 0.05 ) Ranitidine Ach( o.os )

    Fig. 6. Contraction of isolated ileal preparations by ranitidine. Concentration of ranitidine was 29 ,uM. Contraction by ranitidine was blocked by atropine, diphenhydramine and morphine, but not by hexamethonium or pyrilamine. Parentheses indicate the concentration of agents (,uM). Asterisks indicate the washing out of incubated solution in the preparation.

  • 218 M. AONO ET .'IL. Vol. 21, No. 3

    Fig. 7.



    200" o-- Ranitidine o-- Cimetidine * - TZU-0460

    / o- u

    / / t

    100 o ........................................................................................

    0 1 '101 102 103 pM

    Dose-response contraction of electrically stimulated longitudinal muscle strip (0.1 msec, 0.l Hz, 5 V) by H2-antagonists. Basal contraction is expressed as one hundred percent. Data are presented as mean +- S.E. of five experiments.

    antagonists increased the contractile activity dose-dependently. We compared the contrac- tile action of each H2-antagonists. Basal con- tractile activity was expressed as 100%. Raniti- dine stimulated the contraction dose-depen- dently, and was 10 times more potent than the other H2-antagonists. YM-11170 was a very weak stimulant. The highest dose of YM- 11170 used did not elicite a further increase of con- tractile activity.

    Discuss ion

    The present study demonstrated that AChE and PChE were inhibited by H2-receptor antagonists. Ranitidine is the most potent in- hibitor of AChE among the H2-antagonists ex- amined. A previous observation s J2) that the anticholinesterase activity of ranitidine is rather selectively directed toward AChE was confirmed. H2-antagonists may interfere with acetylcholine catabolism. Inhibition of AChE by H~-antagonists might follow the accumula- tion of acetylcholine at cholinergic nerve termi- nals, resulting in cholinergic excitation. As a consequence, several organ functions con- trolled by acetylcholine could be influenced. Histamine as well as acetylcholine receptors are

    important in the control of gastric secretion. Inhibition on gastric secretion by H2-antago- nists might be far more greater than stimula- tion of acetylcholine. Thus, the gastrointestinal functions other than gastric secretion may be affected.

    Furthermore, we observed the action of these drugs during contraction of the isolated guinea-pig ileum. Ranitidine per se induced contraction of the ileum, the pattern of which was similar to that of physostigmine but differ- ent from that of acetylcholine. Doses of cimeti- dine, TZU-0460 and YM-11170 100 times higher than that of ranitidine were needed to elicite even slight contraction.

    The mode of action of ranitidine-induced contraction was examined by using Hi-antago- nists and atropine. Diphenhydramine inhibited the contraction induced by ranitidine. Raniti- dine might have had histamine-Hx activity, however, diphenhydramine inhibited the con- traction induced by acetylcholine. The Hi- blocker, pyrilamine, had no effect. This sug- gests that diphenhydramine acts as an anticho- linergiclS,x4). Pyrilamine did not inhibit the contraction induced by ranitidine, thus raniti- dine has no histamine-Hx activity. Atropine

  • June 1986 Cholinergic Effects of H~-blockers 219

    sulfate inhibi ted the contract ion induced by

    ranit id ine, ind icat ing that rani t id ine has cho-

    l inergic action. The gangl ionic blocker, hexamethon ium,

    had no effect on contract i le effect of ranit id ine.

    pr ior t reatment with morph ine , a blocker of

    endogenous acetylchol ine 11), abol ished the



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