UP S’IW _ ..

Regulation of acetylcholine hydrolysis in canine tracheal smooth m

The regutatiun of acctykhofiue fACh1 lifetime by ~~t~~c~~iue~te~~ fAChE, EC 3.t.i.3 and ~t~k~~~~c~ g&C&E. EC 3.1.1.8) was evafuated in vitro in canine tracheal smooth muscle preparations. Selective inhibition of AChE by Eow concentrations of l.5-bi~~-allyl-N.N-dimcthyi-4-ammoniumphenyl)_pcntaneJ-one dibromide (BW 284C51) ted to increases in the amplitude and half-relaxation time of contractions elicited by electric field stimulation. Maximal responseswere observed in the presence of lo-” M BW 284C51. whew the amplitude and half-relaxation time were increased by 8-t and I%?%. respectively.

Higher concentrations of BW 284CSl. on the other hand. depressed the amplitude and shortened the decay of electric fiikl ~timulat~n-jnduccd contractions by a mechanism involving blockade of muscarinic receptors. Selective inhi~~t~~ of BKhE bc tetraisopr~lpy~p~sphoram~de Go-OMPAl ted to monotonic increases in the electric field stimulation am$itude and duration. These alterations were less marked than those abscrvcd in the presence of BW 284Cjl. Co-application af BW 284CSl (lo-’ M) and iso-OMPG (IO-’ M) resulted in a 13303 prolongation in the decay of electric field stimulation-induced contractions and the development of a sustained contracture. Such contractures were not obsemd with either inhibitor alone at any concentration tested. The results indicate that both hydrolytic enzymes are involved in the rqulation of ACh lifetime at the canine trachea8 neuroeffector junction with AChE exerting the more prominent role. The ftnding that BuChE co-regulates ACb lifetime in canine trachealis mu& demonstrates a functional role for this enzyme. II

Trachea (canine); Smooth muscle: Acetylcholinesterase: Butyrylcholinesterase; BW 284C51: TetraisopropyIpyrophospboramide (iso-OMPA)

2. fntmduction The neurotransmitter acetylcholine (ACh) can be hydrolyzed by two distinct enzymes, acetylcholinesterase (AChE, EC 3.1.1.7) and bu~~rcholinesterase f BuChE, EC 3.f.l.8~. AChE is present at all choiinergic synapses and is responsible for limiting the duration of neurally released ACh. BuChE is found primarily in cardiac tissue. smooth muscle and plasma (Koelle et al.. 1950, 1981; Klingman et al., 1968; Edwards and Brimijoin, 19811, and has no accepted physiological role (Taylor, f985). In most tissues, BuChE accounts

Correspondence to: M. Adler. Nrurotoxicology Branch. USAMRICD. APG. MD ll(llCl-543. U.S.A. Tel. 1.3Of.h71 35f1Z. fax 1.301.67f, ?W. * The opinion or assertions contained herein are the private vie% of the authors and are not to be construed as officiai or as the views of the Army or the Department of Defense. In conducting the research described in this report. the investigators adhered to the ‘Guide for the Care and Use of Laboraton Animals’ as adopted and promulgated by the National Institutes of Health. * * National Resrarch Council Associate Investigator.

for Iess than IO% of the total cholinesterase 58% leads to sustained increases in basal muscle tone (Adler et al., in press). To determine the contribution of the two erwmes to the above phenomena, we examined the actions of the selective inhibitors tetra~~propy~p~oph~phoramide (iso-OMPA) and It5bis(N-allyI-N.N-dimethyi-4ammoniumphenyll-pentane-3-one dibromide (BW 284C51) on baseline tension and electric field stimulation-evoked contractions in canine tracheal smooth muscle. Addition of either BW 284C5t or iso-UMPA

arccs ot. reagents u.sed were as follows: BW atropine. Sigma Chemical Co.. 2,-f. ~~~~~Fmbols represent the meantS.E. of v&es fram I6 strip+ in four animals. Stimulus parameters were 50 V. 1 ms and 10 Hz. Train durations wete Bl s. Drug addition were cumulative with -76)win ktween additions. Significant differences frm cmtroi are indicated by asterisks. P < 0.05.

approximately half-maximal under control conditions; maximal electric field stimulation-elicited tensions were generally obtained at frequencies of 50-60 Hz_ Figure 2 shows concentration-rcs~n~ data from tracheal smooth muscle strips exposed to iso-OMPA ( 10-k-IO-’ MJ by cumulative bath application. Iso-OMPA addition led to increases in the amplitude of electric field stimulation contractions (fig. 2A) and prolongations of the half-relaxation time (fig. 2Bk The effect on amplitude was significant in the presence of iso-0MPA concentrations 2 lo-’ M and the effect on haff-retaxation time was significant with iso-OMPA concentrations 2 3 x lo-’ M. The actions of iso-OMPA were irreversible since no recovery was detected after extensive washout with drug-free sofution (Adler and FiIbert. 1990).

. 0L-d 0

10.8

10’

106

[IS~-OMPA~ M

Fig. 1. Selective inhibition of AChE and BuChE by BW XK51 (A) and iso-OMPA (B). The data were obtained by use of [‘4C]MeCh (0). a substrate specific for AChE or [ ‘%Z]BuCh f l k a substrate specific for BuChE. Experiments were performed on muscle strips and represent surface enzyme activity. The bars indicate +S.E.: n = 12 strips from three animals. The 1005 point corresponds to the hydrolysis of 0.61 *O&l nmol MeCh/h per mg trachealis and 12.3 rt I.1 nmol BuCh/h per mg trachealis. Values that are significantly different from control are indicated by asterisks. P Q 0.05.

3.3. Effect of B W 283c51 on electric field stinrrclatian-indttced cmtractiorn BW 284C51 was found to have a biphasic effect on electric field stimulation contractions_ ~~centration~ of BW 284C51 G lO_’ M increased the amplitude of electric field stimulation responses (fig. 3A) and prolonged their decay (fig. 3B). Higher concentrations depressed the amplitude of electric field stimulation contractions and shortened their decay phase. The

[‘HJONB hinding can presumably account for its inhihitian of muscle tension folIowing electrical stimula&In of cholinergic nerve fibers. The BW 284C514nduced depression of ch~~linergic responses appears to be somewhat selective. since the inhibitor did not alter contractions produced by high K’ (120 mM) (unpublished observations).

on

Tk kfine of cktric field stimuiat~on-elbowed rn5~+& trnsivwr it3 tk prcscncc of high BW ~-lC,Sl ~~~~~~~~~~~ns su_westa that rhe selective AChE inas a LfirrCt hI&i:king effwt on tracheal smooth s preparations. Anxjng the variouls possibilities. an ~~~~rn~~~n~c action appeared likely since in ChE ikir~4 ~~~c~calstrips. an~~mu.~arinic agents produce h a ~~r~~~~~n in electric field s~in~ulation-induced ~~~~~~~~e tension ;md a ~onc~~itsnt shortening in the relaxati0n time k4dler et al.. 1991). To determine whether BW IX4CSI has antimusrties. we ttsteo its ahilit?: to compete with rnu~ar~~jc receptor sites in canine tramuscle homogenaaes. As shown in fig. 4. was hugely effective in displacing [-‘H]QNB pecific binding Sites (K, = I.94 x IO-” NJ. In 3S4GI was considerably more potent than holine in antagonizing [“HMNB binding al.. f%?k The observed action of BW 3XSl

Figure 5 shows data from a representative tracheal smooth muscle strip exposed to BW 28N31 (lo-’ M) and iso-OMPA (l0-c M). alone and in combination, Each inhihitor produced a SF.II increase in the amplitude and duration of electric field stimuIation-induced cont~ctions. however. combined exposure to the two inhibitors resulted in a S-fold greater prolongation in the half-relaxation time of these contractions than was observed with either inhibitor alone. Furthermore. the combined inhibitors also generated a sustained base-

Control

BW 284C51

ISO-OMPA

4 BW 284651 Fig. 6. Records illustrating the sefective and combined inhibition of AChE and BuChE on electric field stimul3ti~n-eyo~ed ~ntr~ctions. The traces wrre obtained from a sin& strip under the indicated conditions. The concentration of each inhihitor was IO-’ M. BW ZXICT I was removed hy wash (7h min) prior to addition of iso-OMPA. The hottom trace shows the consequences of re-application of BW XK31 after irrerrrsihle inhibition of BuChE hy prior iso-OMPA treatment. Note the contraeture following inhihlion of both enzynes. Train durations are indicated hy horizontal bars.

Fig. 6. llidogtams illustraGng the cfkts of progtekre inhibition of BuChE by iw-OMPA on the half-rclaxrtinn time of electric field sGmul;rticm-uliciwd con~r;lcGons in rrlchral strip prcttrakd with BW %lC’.SI Lo inhibit AChE. The hurs denote the mean+S.E. of velues ohlaintrd from IZ strips in three mimllls. Vrrlues that are

significantly different ftum c~mlrnl arc: indicated hy a~ktisl;a

line contracture that was not observed with either inhibitor alone at any concentration tested. These rcsuits suggest that in canine

tracheal

both AChE and BuChE participate ACh foil~ing nerve stimulation.

smooth

muscle,

in the hydrolysis of

results are clearly in accord with the former hypothesis that BuChE plays a significant rote in regulating lifetimes in canine trdchealis muscle.

ACh

Additional evidence in support of the above conclu-

tt may be argued than when high concentrations

of

sion is provided

in table I. The data were obtained

by

iso-OMPA and BW 284C51 are co-applied, the potcntiation observed in muscle tension stems from the small

examining the interaction of equimolar concen:ratiom of BW 2X4CSl and iso-OMPA on electric field stimula-

additional

tion-elicited

inhibition

of AChE

by iso-OMPA.

than from its blockade of BuChE. tween

these

possibilities,

rather

To distinguish

iso-OMPA

was

added

bein

graded increments to strips equilibrated with 10 -5 M BW 284C51 (fig. 6). If BuChE hydrolyzing

ACh

in canine

is indeed important tracheal

smooth

for

muscle,

potentiation should be evident with low concentrations of iso-OMPA. If only AChE can hydrolyze transmitter that is reteased during stimulation,

potentiation

will he

restricted to high concentrations of iso-OMPA where the inhibitor produces a small blockade of AChE (see fig. IA). The data in fig. 6 reveal a marked enhancement in the half-relaxation time beginning with IO-’ M iso-OMPA, where BuChE activity is depressed significantly

TABLE

but AChE

activity

is unaltered.

Thus

the

tensions and ChE activities. Of particular

interest was the &ding that in strips exposed to 3 x 1tl-7 M BW 284CSL the decays of eIectric field stimulation contractions showed a much larger enhancement when an equimolar concentration of iso-OMPA was co-applied (from 16.6 to 43.X s) than when the BW 284C51 concentration was raised to IO-’ M. Addition of 3 x IO-’ M iso-OMPA caused littfe or no inhibition of AChE, whereas raising the BW 284C51 concentration to IO-”

M led to a further

reduction

in AChE

activity from 38.6 to 23.3% of control (table 1). The effect on basal tone of combined BW 28X51 and iso-OMPA administration is depicted in fig. 7. The lower curve was obtained by addition of BW 284C5 1 to tracheal smooth muscle strips pretreated with IO-” M

1

Effectsof iso-OMPA and BW 74HC51 on electric field ~limulatit~n-elicited contractions and ChE actkitic\ * Conditions

Tension h

k) Cant ml iso-OMPA (3 x 10 ’ Ml BW ‘WCS1 (3 x 10 ’ M) iso-OMPA + BW 2X-0 1 ’ iso-OMPA (lo-” M1 5W 28JCSl flU-’ Mf iso-OMPA + BW 24CS 1 p

I?.‘)+ I..l 17.2 f I .3 ,.i.g+ I.1 J 23.-‘,-+ I .5 4 1x.3+ I.4 Ii X3+ 1.7 11 ‘9.7 + 3.2 d

IfRT ‘ lsl

ACbE tci control activi;ity)

5uChE tc; control actkiQt

5.2 ir 0.3 9.6 2 0.4 J 16.3 * 1.7 J 43.x * 3.2 &= 13.3 * 0.Y li 17.1 rt1.2 .l hY.3 & 5.7 kc

I(W) Yh.? *x.4 38.6 + 2.Y d 41.9+3.4 a 92.3 -f 7.’ 22.1 f 2.8 IJ 20.7rt3.1 d

f 00 1h.X* I .3 J 105.3 _t 12.7 11.1+ 1.2 J 2.4 2 03 11 X9.6 i_ 9.7 1.3&O.I J

a AChE and BuChE activities were determined on I?--lh srripa from 3-1 animals using I’HIMeCh and [‘H]BuCh as descnkd in the Materials and methods section. h Tension measurements were prrformed on I6 strips from 1 animals. r‘ Half-relaxation time. ’ Differs signtfkantly frttsn conrrol (P < 0.05). ’ Differs significantly from corresponding values in iso-OMPA or BW 2XKSl alone. t Both inhibiton wzre pwen: ai 3 X 10. ’ M. B Both inhibitors were present at 1K” M.

reeutts of tfiis investigation denxmstrate that in wwh mus&. selective inhibition of of the two ACh hydrolyzing enzymes. AChE or E. ~zu.scwincreases in the amplitude and decay of rbcctti field srimuht~~,n-~\-c~teJ czmtractions with little cl~:\ntirbn in the basal tone. Combined inhibition h enzyntc\s. htwvever. leads to a marked prolongathe decay of clcctric field stimulation responses and a sustained contrxture. Of ttk‘ two ChE inhibitors. the organophosphorus asnt &-OMPA appeared to be more selective and w-as found to be &void of direct actions on muscle _ At the highest concentr&tion tested on electric ~rnu~at~~nrespomsesI IO ’ W. i.so-OMPA produccd complete inhibition of BuChE and only 139 ~~~~~~t~~~of AChE (fig. IBl. With the divalent BW LS-tC51 compound it was not possible to inhibit AChE in muscle strips completely without encountering a loss of sekcti\;iQ (fig. IA). Moreover. BW X-K31 also e~~b~ted a marked depressant effect on trachealis muscies in the same concentration range that it inhibited AChE (figs. 3 and 71. This action was attributed p~mari1y to c-A-u.,peiitive blockade of mubcarinic receptors as revealed by E-‘H]QNB displacement experiments (fig. 4). lhe antimuscatinic action of BW 384C51 made a rigorous assessment of the role of AChE in tracheal h muscle contractility difficult. In principle. it is tbk to evaluate the direct depressant action of BW 22X31 by using soman-treated muscles with com@etch inhtbited ChEs (fig. 7). On such preparations.

CazGzw t~&csf

BW XK~l caused a mruuuonic reduction of muscle tension. since the facilitation expected from ChE blruzkade H’S already maximal due to the soman CX~Osure. It is of interest that in the presence of soman. the direct inhibitory action of BW XlC5I was evident at concentrations zs3 x ItI- ’ M. whereas under conditions in which ChE activity is only partially blocked, the reduction in muscle tension required a 3 to l&fold increase in BW XK5I concentration (figs. 2 and 7). These differences reflect the mutual antagonism between AChE inhibition and muscarinic receptor bloekade on elcctrie field stimulation-elicited tensions. The inability of BW ZS-lCSI to produce total inhibition d AChE without loss of selectivity created additional difficulties for a rigorous assessment of the contribution of AChE in tracheal smooth mu&z function. However, it is clear that reductions in AChE activity alone cxutld not account adequately for the observed actions of anti-ChEs in canine trachea. Thus. in the presence of agents that block both AChE and BuChE. inhibition of total hydrolytic activity by approximately SiFi. using a variety of non-selective inhibitors, resulted in cuntractures of 12-15 g and prolongations of over ltl-fold in the decay of electric field stimulation responses (Adler et al.. in press). Alterations of this magnitude were never observed following exposure to BW Z~S43I alone, even though at a concentration of lo-’ M. BW 34C51 inhibited AChE activity by over ‘s03. it is unlikely that the failure to observe these more pronounced effects can be attributed entirely to the antimuscarinic action of BW 284C51, since the co-application of iso-OMPA led to marked prolongations in the decay of electric field stimulation-induced tensions (fig. 6) and to the appearance of contractures (fig. 7) with no additional inhibition of AChE (fig. 1. table 1). In conclusion, AChE has iong been recognized as an important enzyme for regulating the lifetime of ACh at both nicotinic and muscarinic synapses. The role of BuChE. by contrast. has been poorly understood. In spite of its abundance in vertebrate smooth muscle, the function of BuChE has rem&cd enigmatic. The present study suggests that BuChE has an important role in canine trachealis muscle in co-regulating the lifetime of synaptically released ACh. This represents the first well defined function for smooth muscle BuChE since Koelle sugges:ed a possible role for this enzyme over 40 years ago (Koelle et al., 1950). It remains to be determined whether BuChE has a similar role in other preparations.

Acknowledgements The authors are most grateful to Drs. George B. Koelle. Bert N. LaDu and Robert E. Sheridan for their many helpful suggestions and discussions. We are indebted to Mr. Kenneth Rent for surgical

wpport. We also thank Ms. Sorabc Phwn. Ms. Angel-a M. Nicodcnuts. Mr. Richard Gullick and Ms. Ilelcn excellent rcchnical assistance.

MacFarlane

for their

References Aas. P.. R. Veiteberg and F. Fonnum. 1986. In vitro effects of soman on bronchial smooth muscle, Biochem. Pharmacol. 35, 1793. Adler, M. and M.G. Filbert. The role of butyrylcholinesterase in canine trxheal smooth muscle function, FEBS Lett. 2o7. IU7. Adler. M.. D.H. Moure and M.G. Filbert. 1991. Effects of anticholinesterases on airway smooth muscle. in: Basic and CTinical Toxicology of Grganophosphates and Carbamates. eds. B. Ballantyne and T. Maws (Butterworth. U.K.) (in press). Adler. M.. S.A. Reutter. D.H. Moore and M.G. Filbert, 1987. Actions of soman on isolated tracheal smooth muscle. in: Cellular and Molecular Basis of Cholinergic Function, eds. M.J. Dowdall and J.N. Hawthorne (Ellis Hawood, Derbyshire. U.K.) p. 582. Diegenbach. PC.. 196. Use of inhibitors in cholinesterase histochemistry. Nature 207. 30X. Edwards, J.A. and S. Brimijoin. 19X2, Divergent regulation of acetylcholinesterase and butyrylcholinesterase in tissues of the rat. J. Neurochem. 3X. 1393. Filbert. M.G.. S.A. Reutter. D.H. Moore and M. Adler. 19X7. Effect of acetylcholinesterase inhibitors on tracheal smooth muscle, in: International Symposium on Muscarinic Cholinergic Mechanisms. eds. S. Cohen and M. Sokolovsky (Freund Publishing House Ltd.. U.K.) p. IX.

Klingman. G.I.. J.D. Ktingmwr and A. Pdrw_c&. I%% ilcer~+- and pseudocbolinesteraw activitie\ in sympathetic pugha clt (44%.J Neurorhem. 15. 1121. Koelle. G.B.. ES. Koelle and IS. Fricdewatd. I%it. The rffe